Biological fillers for tissue restoration and regeneration

Abstract

Compositions and methods for filling tissue voids and defects using self-assembling biopolymers configured to form a shape-retaining matrix are described. As described herein, the inventors have developed a flowable tissue filler comprising in situ polymerizable oligomeric collagen and a neutralizing (self-assembling) buffer. After mixing the liquid collagen with the neutralizing buffer, the neutralized collagen solution can be used to fill tissue voids (e.g., surgical wounds) or defects, including deep and / or difficult-to-access irregularly shaped tissue voids. Upon application, the applied solution rapidly forms fibrils (in approximately one minute at body temperature) through molecular self-assembly. The resulting tissue-filling matrix restores and maintains tissue shape and consistency over time and triggers a tissue implant response characterized by cellularization, vascularization, and new tissue formation, without triggering the inflammatory response typically observed in wound healing or the foreign body reaction typically observed in conventional tissue implant responses.

Description

[Technical Field]

[0001] Related applications This application claims the advantages of U.S. Provisional Patent Application No. 62 / 966,398, filed on 27 January 2020, and U.S. Provisional Patent Application No. 63 / 015,946, filed on 27 April 2020. The entire contents of both are incorporated herein by reference, however, in the event of any conflict between the disclosures or definitions herein and those herein, the disclosures or definitions herein shall prevail.

[0002] This instruction relates, in general, to methods, compositions, and kits for treating tissue voids and defects using tissue fillers to restore and regenerate tissue. [Background technology]

[0003] Breast cancer is the most commonly diagnosed cancer in women, with over 2 million new cases reported worldwide each year, and approximately 330,000 cases in the United States alone. It is estimated that 60-70% of these cases (approximately 1.3 million worldwide) are treated with breast-conserving surgery (BCS; or commonly known as mammary tumor removal), which is typical of the standard treatment for early-stage breast cancer. Conventional BCS involves the removal of healthy tissue that does not contain the tumor or cancer (negative margin) through a small, cosmetically defined incision. BCS with adjuvant radiation is preferred over total mastectomy (i.e., removal of the entire breast) for eligible patients because it offers comparable survival rates while preserving the patient's breast and reducing surgical time, recovery time, and complications. Long-term outcomes and survivorship are particularly important in the treatment of this disease, given the relatively high survival rate for breast cancer (approximately 90%). In particular, with regard to breast cancer sculpting (BCS), it is crucial to completely remove cancerous tissue in a single surgical procedure (achieving a negative margin) while preserving the shape, appearance, and consistency of the breast (i.e., achieving satisfactory breast cosmetic surgery) in order to achieve a good outcome and a good quality of life for the patient.

[0004] One of the main challenges of breast cancer surgery (BCS) is to remove all cancerous tissue while simultaneously maintaining an acceptable cosmetic outcome. Standard guidelines for BCS involve "closing the excision (surgical) defect in layers as cosmetically as possible" after tumor removal. This is followed by the healing of the complex surgical wound, and the subsequent filling of the remaining tissue void with serous fluid and / or blood, initially forming a seroma or hematoma, then scarring and atrophy. For surgeons, predicting the cosmetic outcome of BCS is extremely difficult, if not impossible, given the significant patient variability in breast tumor size, shape, and location, as well as the unpredictable nature of the tissue repair process, which is exacerbated by the effects of adjuvant radiotherapy. As a result, a relatively high level of BCS-related breast deformity remains, with approximately one-third of women experiencing unsatisfactory cosmetic results (e.g., hollowing, distortion, and intermammary asymmetry). Such outcomes have ultimately been shown to reduce the overall quality of life of breast cancer survivors, increasing breast pain and discomfort due to scarring and atrophy, feelings of depression, anxiety, and anxiety disorder, and negatively impacting self-esteem, body image, and intimacy. Furthermore, the need for secondary surgical procedures (e.g., re-excision of residual tumors and corrective / reconstructive procedures to repair depressions / deformities) remains high with respect to BCS, estimated to be in the range of 20–40%. This includes re-excision due to positive margins, as well as corrective and reconstructive procedures to repair breast deformities. Overall, it is estimated that reoperations after the initial BCS due to these poor outcomes and complications add an average of over $16,000 to medical costs per additional procedure. Based on these challenges and concerns, BCS may not be an option for all women, especially those with tumors that are large relative to breast size (diameter > 5 cm; tumor: breast volume > 1.5 percent) or within the lower quadrant of breast size.Therefore, breast surgeons need novel treatment options to further optimize the oncological and cosmetic outcomes of BCS and enable the confident application of this conservative treatment to a wider range of patients with desirable outcomes.

[0005] Currently, there are no additional tissue products that would allow surgeons to restore, reconstruct, or regenerate tissue, such as breast tissue, as predicted. Furthermore, it is clear that surgeons are actively seeking solutions for this challenge. In particular, many surgeons have been trying to use a relatively new three-dimensional helical-shaped tumor bed bioabsorbable marker called BioZorb, which is primarily intended to mark surgical cavities or defects from mammary tumor removal for targeted adjuvant radiotherapy. Breast surgeons have applied this implantable device with the hope that it would not only serve as a marker but also fill tissue cavities and improve cosmetic outcomes. However, both surgeons and patients are not uniformly satisfied with BioZorb, especially since it results in a hard, palpable implant that lasts up to 2.8 years and increases patient pain and discomfort. In addition, surgeons have shown that it is expensive compared to other radiomarkers and does not significantly improve outcomes. [Overview of the project] [Problems that the invention aims to solve]

[0006] On the other hand, there are two experimental surgical reconstruction options: autologous lipid (fat) grafting (also known as lipofilling or lipid transplantation) and oncoplastic surgery, which aim to improve the cosmetic outcomes of BCS and potentially expand the BCS-eligible patient population. Lipid grafting involves harvesting lipids (adipose tissue) from one area of ​​the body via liposuction and reinjecting minimally processed lipid tissue into another area (e.g., tissue cavities). Originally, lipid grafting was used for delayed breast reconstruction procedures, but recently it has been investigated for use immediately after BCS. Problems associated with this approach include rapid reabsorption leading to significant volume loss (ranging from 25% to 80%), lipid necrosis, oil cyst formation, microcalcification, and questions regarding oncological safety (i.e., cancer recurrence). On the other hand, oncoplastic surgery is used in combination with surgical oncology and plastic surgery techniques to perform breast reconstruction at the time of mammary gland tumor removal. Oncoplastic procedures encompass both volume replacement (rearrangement of remaining healthy breast tissue) and volume replenishment (reconstruction using various autologous tissue flaps). While both surgical reconstruction procedures demonstrate the advantages of using the patient's own tissue and have achieved some success, they require specialized training, frequent involvement of multiple surgeons, and lengthy surgical procedures, thus limiting their availability and increasing costs. Currently, these techniques are not yet widely adopted due to the required specialized training and lingering concerns about sacrificing oncological safety and efficacy to improve cosmetic outcomes. [Means for solving the problem]

[0007] The present invention describes a regenerative and regenerative tissue filler that may be applied as a liquid to any type of tissue void or defect—including, but not limited to, tissue voids resulting from surgical wounds (e.g., surgical wounds resulting from BCS, but not limited to), physical defects (e.g., scars, depressions, birth defects, etc.), injuries, disease progression, and / or similar—before transitioning to a fibrous collagen matrix having tissue consistency.

[0008] As described herein, the inventors have developed a fluid tissue filler comprising in-situ polymerizable oligomeric collagen and a neutralizing (self-assembling) buffer. After mixing the liquid collagen and the neutralizing buffer, the neutralized collagen solution can be used to fill tissue voids (e.g., surgical wounds) or defects, including tissue voids that are deep and / or difficult to access and have an irregular shape. Upon application, the applied solution rapidly forms fibrils (in about 1 minute at body temperature) via molecular self-assembly. The resulting tissue filler matrix restores and maintains tissue shape and consistency over time, eliciting a tissue embedding response characterized by cellularization, angiogenesis, and novel tissue formation, without causing the inflammatory response typically observed in wound healing or the foreign body reaction typically observed in conventional tissue embedding responses.

[0009] In some embodiments, the tissue fillers according to this teaching may offer one or more of the following advantages: (1) less or no scarring (i.e., compared to conventional non-filling procedures); (2) less or no defect atrophy (i.e., compared to conventional non-filling procedures); (3) less or no inflammatory mediators or inflammatory response (i.e., compared to conventional non-filling procedures); (4) tissue consistency similar to that of natural tissue (e.g., compressibility or range of compressibility similar to that of natural tissue); (5) recovery and generation of breast tissue, including adipose tissue, mammary gland tissue, etc.; (6) recovery and generation of skeletal muscle; (7) tissue implantation response does not interfere with a given clinical procedure, including re-excision, ultrasound, or radiography; and / or (8) less tissue implantation response (i.e., compared to conventional procedures) or is not adversely affected by adjuvant irradiation (e.g., fewer or no areas of lipid cysts, microcalcifications, lesions, and / or high opacity, any of which may interfere with imaging).

[0010] In some embodiments, the method for filling tissue voids or defects according to this teaching comprises (a) introducing a self-assembling biopolymer into the tissue voids or defects, and (b) polymerizing the self-assembling biopolymer to form a shape-retaining matrix.

[0011] In some embodiments, a method for filling tissue voids or defects resulting from a mammary tumor resection or mastectomy procedure according to this teaching comprises (a) introducing a mixture comprising an oligomeric collagen solution and a neutralizing solution into the tissue voids or defects; and (b) polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix. The oligomeric collagen solution may comprise lyophilized type I oligomeric collagen and an acid.

[0012] In some embodiments, a method for filling tissue voids or defects resulting from a mammary tumor removal or mastectomy procedure comprises (a) introducing a mixture comprising an oligomeric collagen solution and a neutralizing solution into the tissue voids or defects; and (b) polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix. In some embodiments, the oligomeric collagen solution may comprise lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid. In some embodiments, the concentration of the oligomeric collagen solution is about 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen. In some embodiments, the ratio of oligomeric collagen solution to neutralizing solution is about 9:1.

[0013] In other embodiments, a collagen matrix prepared according to any of the methods described above is provided. In further embodiments, a kit comprising a collagen composition and a buffer is provided. In further embodiments, a kit comprising lyophilized type I oligomeric collagen, a hydrochloric acid solution, and a buffer is provided. In further embodiments, one or more therapeutic agents acceptable to the collagen matrix are provided—including, but not limited to, chemotherapeutic agents, anti-inflammatory agents, antibiotics, analgesics, and / or similar, and combinations thereof—so that one or more therapeutic agents are delivered into the matrix at the site of a tissue void or defect. In some embodiments, one or more therapeutic agents are configured to be delivered locally into the matrix at the site of a tissue void or defect.

[0014] Additional features and advantages of this teaching may be described by embodiments shown in any of the following listed sections. It should be understood that any embodiment described herein may be used in relation to other embodiments described herein, to the extent that the embodiments do not contradict each other. Therefore, any applicable combination of the following listed sections should be considered.

[0015] 1. A method for filling tissue voids or defects in a patient, comprising: introducing a self-assembling biopolymer into the tissue voids or defects; and polymerizing the self-assembling biopolymer to form a shape-retaining matrix.

[0016] 2. The method according to item 1, wherein the self-assembling biopolymer comprises in-situ polymerizable oligomeric collagen.

[0017] 3. The method according to any one of the preceding items, wherein the in-situ polymerizable oligomeric collagen contains collagen molecules.

[0018] 4. The method according to any one of the preceding items, wherein at least a portion of the collagen molecules are covalently bonded by one or more intermolecular crosslinks.

[0019] 5. The method according to any one of the preceding items, wherein the patient is a mammal.

[0020] 6. The method described in any one of the preceding items, wherein the patient is human.

[0021] 7. The method described in any one of the preceding items, wherein the introduction is achieved under sterile conditions.

[0022] 8. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises in-situ polymerizable collagen and the shape-retaining matrix comprises a collagen fibril matrix.

[0023] 9. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises liquid type I collagen.

[0024] 10. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises type I oligomeric collagen derived from porcine dermis.

[0025] 11. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and an acid.

[0026] 12. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid.

[0027] 13. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution further comprising a buffer.

[0028] 14. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises an oligomeric collagen solution and a buffer, and the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid.

[0029] 15. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises an oligomeric collagen solution and a buffer, the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid, and the ratio of the oligomeric collagen solution to the buffer is approximately 9:1.

[0030] 16. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid, the concentration of the solution being about 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen.

[0031] 17. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation.

[0032] 18. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation and then filtered through a sterile membrane filter.

[0033] 19. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing freeze-dried type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation, irradiated with ultraviolet light, and then filtered through a sterile membrane filter.

[0034] 20. The self-assembling biopolymer contains a solution comprising freeze-dried type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation to 500 mJ / cm³. 2 The method according to any one of the preceding items, wherein the object is irradiated with ultraviolet light and then filtered through a sterile membrane filter.

[0035] 21. The method according to any one of the preceding items, wherein the introduction involves injecting a biopolymer that self-assembles into a tissue cavity or defect via a syringe.

[0036] 22. The method according to any one of the preceding paragraphs, wherein tissue cavities or defects are produced by the procedure for removing a mammary tumor.

[0037] 23. The method described in any one of the preceding paragraphs, wherein a tissue void or defect is created by the mastectomy procedure.

[0038] 24. The method according to any one of the preceding items, wherein filling a void or defect in the tissue does not result in defect atrophy or scar tissue formation.

[0039] 25. The method according to any one of the preceding items, wherein filling a void or defect in tissue does not result in an inflammatory mediator, inflammatory response, or foreign body reaction.

[0040] 26. The method according to any one of the preceding items, wherein filling voids or defects in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0041] 27. The method according to any one of the preceding items, wherein filling voids or defects in the tissue results in the formation of breast tissue, including adipose tissue, mammary gland tissue, or a combination thereof.

[0042] 28. The method according to any one of the preceding items, wherein the tissue embedding response to filling tissue voids or defects is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesion masses, and / or areas of high opacity.

[0043] 29. A method for filling tissue voids or defects in a patient, wherein the tissue voids or defects are caused by a mammary tumor removal or mastectomy procedure, and the method comprises: introducing a mixture comprising an oligomeric collagen solution and a buffer into the tissue voids or defects; polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid.

[0044] 30. The method according to item 29, wherein the ratio of oligomeric collagen solution to buffer solution is approximately 9:1.

[0045] 31. The method according to item 29 or 30, wherein the acid contains 0.01 N hydrochloric acid and the concentration of the oligomeric collagen solution is approximately 8 mg / mL based on the dry weight of lyophilized type I oligomeric collagen.

[0046] 32. A method for filling tissue voids or defects in a patient, wherein the tissue voids or defects are caused by a mammary tumor removal or mastectomy procedure, and the method comprises: introducing a mixture comprising an oligomeric collagen solution and a buffer into the tissue voids or defects; polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid; the concentration of the oligomeric collagen solution is about 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen; and the ratio of the oligomeric collagen solution to the buffer is about 9:1.

[0047] 33. The method according to paragraph 32, wherein the oligomeric collagen solution is clarified using ultracentrifugation, filtered through a sterile membrane filter, irradiated with ultraviolet light, or a combination thereof.

[0048] 34. The method described in any one of paragraphs 32 to 33, wherein the tissue void or defect includes a wound.

[0049] 35. The method described in any one of paragraphs 32 to 34, wherein the tissue void or defect includes a surgical wound.

[0050] 36. The method according to any one of paragraphs 32 to 35, wherein tissue voids or defects result from the removal of the tumor.

[0051] 37. The method according to any one of paragraphs 32 to 36, wherein tissue cavities or defects result from the removal of a breast tumor.

[0052] 38. The method according to any one of the claims 32 to 37, wherein a self-assembling biopolymer comprises a tissue filler.

[0053] 39. The method according to any one of paragraphs 32 to 38, wherein filling a void or defect in the tissue does not result in defect atrophy or scar tissue formation.

[0054] 40. The method according to any one of paragraphs 32 to 39, wherein filling a void or defect in tissue does not result in an inflammatory mediator, inflammatory response, or foreign body reaction.

[0055] 41. The method according to any one of claims 32 to 40, wherein filling voids or defects in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0056] 42. The method according to any one of paragraphs 32 to 41, wherein filling a void or defect in the tissue results in the formation of breast tissue, including adipose tissue, mammary gland tissue, or a combination thereof.

[0057] 43. The method according to any one of paragraphs 32 to 42, wherein the tissue embedding response to filling tissue voids or defects is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesions, and / or areas of high opacity.

[0058] 44. A method for filling a wound, comprising: introducing a mixture comprising an oligomeric collagen solution and a buffer into the wound; polymerizing the oligomeric collagen solution to form a collagen-protofibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized oligomeric collagen and an acid.

[0059] 45. The method according to paragraph 44, wherein the freeze-dried oligomeric collagen comprises freeze-dried type I oligomeric collagen.

[0060] 46. ​​The method described in paragraph 44 or 45, wherein the wound includes a surgical wound.

[0061] 47. The method described in any one of paragraphs 44 to 46, wherein the surgical wound results from the removal of a tumor.

[0062] 48. The method according to any one of paragraphs 44 to 47, wherein the surgical wound results from the removal of a breast tumor.

[0063] 49. The method according to any one of items 44 to 48, wherein the oligomeric collagen solution comprises a tissue filler.

[0064] 50. The method according to any one of paragraphs 44 to 49, wherein filling the wound does not result in defect atrophy and scar tissue formation.

[0065] 51. The method according to any one of paragraphs 44-50, wherein filling the wound does not result in an inflammatory mediator, inflammatory response, or foreign body reaction.

[0066] 52. The method according to any one of paragraphs 44 to 51, wherein filling a wound results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0067] 53. The method according to any one of paragraphs 44 to 52, wherein filling a wound results in the formation of breast tissue, including adipose tissue, mammary gland tissue, or a combination thereof.

[0068] 54. The method according to any one of paragraphs 44 to 53, wherein the tissue implantation response to wound filling is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesions, and / or areas of high opacity.

[0069] 55. A method for restoring and regenerating skeletal muscle tissue in a void or defect in a patient's tissue, comprising: introducing a self-assembling biopolymer into the void or defect in the tissue; and polymerizing the self-assembling biopolymer to form a shape-retaining matrix.

[0070] 56. The method described in paragraph 55, wherein the tissue void or defect includes a wound.

[0071] 57. The method described in paragraph 55 or 56, wherein the tissue void or defect includes a surgical wound.

[0072] 58. The method according to any one of paragraphs 55 to 57, wherein tissue cavities or defects result from the removal of the tumor.

[0073] 59. The method according to any one of paragraphs 55 to 58, wherein the restoration and regeneration of skeletal muscle tissue in a tissue void or defect does not result in defect atrophy or scar tissue formation.

[0074] 60. The method according to any one of paragraphs 55-59, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects does not result in inflammatory mediators, inflammatory responses, or foreign body reactions.

[0075] 61. The method according to any one of paragraphs 55 to 60, wherein the restoration and regeneration of skeletal muscle tissue in a void or defect in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0076] 62. The method according to any one of paragraphs 55 to 61, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects results in the generation of skeletal muscle together with adipose tissue.

[0077] 63. The method according to any one of paragraphs 55 to 62, wherein the tissue implantation response to the restoration and regeneration of skeletal muscle tissue is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesions, and / or areas of high opacity.

[0078] 64. A method for restoring and regenerating skeletal muscle tissue in a tissue void or defect, comprising: introducing a mixture comprising an oligomeric collagen solution and a buffer into the tissue void or defect; polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid.

[0079] 65. The method described in paragraph 64, wherein the tissue void or defect includes a wound.

[0080] 66. The method described in paragraph 64 or 65, wherein the tissue void or defect includes a surgical wound.

[0081] 67. The method according to any one of paragraphs 64 to 66, wherein tissue voids or defects result from the removal of the tumor.

[0082] 68. The method according to any one of paragraphs 64 to 67, wherein the restoration and regeneration of skeletal muscle tissue in a tissue void or defect does not result in defect atrophy or scar tissue formation.

[0083] 69. The method according to any one of paragraphs 64-68, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects does not result in inflammatory mediators, inflammatory responses, or foreign body reactions.

[0084] 70. The method according to any one of paragraphs 64 to 69, wherein the restoration and regeneration of skeletal muscle tissue in a void or defect in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0085] 71. The method according to any one of paragraphs 64 to 70, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects results in the generation of skeletal muscle together with adipose tissue.

[0086] 72. The method according to any one of paragraphs 64 to 71, wherein the tissue implantation response to the restoration and regeneration of skeletal muscle tissue is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesions, and / or areas of high opacity.

[0087] 73. A method for preparing a matrix in a tissue void or defect, comprising using a single mixing step to polymerize collagen, wherein the single mixing step comprises mixing a collagen composition with a buffer to form a collagen solution, and polymerizing the collagen in the collagen solution to form a matrix.

[0088] 74. The method according to claim 73, further comprising incubating the collagen solution at a temperature above approximately 25°C to promote the polymerization of collagen in the collagen solution.

[0089] 75. The method according to paragraph 73 or 74, further comprising incubating the collagen solution at a temperature of approximately 37°C to promote the polymerization of collagen in the collagen solution.

[0090] 76. The method according to any one of items 73 to 75, wherein the collagen comprises collagen oligomers.

[0091] 77. The method according to any one of items 73 to 76, wherein the collagen comprises collagen molecules.

[0092] 78. The method according to any one of items 73 to 77, wherein the collagen consists of collagen oligomers.

[0093] 79. The method according to any one of paragraphs 73 to 78, wherein the collagen consists of intermolecularly cross-linked collagen molecules.

[0094] 80. The method according to any one of items 73 to 79, wherein the collagen substantially consists of intermolecularly cross-linked collagen molecules.

[0095] 81. The method according to any one of items 73 to 80, wherein the collagen further comprises telocollagen.

[0096] 82. The method according to any one of items 73 to 81, wherein the collagen further comprises atelocollagen.

[0097] 83. The method according to any one of claims 73 to 82, wherein collagen containing collagen oligomers is obtained from tissue containing collagen oligomers, from cells that produce collagen oligomers, or by chemically crosslinking collagen to obtain collagen oligomers.

[0098] 84. The method according to any one of items 73 to 83, wherein the collagen is derived from porcine skin tissue.

[0099] 85. The method according to any one of claims 73 to 84, wherein the collagen composition further comprises an acid.

[0100] 86. The method according to any one of claims 73 to 85, wherein the acid is selected from the group consisting of hydrochloric acid, acetic acid, lactic acid, formic acid, citric acid, sulfuric acid, and phosphoric acid.

[0101] 87. The method according to any one of headings 73 to 86, wherein the acid is hydrochloric acid.

[0102] 88. The method according to any one of the items 73 to 87, wherein the hydrochloric acid is hydrochloric acid of about 0.005 N to about 0.1 N.

[0103] 89. The method according to any one of the items 73 to 88, wherein the hydrochloric acid is approximately 0.01 N hydrochloric acid.

[0104] 90. The method according to any one of items 73 to 89, wherein the concentration of collagen in the collagen solution is approximately 0.1 mg / ml to approximately 40 mg / ml.

[0105] 91. The method according to any one of items 73 to 90, wherein the concentration of collagen in the collagen solution is approximately 7 mg / mL to approximately 8 mg / mL.

[0106] 92. The method according to any one of items 73 to 91, wherein the concentration of collagen in the mixture of collagen solution and buffer is approximately 6.3 to approximately 7.2 mg / mL.

[0107] 93. The method according to any one of items 73 to 92, wherein the collagen composition is sterilized.

[0108] 94. The method according to any one of claims 73 to 93, wherein a collagen composition, collagen solution, or collagen matrix is ​​sterilized by a method selected from the group consisting of exposure to chloroform, viral filtration, sterile filtration, gamma irradiation, ultraviolet irradiation, electron beam, and combinations thereof.

[0109] 95. The method according to any one of items 73 to 94, wherein the collagen composition is sterilized by filtration.

[0110] 96. The method according to any one of items 73 to 95, wherein the buffer solution contains approximately 0.03 mM to approximately 0.2 mM MgCl2.

[0111] 97. The method according to any one of items 73 to 96, wherein the buffer solution contains approximately 0.002 mM to approximately 0.02 mM MgCl2.

[0112] 98. The method according to any one of items 73 to 97, wherein the buffer solution contains less than approximately 0.02 mM MgCl2.

[0113] 99. The method according to any one of items 73 to 98, wherein the buffer solution does not contain MgCl2.

[0114] 100. The method according to any one of paragraphs 73 to 99, wherein the buffer further comprises approximately 0.3 mM to approximately 3 mM KH2PO4.

[0115] 101. The method according to any one of items 73 to 100, wherein the buffer further comprises approximately 1 mM to approximately 10 M Na2HPO4.

[0116] 102. The method according to any one of items 73 to 101, wherein the buffer solution further comprises approximately 0.1 mM to approximately 4 mM KCl.

[0117] 103. The method according to any one of items 73 to 102, wherein the buffer further comprises approximately 0.02 M to approximately 0.3 M of NaCl.

[0118] 104. The method according to any one of items 73 to 103, wherein the buffer further comprises NaOH at a concentration of approximately 0.002 N to approximately 0.02 N.

[0119] 105. The method according to any one of items 73 to 104, wherein the buffer further comprises about 0.5 weight percent to about 5 weight percent glucose.

[0120] 106. The method according to any one of items 73 to 105, wherein the buffer solution contains glucose at a rate of approximately 0.5 weight percent or less.

[0121] 107. The method according to any one of items 73 to 106, wherein the buffer solution does not contain glucose.

[0122] 108. The method according to any one of claims 73 to 107, further comprising adding cells to a collagen solution.

[0123] 109. The method according to any one of items 73 to 108, wherein the matrix comprises collagen fibrils.

[0124] 110. The method according to any one of items 73 to 109, wherein the collagen is soluble collagen.

[0125] 111. The method according to any one of paragraphs 73 to 110, wherein a collagen composition, collagen solution, and / or matrix are sterilized using UVC irradiation.

[0126] 112. The method according to any one of claims 73 to 111, wherein a collagen composition, a collagen solution, and / or a matrix are sterilized using UVC irradiation and sterile filtration.

[0127] 113. The method according to any one of claims 73 to 112, wherein the matrix obtained from the polymerization of a collagen solution maintains polymerization properties compared to an unirradiated collagen composition or unirradiated freeze-dried collagen.

[0128] 114. The method according to any one of items 73 to 113, wherein the polymerization property is the shear storage coefficient.

[0129] 115. The radiation dose is approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 The method described in any one of paragraphs 73 to 114, which falls within the range of the specified paragraph.

[0130] 116. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2 The method described in any one of paragraphs 73 to 115, which falls within the scope of the above.

[0131] 117. The method described in any one of paragraphs 73 to 116, wherein sterilization inactivates the virus.

[0132] 118. A method for preparing a matrix in a tissue defect or void, comprising polymerizing collagen by mixing a collagen composition with a buffer to form a collagen solution, and polymerizing the collagen in the collagen solution to form a matrix, wherein the buffer does not contain magnesium ions or manganese ions.

[0133] 119. The method according to item 118, further comprising incubating the collagen solution at a temperature above approximately 25°C to promote the polymerization of collagen in the collagen solution.

[0134] 120. The method according to paragraph 118 or 119, further comprising incubating the collagen solution at a temperature of approximately 37°C to promote the polymerization of collagen in the collagen solution.

[0135] 121. The method according to any one of items 118 to 120, wherein the collagen comprises collagen oligomers.

[0136] 122. The method according to any one of items 118 to 121, wherein the collagen comprises collagen molecules.

[0137] 123. The method according to any one of items 118 to 122, wherein the collagen consists of collagen oligomers.

[0138] 124. The method according to any one of items 118 to 123, wherein the collagen consists of intermolecularly cross-linked collagen molecules.

[0139] 125. The method according to any one of items 118 to 124, wherein the collagen substantially consists of intermolecularly cross-linked collagen molecules.

[0140] 126. The method according to any one of items 118 to 125, wherein the collagen further comprises telocollagen.

[0141] 127. The method according to any one of items 118 to 126, wherein the collagen further comprises atelocollagen.

[0142] 128. The method according to any one of claims 118 to 127, wherein collagen containing collagen oligomers is obtained from tissue containing collagen oligomers, from cells that produce collagen oligomers, or by chemically crosslinking collagen to obtain collagen oligomers.

[0143] 129. The method according to any one of items 118-128, wherein the collagen is derived from porcine skin tissue.

[0144] 130. The method according to any one of claims 118 to 129, wherein the collagen composition further comprises an acid.

[0145] 131. The method according to any one of claims 118 to 130, wherein the acid is selected from the group consisting of hydrochloric acid, acetic acid, lactic acid, formic acid, citric acid, sulfuric acid, and phosphoric acid.

[0146] 132. The method according to any one of headings 118 to 131, wherein the acid is hydrochloric acid.

[0147] 133. The method according to any one of the items 118 to 132, wherein the hydrochloric acid is hydrochloric acid of about 0.005 N to about 0.1 N.

[0148] 134. The method according to any one of the items 118 to 133, wherein the hydrochloric acid is approximately 0.01 N hydrochloric acid.

[0149] 135. The method according to any one of items 118 to 134, wherein the concentration of collagen in the collagen solution is approximately 0.1 mg / ml to approximately 40 mg / ml.

[0150] 136. The method according to any one of items 118 to 135, wherein the concentration of collagen in the collagen solution is approximately 7 mg / mL to approximately 8 mg / mL.

[0151] 137. The method according to any one of items 118 to 136, wherein the concentration of collagen in the mixture of collagen solution and buffer is approximately 6.3 to approximately 7.2 mg / mL.

[0152] 138. The method according to any one of the items 118 to 137, wherein the collagen composition is sterilized.

[0153] 139. The method according to any one of claims 118 to 138, wherein a collagen composition, collagen solution, or collagen matrix is ​​sterilized by a method selected from the group consisting of exposure to chloroform, viral filtration, sterile filtration, gamma irradiation, ultraviolet irradiation, electron beam, and combinations thereof.

[0154] 140. The method according to any one of items 118 to 139, wherein the collagen composition is sterilized by filtration.

[0155] 141. The method according to any one of items 118 to 140, wherein the buffer solution contains approximately 0.03 mM to approximately 0.2 mM MgCl2.

[0156] 142. The method according to paragraph 141, wherein the buffer solution contains approximately 0.002 mM to approximately 0.02 mM MgCl2.

[0157] 143. The method according to any one of items 118 to 142, wherein the buffer solution contains less than approximately 0.02 mM MgCl2.

[0158] 144. The method according to any one of paragraphs 118 to 143, wherein the buffer solution does not contain MgCl2.

[0159] 145. The method according to any one of paragraphs 118 to 144, wherein the buffer further comprises approximately 0.3 mM to approximately 3 mM KH2PO4.

[0160] 146. The method according to any one of items 118 to 145, wherein the buffer further comprises approximately 1 mM to approximately 10 M Na2HPO4.

[0161] 147. The method according to any one of items 118 to 146, wherein the buffer solution further comprises approximately 0.1 mM to approximately 4 mM KCl.

[0162] 148. The method according to any one of items 118 to 147, wherein the buffer further comprises approximately 0.02 M to approximately 0.3 M of NaCl.

[0163] 149. The method according to any one of items 118 to 148, wherein the buffer further comprises NaOH at a concentration of approximately 0.002 N to approximately 0.02 N.

[0164] 150. The method according to any one of items 118 to 149, wherein the buffer further comprises about 0.5 weight percent to about 5 weight percent glucose.

[0165] 151. The method according to any one of items 118 to 150, wherein the buffer solution contains glucose at a rate of approximately 0.5 weight percent or less.

[0166] 152. The method according to any one of items 118 to 151, wherein the buffer solution does not contain glucose.

[0167] 153. The method according to any one of items 118 to 152, further comprising adding cells to a collagen solution.

[0168] 154. The method according to any one of claims 118 to 153, wherein the matrix contains collagen fibrils.

[0169] 155. The method according to any one of claims 118 to 154, wherein the collagen is soluble collagen.

[0170] 156. The method according to any one of claims 118 to 155, wherein the collagen composition, the collagen solution, and / or the collagen matrix is sterilized using ultraviolet irradiation.

[0171] 157. The method according to any one of claims 118 to 156, wherein the collagen composition, the collagen solution, and / or the matrix is sterilized using UVC irradiation and sterile filtration.

[0172] 158. The method according to any one of claims 118 to 157, wherein the matrix obtained from the polymerization of the collagen solution maintains polymerization characteristics as compared to the non-irradiated collagen composition or the non-irradiated lyophilized collagen.

[0173] 159. The method according to any one of claims 118 to 158, wherein the polymerization characteristic is the shear storage modulus.

[0174] 160. The irradiation dose is in the range of about 5 mJ / cm 2 to about 800 mJ / cm 2 The method according to any one of claims 118 to 159.

[0175] 161. The irradiation dose is in the range of about 30 mJ / cm 2 to about 300 mJ / cm 2 The method according to any one of claims 118 to 160.

[0176] 162. The method according to any one of claims 118 to 161, wherein the sterilization inactivates the virus.

[0177] 163. A collagen matrix prepared according to the method described in any one of paragraphs 1 to 162.

[0178] 164. A collagen matrix as described in paragraph 163, which is a medical graft.

[0179] 165. The collagen matrix according to paragraph 163 or 164, wherein the medical graft has a use selected from the group consisting of tissue graft material, injectable graft material, wound dressing material, hemostatic dressing material, delivery medium for therapeutic cells, and delivery medium for therapeutic agents.

[0180] 166. A collagen matrix as described in any one of paragraphs 163-165, for use in research.

[0181] 167. A collagen matrix as described in any one of the paragraphs 163 to 166, used for drug toxicity testing or drug development.

[0182] 168. A collagen matrix as described in any one of paragraphs 163 to 167, sterilized using ultraviolet irradiation.

[0183] 169. A collagen matrix according to any one of paragraphs 163 to 168, which maintains polymerization properties compared to an unirradiated collagen matrix.

[0184] 170. A collagen matrix according to any one of paragraphs 163 to 169, wherein the polymerization property is the shear storage coefficient.

[0185] 171. The radiation dose was approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 A collagen matrix as described in any one of paragraphs 163 to 170, which falls within the range of the specified paragraphs.

[0186] 172. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2A collagen matrix as described in any one of paragraphs 163 to 171, which falls within the range of the specified paragraphs.

[0187] 173. A collagen matrix as described in any one of paragraphs 163-172, wherein sterilization has inactivated the virus.

[0188] 174. A collagen matrix as described in any one of paragraphs 163 to 173, sterilized using UVC irradiation.

[0189] 175. A collagen matrix as described in any one of paragraphs 163 to 174, sterilized using UVC irradiation and sterile filtration.

[0190] 176. A collagen matrix prepared by introducing a self-assembling biopolymer into a void or defect in a tissue and polymerizing the self-assembling biopolymer to form a shape-retaining matrix, wherein the pH of the self-assembling biopolymer is in the range of about 5.5 to about 8.5, the self-assembly time of the self-assembling biopolymer is in the range of about 0.2 minutes to about 1.5 minutes, the shear storage coefficient (G') of the collagen matrix is ​​in the range of about 2.0 kPa to about 4.0 kPa, the shear loss coefficient (G”) of the collagen matrix is ​​in the range of about 0.1 kPa to about 0.7 kPa, and the compressibility coefficient of the collagen matrix is ​​in the range of about 5.0 kPa to about 10.0 kPa.

[0191] 177. The collagen matrix described in paragraph 176, wherein the pH of the self-assembling biopolymer is approximately 7.25 ± approximately 0.25, the self-assembly time of the self-assembling biopolymer is approximately 0.8 min ± approximately 0.3 min, the shear storage coefficient (G') of the collagen matrix is ​​approximately 3.1 kPa ± approximately 0.4 kPa, the shear loss coefficient (G”) of the collagen matrix is ​​approximately 0.4 kPa ± approximately 0.1 kPa, and the compressibility coefficient of the collagen matrix is ​​approximately 7.7 kPa ± approximately 1.9 kPa.

[0192] 178. A collagen matrix as described in paragraph 176 or 177, which is a medical graft.

[0193] 179. A collagen matrix according to any one of the claims 176 to 178, wherein the medical graft has a use selected from the group consisting of tissue graft material, injectable graft material, wound dressing material, hemostatic dressing material, delivery medium for therapeutic cells, and delivery medium for therapeutic agents.

[0194] 180. A collagen matrix as described in any one of paragraphs 176-179, for use in research.

[0195] 181. A collagen matrix as described in any one of the paragraphs 176-180, used for drug toxicity testing or drug development.

[0196] 182. A collagen matrix as described in any one of paragraphs 176-181, sterilized using ultraviolet irradiation.

[0197] 183. A collagen matrix according to any one of paragraphs 176 to 182, which maintains polymerization properties compared to an unirradiated collagen matrix.

[0198] 184. A collagen matrix according to any one of paragraphs 176 to 183, wherein the polymerization property is the shear storage coefficient.

[0199] 185. The radiation dose is approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 A collagen matrix as described in any one of paragraphs 176 to 184, which falls within the range of the specified paragraphs.

[0200] 186. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2 A collagen matrix as described in any one of paragraphs 176 to 185, which falls within the range of the specified paragraphs.

[0201] 187. A collagen matrix as described in any one of paragraphs 176-186, wherein sterilization has inactivated the virus.

[0202] 188. A collagen matrix as described in any one of paragraphs 176-187, sterilized using UVC irradiation.

[0203] 189. A collagen matrix as described in any one of paragraphs 176-188, sterilized using UVC irradiation and sterile filtration.

[0204] 190. A kit for restoring and regenerating tissue in tissue voids or defects, comprising an in-situ polymerizable collagen composition and a buffer.

[0205] 191. The kit according to item 190, wherein the in situ polymerizable collagen composition comprises liquid type I collagen.

[0206] 192. A kit according to paragraph 190 or 191, wherein the in-situ polymerizable collagen composition comprises type I oligomeric collagen derived from porcine dermis.

[0207] 193. A kit according to any one of items 190 to 192, wherein the in-situ polymerizable collagen composition comprises a solution containing freeze-dried oligomeric collagen and an acid.

[0208] 194. A kit according to any one of the following paragraphs, 190 to 193, comprising an in-situ polymerizable collagen composition containing a solution comprising lyophilized type I oligomeric collagen and an acid.

[0209] 195. A kit according to any one of claims 190 to 194, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid.

[0210] 196. A kit according to any one of items 190 to 195, wherein the ratio of in situ polymerizable collagen composition to buffer solution is approximately 9:1.

[0211] 197. A kit according to any one of items 190 to 196, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid, and the collagen concentration in the solution of the in-situ polymerizable collagen composition is approximately 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen.

[0212] 198. A kit according to any one of claims 190 to 197, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, and the solution of the in-situ polymerizable collagen composition is clarified using ultracentrifugation.

[0213] 199. The kit according to any one of the following paragraphs, 190 to 198, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution of the in-situ polymerizable collagen composition being clarified using ultracentrifugation and then filtered through a sterile membrane filter.

[0214] 200. The kit according to any one of the following paragraphs, 190 to 199, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution of the in-situ polymerizable collagen composition being clarified using ultracentrifugation, irradiated with ultraviolet light, and then filtered through a sterile membrane filter.

[0215] 201. The in-situ polymerizable collagen composition comprises a solution containing freeze-dried type I oligomeric collagen and hydrochloric acid, and the solution of the in-situ polymerizable collagen composition is clarified using ultracentrifugation to 500 mJ / cm³. 2 A kit as described in any one of paragraphs 190 to 200, which is irradiated with ultraviolet light and then filtered through a sterile membrane filter.

[0216] 202. The kit according to any one of claims 190 to 201, further comprising a syringe configured for delivering a mixture of an in-situ polymerizable collagen composition and a buffer to a void or defect in tissue.

[0217] 203. A kit as described in any one of sections 190 to 202, wherein the buffer solution contains approximately 0.03 mM to approximately 0.2 mM MgCl2.

[0218] 204. A kit as described in any one of items 190 to 203, wherein the buffer solution contains approximately 0.002 mM to approximately 0.02 mM MgCl2.

[0219] 205. A kit described in any one of items 190-204, containing a buffer solution of less than approximately 0.02 mM MgCl2.

[0220] 206. A kit described in any one of sections 190-205, wherein the buffer solution does not contain MgCl2.

[0221] 207. A kit as described in any one of sections 190 to 206, wherein the buffer further contains approximately 0.003 M to approximately 0.03 M of KH2PO4.

[0222] 208. A kit as described in any one of sections 190 to 207, wherein the buffer solution further contains approximately 0.01 M to approximately 0.1 M of Na2HPO4.

[0223] 209. A kit as described in any one of sections 190 to 208, wherein the buffer solution further contains approximately 0.001 M to approximately 0.04 M of KCl.

[0224] 210. A kit as described in any one of sections 190 to 209, wherein the buffer solution further contains approximately 0.2 M to approximately 3.0 M of NaCl.

[0225] 211. A kit as described in any one of sections 190 to 210, wherein the buffer solution further contains NaOH at a concentration of approximately 0.02 N to approximately 0.2 N.

[0226] 212. A kit according to any one of items 190 to 211, wherein the buffer further comprises approximately 0.2 to 5 weight percent glucose.

[0227] 213. A kit as described in any one of sections 190 to 212, wherein the buffer solution contains glucose at a rate of approximately 0.5 weight percent or less.

[0228] 214. A kit described in any one of sections 190-213, wherein the buffer solution does not contain glucose.

[0229] 215. A kit according to any one of items 190 to 214, wherein the concentration of collagen in the in-situ polymerizable collagen composition is approximately 0.1 mg / ml to approximately 40 mg / ml.

[0230] 216. A kit according to any one of items 190 to 215, wherein the concentration of collagen in the in situ polymerizable collagen composition is approximately 7 mg / mL to approximately 8 mg / mL.

[0231] 217. A kit according to any one of items 190 to 216, wherein the concentration of collagen in a neutralized collagen filler comprising an in-situ polymerizable collagen composition and a buffer is approximately 6.3 to approximately 7.2 mg / mL.

[0232] 218. A kit according to any one of items 190 to 217, wherein the collagen solution contains hydrochloric acid ranging from approximately 0.005 N to approximately 0.1 N.

[0233] 219. The kit according to any one of claims 190 to 218, wherein the buffer is configured to polymerize the in situ polymerizable collagen composition in a single mixing step comprising mixing the in situ polymerizable collagen composition with the buffer.

[0234] 220. A kit according to any one of items 190 to 219, wherein the in situ polymerizable collagen composition and buffer are contained in separate containers.

[0235] 221. A kit as described in any one of the items 190 to 220, wherein each individual container contains a sterile vial.

[0236] 222. A kit as described in any one of the headings 190 to 221, wherein each individual container contains a separate compartment for a dual-barrel syringe.

[0237] 223. A kit according to any one of the following sections, 190 to 222, comprising a dual-barrel syringe containing a mixing element.

[0238] 224. A kit described in any one of sections 190-223, wherein the dual-barrel syringe is sterile.

[0239] 225. The kit described in any one of sections 190 to 224, further including instructions for using the components of the kit.

[0240] 226. A kit according to any one of the paragraphs 190 to 225, further comprising at least one therapeutic agent configured for local delivery to a tissue void or defect.

[0241] 227. A kit according to any one of the paragraphs 190 to 226, wherein at least one therapeutic agent comprises a chemotherapeutic agent, an anti-inflammatory agent, an antibiotic, an analgesic, or a combination thereof.

[0242] 228. A kit according to any one of the paragraphs 190 to 227, wherein the tissue void or defect includes a wound.

[0243] 229. A kit according to any one of the paragraphs 190 to 228, wherein the tissue void or defect includes a surgical wound.

[0244] 230. A kit according to any one of paragraphs 190-229, wherein tissue voids or defects result from tumor removal.

[0245] 231. A kit according to any one of paragraphs 190-230, wherein tissue voids or defects result from the removal of a breast tumor.

[0246] 232. A kit for regenerating tissue after breast-conserving surgery, as described in any one of paragraphs 190 to 231.

[0247] 233. A kit for preparing a matrix in a tissue void or defect, as described in any one of the paragraphs 190 to 232.

[0248] 234. A kit according to any one of the paragraphs 190 to 233, wherein the in-situ polymerizable collagen composition or freeze-dried oligomeric collagen is sterilized using ultraviolet irradiation.

[0249] 235. The kit according to any one of items 190 to 234, wherein the collagen matrix obtained from the polymerization of an in-situ polymerizable collagen composition maintains its polymerization properties compared to an unirradiated collagen composition or unirradiated lyophilized collagen.

[0250] 236. A kit described in any one of sections 190 to 235, wherein the polymerization property is the shear storage coefficient.

[0251] 237. The radiation dose was approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 A kit described in any one of paragraphs 190 to 236, which falls within the range of the specified items.

[0252] 238. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2 A kit as described in any one of paragraphs 190 to 237, which falls within the range of [the specified area].

[0253] 239. A kit described in any one of paragraphs 190-238, wherein sterilization has inactivated the virus.

[0254] 240. A kit according to any one of paragraphs 190 to 239, wherein an in-situ polymerizable collagen composition or lyophilized oligomeric collagen is sterilized using UVC irradiation.

[0255] 241. A kit according to any one of the paragraphs 190 to 240, wherein the collagen composition or freeze-dried oligomeric collagen is sterilized using UVC irradiation and sterile filtration. [Brief explanation of the drawing]

[0256] [Figure 1] Figure 1 shows an overview of a liquid tissue filler that has soft tissue-like properties and forms a viscoelastic matrix in situ. Figure 1A shows a kit including a syringe containing a sterile type I oligomeric collagen solution, a syringe of appropriate neutralizing (self-assembling) buffer, a Luer lock adapter, and an applicator tip. Figure 1B shows mixing the two reagents and subsequently injecting them into a plastic mold maintained at body temperature (37°C), whereupon the liquid migrates into a stable, shape-retaining fibrous collagen matrix. Figure 1C shows 4–20% and 6% SDS-PAGE gels recording the purity and characteristic band patterns of type I oligomeric collagen, with lane 1 corresponding to molecular weight standards and lane 2 corresponding to type I oligomeric collagen. Figure 1D shows a table summarizing the tissue filler polymerization kinetics and performance specifications (mean ± SD; N=4, n=6–8) of the matrix formed by the tissue filler. [Figure 2]Figure 2 shows an overview of the simulated mammary tumor excision procedure. Figure 2A shows a table summarizing the surgically excised mammary tissue volume, which represents approximately one-quarter of the total mammary tissue volume [data compiled from both long-term and radiation studies (mean ± SD) (1 week: collagen filler: n=12, unfilled=6; 4 weeks: collagen filler: n=18, unfilled: n=9; 16 weeks: collagen filler: n=18, unfilled: n=9)]. Figure 2B shows the surgical space before the application of tissue filler. Figure 2C shows the surgical space after the application of tissue filler. Figure 2D shows the application of tissue filler. Figure 2E shows the excised pig mammary tissue. Figure 2F shows the surgical site immediately after surgery, including bandaging. Figure 2G shows the surgical site 16 weeks after the simulated mammary tumor excision and irradiation. [Figure 3] Figure 3 outlines how long tissue fillers persist without causing a typical inflammatory or foreign body reaction and induce a tissue implantation response that supports breast tissue formation. Figure 3A shows a graph of breast uniformity / concentration scores (mean ± SD; collagen tissue fillers: n=12; unfilled: n=6) assigned by breast surgeons for collagen tissue-filled and unfilled (negative control) treated spaces at various time points after mammary tumor excision in a simulated experiment. All unsurgered breasts were scored as 0. Figure 3B shows cross-sections of postoperative spaces treated with collagen tissue fillers or unfilled, compared to normal breast tissue. Arrows represent surgical clips placed to mark the boundaries of the postoperative spaces. [Figure 4]Figure 4 outlines the extent to which the tissue filler supports breast tissue formation without inducing an inflammatory response or foreign body reaction. Figure 4A shows histological cross-sections (H&E) of collagen-filled cavities at 1, 4, and 16 weeks post-mastectomy in a simulated experiment. Low-magnification images show the tissue filler within the cavity and its boundary with the surrounding host tissue (large arrows indicate surgical clip locations). High-magnification images characterize the central region of the tissue filler and the tissue filler-host tissue boundary. Cellular infiltration, angiogenesis, and breast tissue formation within the matrix implants occurred over time, without evidence of an inflammatory response typically seen in the healing of untreated tissue cavities (i.e., neutrophil and macrophage infiltration) or a foreign body reaction typically observed in tissue implantation responses (i.e., macrophage activation, giant cell formation, phagocytosis, and fibrous capsule formation). By 16 weeks, the tissue filler was fully cellularized and vascularized (small arrows indicate blood vessels), and evidence of mammary (RG) and adipose tissue (RF) formation was observed. Onc: Tissue filler matrix without cellular infiltration; Oc: Tissue filler matrix with cellular infiltration. Figure 4B shows cross-sections (H&E) of untreated (unfilled) postoperative cavities at 1, 4, and 16 weeks post-mastectomy in a simulated experiment. Low-magnification images show the cavity and surrounding host tissue. High-magnification images characterize the central region of the cavity and the cavity / host tissue boundary. The hematoma (H) was typical at 1 week, followed by a gradual defect atrophy and healing response, resulting in scar tissue formation (S). [Figure 5] Figure 5 provides an overview of how little the tissue filler interferes with radiographic or ultrasound procedures. Figure 5A shows representative ultrasound images of postoperative spaces treated with or not treated with collagen tissue filler compared to normal breast tissue at 1, 4, and 16 weeks, and Figure 5B shows representative radiographs of the same. Clear radiopaque marker clips in the radiographs indicate the boundaries of the postoperative spaces and provide evidence of reduced wound atrophy with respect to spaces treated with tissue filler compared to unfilled spaces. [Figure 6]Figure 6 provides an overview of the extent to which radiation has little to no effect on tissue fillers and associated tissue implantation responses. Figure 6A shows a graph of breast uniformity / concentration scores (mean ± SD; collagen: n=6; unfilled: n=3) assigned by breast surgeons for collagen tissue-filled and unfilled (negative control) treated spaces at various time points after mammary tumor excision and radiation in a simulated experiment. All breasts that did not undergo surgery were scored as 0. Figure 6B shows cross-sections of postoperative spaces after irradiation with or without tissue fillers, compared to unsurgically treated normal breast tissue. Arrows represent surgical clips placed to mark the boundaries of the postoperative spaces. Figure 6C shows histological cross-sections (H&E) of collagen-filled spaces at 4 and 16 weeks after mammary tumor excision and adjuvant irradiation in a simulated experiment. Low-magnification images show the tissue filler within the space and its boundary with the surrounding host tissue. High-magnification images characterize the central region of the tissue filler and the tissue filler-host tissue boundary. Cellular infiltration, angiogenesis, and mammary tissue formation within the matrix implant occur over time, albeit at a slower rate than in areas of unirradiated animals. By 16 weeks, the tissue filler is fully cellularized and angiogenic (small arrows indicate blood vessels), and evidence of adipose tissue (RF) formation is observed. Onc: Tissue filler matrix without cellular infiltration; Oc: Tissue filler matrix with cellular infiltration. Figure 6D shows cross-sections (H&E) of untreated (unfilled) postoperative spaces at 4 and 16 weeks after mammary tumor excision and radiation in a simulated experiment. Low-magnification images show the space and surrounding host tissue, with scar tissue (S) and conjugation-associated granulomas (G) evident at 4 weeks (large arrows indicate surgical clip locations). High-magnification images characterize the central region of the inflammatory response and the scar-host tissue boundary, which is scar tissue formed within the space. [Figure 7]Figure 7 provides an overview of how tissue fillers do not impair the interpretation of diagnostic images of breast tissue, even after adjuvant irradiation. Figure 7A shows representative ultrasound images of postoperative spaces treated with or without tissue fillers compared to normal breasts at 4 and 16 weeks, and Figure 7B shows representative radiographs of the same. Clear radiopaque marker clips in the radiographs indicate the boundaries of the postoperative spaces and show reduced wound atrophy with respect to spaces treated with tissue fillers compared to unfilled spaces. [Figure 8] Figure 8 shows the timeline and process of the healing response observed in a simulated mammary gland tumor excision model in pigs. Figure 8A shows a schematic diagram comparing and contrasting the phases and processes associated with a typical repair healing response observed in the unfilled case. Figure 8B shows a schematic diagram comparing and contrasting the phases and processes associated with the regenerative and regenerative healing response observed with collagen tissue fillers. [Figure 9] Figure 9 shows the multi-tissue type composition of normal mammary tissue and the skin above it, as well as the effects of irradiation. Cross-sections (H&E) of normal mammary tissue and associated skin from unirradiated pigs and from pigs 4 and 16 weeks after mammary tumor excision and radiation are shown. Mammary tissue consists of collagenous connective tissue (C), mammary lobules (M), mammary ducts (D), and adipose (lipid) tissue (F). Skin includes the multicellular epidermis (E) and the underlying collagenous dermis (C). [Figure 10] Figure 10 shows the semi-quantitative scoring used for postoperative evaluation of pig mammary glands. The mammary glands and surgical sites were evaluated based on macroscopic appearance, including evidence of erythema and crusting, as well as edema. Palpation was used to assess the uniformity and consistency of the mammary glands. [Figure 11]Figure 11 shows an overview of how well the collagen tissue filler supports mammary gland formation, and at the same time, gland necrosis is evident in the unfilled voids. Cross-sections of corresponding H&E (Figures 11A and 11C) and pan-cytokeratin staining (Figures 11B and 11D) show the postoperative voids 16 weeks after treatment with the collagen tissue filler (Figures 11A and 11B) or without filling (Figures 11C and 11D). The selected areas represent the periphery of the collagen tissue filler and the formed scar tissue, respectively. Pan-cytokeratin highlights the epithelial cells covering the lobules and the inner walls of the ducts within the collagen tissue-filled and unfilled groups. The unfilled group also shows evidence of necrotic glands (black and white arrows). The immunofluorescence images show pan-cytokeratin (green), and the nuclei are counterstained with DAPI (blue). [Figure 12] Figure 12 shows an overview of creating defects in the skeletal muscle and adipose tissue in the back of the pig's neck. The defects were filled with liquid collagen that conformed to the outer shape of the voids. Within approximately 1 minute after application, the liquid collagen polymerized in situ, forming a collagen matrix that restored the tissue's morphology and continuity. [Figure 13] Figure 13 shows an overview of newly formed skeletal muscle and adipose (lipid) tissue within the collagen matrix 11 weeks after implantation. Typically, no evidence of the inflammatory response (i.e., infiltration of neutrophils and macrophages) observed in the healing of the voids in untreated tissue or the foreign body response (i.e., macrophage activation, giant cell formation, phagocytosis, and fibrous capsule formation) typically observed in the tissue implantation response is seen. C: Collagen tissue-filled matrix; F: Lipid; M: Skeletal muscle; Arrow: Associated microvascular system. [Figure 14] Figure 14 shows a schematic diagram of the typical components of a collagen tissue filler kit.

Mode for Carrying Out the Invention

[0257] This instruction describes a regenerative and regenerative tissue filler that may be applied as a liquid to any type of tissue void or defect—including, but not limited to, tissue voids resulting from surgical wounds (e.g., surgical wounds arising from BCS, but not limited to, but not limited to), physical defects (e.g., scars, depressions, birth defects, etc.), injury, disease progression, and / or similar—before transitioning to a fibrous collagen matrix with tissue consistency. Using a simulated BCS model in pigs, the collagen filler according to this instruction has been shown to induce a regenerative healing response characterized by rapid cellularization, angiogenesis, and progressive mammary tissue regeneration, including adipose tissue and mammary glands and ducts. In contrast to conventional biomaterials, no xenobiotic response or inflammation-mediated "active" biodegradation was observed with respect to the collagen filler according to this instruction. Furthermore, the collagen filler according to this instruction also did not impair the simulated surgical re-excision, radiography, or ultrasound procedures, which are important features for clinical interpretation. Furthermore, when postoperative radiation therapy was applied, the tissue response to the collagen filler according to this instruction was almost identical to that under non-irradiated conditions (however, as expected, healing was slightly slower). The in-situ matrix-forming collagen according to this instruction is easy to apply, conforms to patient-specific defects, and generates complex tissue in the absence of inflammation. Therefore, the collagen filler according to this instruction has remarkable translational potential as a first regenerative tissue filler for BCS, as well as other soft tissue recovery and reconstruction needs.

[0258] In some embodiments, the regenerative tissue fillers according to this teaching may offer one or more of the following advantages: (1) less or no scarring (i.e., compared to conventional non-filling procedures); (2) less or no defect atrophy (i.e., compared to conventional non-filling procedures); (3) less or no inflammatory mediators or inflammatory response (i.e., compared to conventional non-filling procedures); (4) tissue consistency similar to that of natural tissue (e.g., compressibility coefficient or range of compressibility similar to that of natural tissue); (5) recovery and generation of breast tissue, including adipose tissue, mammary gland tissue, etc.; (6) recovery and generation of skeletal muscle; (7) tissue implantation response does not interfere with a given clinical procedure, including re-excision, ultrasound, or radiography; and / or (8) less tissue implantation response (i.e., compared to conventional procedures) or is not adversely affected by adjuvant irradiation (e.g., no lipid cysts, microcalcifications, lesions, and / or areas of high opacity, any of which may interfere with imaging).

[0259] According to this teaching, the inventors are attempting a tissue filler that (i) restores and regenerates damaged tissue and tissue cavities as expected, (ii) is easily applied, (iii) conforms to patient-specific defects of varying sizes and shapes, and (iv) does not interfere with or impair given clinical processes and procedures. In some embodiments, the tissue filler is introduced into the tissue cavities or defects under sterile conditions. In some embodiments, the introduction of the tissue filler into the tissue cavities or defects may be achieved by injection (e.g., using one or more single-barrel syringes, dual-barrel syringes, and / or similar, as well as combinations thereof). In other embodiments, the tissue filler may first be applied to an external mold (e.g., in a surgical setting) to form a molded part, which may then be removed from the mold and implanted in the patient. Regenerative medicine approaches involving adjustable in-situ forming biomaterials have potential to address many of these design considerations. In particular, type I oligomeric collagen (oligomers), a highly purified molecular form of collagen that readily dissolves in dilute acids, represents a tunable in-situ-forming biomaterial with the potential to address many of these design considerations. Unlike conventional monomeric collagen preparations, namely telocollagen and atelocollagen, oligomers represent small aggregates of full-length triple-helical collagen molecules (i.e., tropocollagen) with complete carboxy- and amino-terminal telopeptides, held together by spontaneously occurring intermolecular crosslinks. The preservation of these key molecular features, including the carboxy- and amino-terminal telopeptide regions and associated intermolecular crosslinks, provides this natural polymer and the collagen material that forms it with desirable but rare properties. More specifically, oligomers retain the fibrillation (self-assembly) ability inherent in fibrous collagen proteins. When oligomers are neutralized to physiological conditions (e.g., pH and ionic strength), this liquid form can be readily injected and completely fill in complex contours and shapes.At body temperature, the liquid rapidly transforms into a fibrous collagen matrix, replicating the structural and biological signaling properties of the collagen matrix found in the extracellular matrix (ECM) components of tissues. Upon in vivo implantation, these matrices persist, exhibiting slow turnover and remodeling, resistance to proteolysis, and no active biodegradation or xenobiotic response. This natural polymer supports the creation of materials with broadly modifiable physical properties, including morphology, structure (random or aligned fibrils, continuous fibril density gradients), and mechanical integrity, providing a viable platform for personalized regenerative medicine. In-situ forming collagen matrix shows promise as a regenerative tissue filler for breast-conserving surgery and other soft tissue recovery requirements.

[0260] In some embodiments, regenerative tissue fillers are provided that may be applied to wounds—including, but not limited to, defects or contours in BCS—as a liquid before transitioning to a fibrous collagen matrix with tissue consistency. As further described below using a BCS model of a swine simulated experiment, the collagen fillers were shown to induce a regenerative healing response characterized by rapid cellularization, angiogenesis, and progressive mammary tissue regeneration, including adipose tissue and mammary glands and ducts. Unlike conventional biomaterials, no foreign body response or inflammation-mediated "active" biodegradation was observed. The collagen fillers also did not impair surgical re-excision, radiography, or ultrasound procedures in the simulated experiment, which are important features for clinical interpretation. When radiation was applied after BCS, the collagen fillers and their associated tissue responses were nearly similar to those under non-irradiated conditions. However, as expected, the healing rate was somewhat slower. This in-situ matrix-forming collagen is easy to apply, conforms to patient-specific defects / contours, and regenerates complex tissues in the absence of inflammation. It has remarkable translational potential as a first regenerative tissue filler for the recovery and reconstruction of BCS, as well as other soft tissues and skeletal muscle tissues.

[0261] The collagen fillers described herein are fundamentally different from conventional, fluid, injectable collagen products that have been used or previously used for soft tissue augmentation (e.g., cosmetic procedures), management of skin wounds (e.g., ulcers), and tissue volume increase (bulking) (e.g., urinary incontinence). Such products, including Zyderm®, Zyplast®, Integra Flowable®, and Contigen®, are made from reconstituted, enzymatically treated collagen (atelocollagen) or granulated tissue microparticles derived from bovine, porcine, or human tissue sources. To make these materials injectable, insoluble fibrous collagen or tissue microparticles are suspended in physiological saline solution to produce dispersions or suspensions. All of these implantable collagens are transient and exhibit rapid biodegradation (reabsorption; 1–6 months), and are actively degraded through inflammatory processes, including phagocytosis by macrophages / giant cells and proteolysis by secreted matrix metalloproteinases. To slow down degradation and improve persistence, many of these products are treated with glutaraldehyde or other exogenous cross-linking processes.

[0262] In contrast, oligomeric collagen represents molecular subdomains found in collagen fibers of native tissues (e.g., porcine dermis), which may be extracted and purified to remove cellular and other immunogenic tissue components. The type I collagen proteins and crosslinking chemistry that constitute this subdomain are highly conserved across species, documenting the importance of this major structural element in the body. Physiological conditions induce fibrillation, in which oligomeric molecules assemble in an alternating pattern, resulting in an interconnected network or matrix of fibrils. Published studies have shown that the formed matrix closely resembles that naturally found in the extracellular matrix, containing fibrils with a regular D-banding pattern and readily involved in biosignals. The chemistry of native crosslinking present in oligomers but not in polymerizable monomeric collagen is a major contributor to the rapid matrix formation reaction, as well as the improved mechanical integrity, slower turnover, and resistance to proteolysis exhibited by the oligomeric matrix. In summary, these distinctive features contribute to the unique mechanisms of action and regenerative tissue responses exhibited by the oligomeric matrix compared to conventional biodegradable collagen materials.

[0263] The ability to restore and regenerate pathological, injured, or dysfunctional tissue has been one of the major challenges in pharmaceuticals. Indeed, researchers have been working to identify biomaterials and / or anti-inflammatory agents with the goal of achieving more desirable healing outcomes (i.e., regeneration) or biomaterial / device implantation responses. In the case of the breast, this challenge is particularly difficult because it is composed of a multi-tissue type with different functions, including secretory (i.e., milk-producing) glands and tubules, supporting collagen connective tissue, and volume-filling adipose tissue. Currently, tissue engineering and regenerative medicine strategies for soft tissue and breast reconstruction remain in their early stages, with only a few strategies evaluated so far in large animal models. Most approaches have focused on engineered adipose tissue from biological or synthetic scaffolds incorporating lipofilling to promote adipogenesis and angiogenesis, patient-derived cell populations, and growth factors. The main drawback of conventional synthetic scaffold approaches is that the material cannot signal to cells, resulting in a xenobiotic response and slow cellularization and angiogenesis.

[0264] In some embodiments, the tissue filler may include purified fibril-forming liquid type I collagen, for example, derived from porcine dermis. In some embodiments, the in-situ forming collagen device may be supplied as a disposable kit including a sterile glass vial containing a collagen solution (10 mL) in dilute (0.01 N) hydrochloric acid, a sterile glass vial containing a neutralizing (self-assembly) reagent (2 mL) having a registered trademark, two sterile 10 mL syringes, a sterile Luer lock connector, and a sterile applicator tip. After drawing up 9 mL of liquid collagen in one syringe and 1 mL of neutralizing buffer in the other, the user connects the two syringes using the Luer lock connector and mixes the two reagents. After mixing, the neutralized collagen solution may be injected and filled into tissue voids or defects, including those that are deep, difficult to access, and irregularly shaped. Upon application, fibrils are rapidly formed (in about 1 minute at body temperature) via molecular self-assembly. The resulting tissue-filling matrix restores and maintains tissue shape and soft tissue consistency over time, triggering a tissue implantation response characterized by cellularization, angiogenesis, and novel tissue formation, without triggering the inflammatory response typically observed in wound healing or the foreign body reaction typically observed in conventional tissue implantation responses.

[0265] The specially formulated liquid collagen format easily fills and conforms to the patient's unique defect shape and contour, making it suitable for minimally invasive procedures. Upon application to the site, the collagen solution polymerizes (molecular self-assembly reaction) to form a physically stable fibrous collagen matrix, whose volume is sustained and maintained. The matrix provides essential biochemical and biomechanical signaling to cells, supporting cellularization, angiogenesis, and tissue formation, is site-appropriate, and does not cause inflammatory or foreign body reactions. It involves complex tissue composition with different functions, such as those observed in the breast. The material is compatible with numerous standard clinical procedures, including irradiation, radiography, ultrasound, and surgical re-excision.

[0266] Throughout this entire description and the attached claims, the following definitions should be understood:

[0267] The phrase "oligomeric collagen" refers to collagen containing cross-linked collagen molecules. A collagen molecule is composed of three separate polypeptide chains, with non-helical telopeptide ends adjacent to each other, collectively giving rise to its triple-helical quaternary structure. As used herein, the phrase "oligomeric collagen" should be understood to refer to a collagen molecule in which at least one of its parts is covalently linked through intermolecular crosslinking. Oligomeric collagen may be polymerized, for example, as described herein. Polymerizable oligomeric collagen is oligomeric collagen that can be polymerized. One example of a cross-linked collagen molecule that can be found in oligomeric collagen is tropocollagen.

[0268] The term “sterilized” refers to the removal of contaminants, including but not limited to infectious pathogens. For example, contaminants (e.g., bacteria, viruses) may be removed by inactivation, reduction in number or quantity, or inhibition of the activity of the contaminating pathogens, whether infectious or not.

[0269] The term "purified" refers to the removal of contaminants, including but not limited to cellular contaminants, nucleotide contaminants, and endotoxins.

[0270] The term "external body reaction" refers to a localized inflammatory response triggered by any material that would not normally be found in the body. This reaction may be characterized by protein adsorption and inflammatory processes, such as macrophage activation, giant cell formation and fibrous capsule formation and / or degradation, or phagocytosis of the exogenous material.

[0271] The phrase "tissue implantation response" refers specifically to a part of the "foreign body reaction" that arises from the implantation of materials into a patient's body.

[0272] The term “patient” refers to any animal that will be treated for a tissue void or defect, including but not limited to vertebrates. As used herein, the term “patient” includes, but is not limited to, mammals, reptiles, amphibians, birds, and fish. In some embodiments, patient refers to a mammal (e.g., human, dog, cat, horse, rabbit, pig, etc.). In the exemplary embodiment, patient is human.

[0273] The terms “void” and “defect” refer to all forms of tissue abnormalities, but are not limited to wounds, surgical wounds (e.g., surgical wounds resulting from BCS, but are not limited to these), physical defects (e.g., scars, depressions, birth defects, etc.), injuries, disease progression (e.g., muscle atrophy), and / or similar, as well as combinations thereof. As used herein, the term “tissue” includes both hard tissue (e.g., skeletal bone) and soft tissue. Thus, the phrase “tissue abnormality” encompasses all forms of abnormalities in both hard and soft tissue.

[0274] When used in relation to tissue, the terms “recover” and “regenerate” refer to the re-establishment of tissue presence in a patient area already characterized by tissue voids or defects, and the regrowth of tissue in this same area, respectively. In some embodiments, the recovered and / or regenerated tissue may reflect one or more of the appearance, structure, and function of the original tissue being replaced.

[0275] When used in relation to tissue, the term "atrophy" refers to a type of scarring characterized by a reduction in tissue area, which is typically due to the body's healing response.

[0276] The term "matrix" refers to a collagen fibril scaffold-like structure configured to provide a platform on which tissues can originate, develop, and / or grow, both on top of, around, and within.

[0277] In some embodiments, a method for filling a tissue void or defect according to the present teachings includes (a) introducing a self-assembling biopolymer into the tissue defect or void and (b) polymerizing the self-assembling biopolymer to form a shape-retaining matrix.

[0278] In some embodiments, a method for filling a tissue void or defect created by a breast tumor excision or mastectomy procedure according to the present teachings includes (a) introducing a mixture comprising an oligomeric collagen solution and a neutralizing solution into the surgical wound site and (b) polymerizing the oligomeric collagen solution to form a collagen-fibril matrix. The oligomeric collagen solution may comprise lyophilized type I oligomeric collagen and an acid.

[0279] In some embodiments, a method for filling a tissue void or defect created by a breast tumor excision or mastectomy procedure according to the present teachings includes (a) introducing a mixture comprising an oligomeric collagen solution and a neutralizing solution into the surgical wound site and (b) polymerizing the oligomeric collagen solution to form a collagen-fibril matrix. In some embodiments, the oligomeric collagen solution may comprise lyophilized type I oligomeric collagen and 0.01N hydrochloric acid. In some embodiments, the concentration of the oligomeric collagen solution is about 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen. In some embodiments, the ratio of the oligomeric collagen solution to the neutralizing solution is about 9:1.

[0280] In other embodiments, a collagen matrix prepared according to any of the above methods is provided. In further embodiments, a kit comprising a collagen composition and a buffer is provided. In further embodiments, a kit comprising lyophilized type I oligomeric collagen, a hydrochloric acid solution, and a buffer is provided.

[0281] Several additional embodiments are described by the following enumerated sections. Any applicable combination of these embodiments will be considered, as will any applicable combination of the embodiments described in this detailed description section of this application.

[0282] 1. A method for filling tissue voids or defects in a patient, comprising: introducing a self-assembling biopolymer into the tissue voids or defects; and polymerizing the self-assembling biopolymer to form a shape-retaining matrix.

[0283] 2. The method according to item 1, wherein the self-assembling biopolymer comprises in-situ polymerizable oligomeric collagen.

[0284] 3. The method according to any one of the preceding items, wherein the in-situ polymerizable oligomeric collagen contains collagen molecules.

[0285] 4. The method according to any one of the preceding items, wherein at least a portion of the collagen molecules are covalently bonded by one or more intermolecular crosslinks.

[0286] 5. The method according to any one of the preceding items, wherein the patient is a mammal.

[0287] 6. The method described in any one of the preceding items, wherein the patient is human.

[0288] 7. The method described in any one of the preceding items, wherein the introduction is achieved under sterile conditions.

[0289] 8. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises in-situ polymerizable collagen and the shape-retaining matrix comprises a collagen fibril matrix.

[0290] 9. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises liquid type I collagen.

[0291] 10. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises type I oligomeric collagen derived from porcine dermis.

[0292] 11. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and an acid.

[0293] 12. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid.

[0294] 13. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution further comprising a buffer.

[0295] 14. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises an oligomeric collagen solution and a buffer, and the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid.

[0296] 15. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises an oligomeric collagen solution and a buffer, the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid, and the ratio of the oligomeric collagen solution to the buffer is approximately 9:1.

[0297] 16. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid, the concentration of the solution being approximately 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen.

[0298] 17. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation.

[0299] 18. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation and then filtered through a sterile membrane filter.

[0300] 19. The method according to any one of the preceding items, wherein the self-assembling biopolymer comprises a solution containing freeze-dried type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation, irradiated with ultraviolet light, and then filtered through a sterile membrane filter.

[0301] 20. The self-assembling biopolymer contains a solution comprising freeze-dried type I oligomeric collagen and hydrochloric acid, the solution being clarified using ultracentrifugation to 500 mJ / cm³. 2 The method according to any one of the preceding items, wherein the object is irradiated with ultraviolet light and then filtered through a sterile membrane filter.

[0302] 21. The method according to any one of the preceding items, wherein the introduction involves injecting a biopolymer that self-assembles into a tissue cavity or defect via a syringe.

[0303] 22. The method according to any one of the preceding paragraphs, wherein tissue cavities or defects are produced by the procedure for removing a mammary tumor.

[0304] 23. The method described in any one of the preceding paragraphs, wherein a tissue void or defect is created by the mastectomy procedure.

[0305] 24. The method according to any one of the preceding items, wherein filling a void or defect in the tissue does not result in defect atrophy or scar tissue formation.

[0306] 25. The method according to any one of the preceding items, wherein filling a void or defect in tissue does not result in an inflammatory mediator, inflammatory response, or foreign body reaction.

[0307] 26. The method according to any one of the preceding items, wherein filling voids or defects in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0308] 27. The method according to any one of the preceding items, wherein filling voids or defects in the tissue results in the formation of breast tissue, including adipose tissue, mammary gland tissue, or a combination thereof.

[0309] 28. The method according to any one of the preceding items, wherein the tissue embedding response to filling tissue voids or defects is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesion masses, and / or areas of high opacity.

[0310] 29. A method for filling tissue voids or defects in a patient, wherein the tissue voids or defects are caused by a mammary tumor removal or mastectomy procedure, and the method comprises: introducing a mixture comprising an oligomeric collagen solution and a buffer into the tissue voids or defects; polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid.

[0311] 30. The method according to item 29, wherein the ratio of oligomeric collagen solution to buffer solution is approximately 9:1.

[0312] 31. The method according to item 29 or 30, wherein the acid contains 0.01 N hydrochloric acid and the concentration of the oligomeric collagen solution is approximately 8 mg / mL based on the dry weight of lyophilized type I oligomeric collagen.

[0313] 32. A method for filling tissue voids or defects in a patient, wherein the tissue voids or defects are caused by a mammary tumor removal or mastectomy procedure, and the method comprises: introducing a mixture comprising an oligomeric collagen solution and a buffer into the tissue voids or defects; polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid; the concentration of the oligomeric collagen solution is about 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen; and the ratio of the oligomeric collagen solution to the buffer is about 9:1.

[0314] 33. The method according to paragraph 32, wherein the oligomeric collagen solution is clarified using ultracentrifugation, filtered through a sterile membrane filter, irradiated with ultraviolet light, or a combination thereof.

[0315] 34. The method described in any one of paragraphs 32 to 33, wherein the tissue void or defect includes a wound.

[0316] 35. The method described in any one of paragraphs 32 to 34, wherein the tissue void or defect includes a surgical wound.

[0317] 36. The method according to any one of paragraphs 32 to 35, wherein tissue voids or defects result from the removal of the tumor.

[0318] 37. The method according to any one of paragraphs 32 to 36, wherein tissue cavities or defects result from the removal of a breast tumor.

[0319] 38. The method according to any one of the claims 32 to 37, wherein a self-assembling biopolymer comprises a tissue filler.

[0320] 39. The method according to any one of paragraphs 32 to 38, wherein filling a void or defect in the tissue does not result in defect atrophy or scar tissue formation.

[0321] 40. The method according to any one of paragraphs 32 to 39, wherein filling a void or defect in tissue does not result in an inflammatory mediator, inflammatory response, or foreign body reaction.

[0322] 41. The method according to any one of claims 32 to 40, wherein filling voids or defects in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0323] 42. The method according to any one of paragraphs 32 to 41, wherein filling a void or defect in the tissue results in the formation of breast tissue, including adipose tissue, mammary gland tissue, or a combination thereof.

[0324] 43. The method according to any one of paragraphs 32 to 42, wherein the tissue embedding response to filling tissue voids or defects is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesional masses, and / or areas of high opacity.

[0325] 44. A method for filling a wound, comprising: introducing a mixture comprising an oligomeric collagen solution and a buffer into the wound; polymerizing the oligomeric collagen solution to form a collagen-protofibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized oligomeric collagen and an acid.

[0326] 45. The method according to paragraph 44, wherein the freeze-dried type of oligomeric collagen comprises freeze-dried type I oligomeric collagen.

[0327] 46. ​​The method described in paragraph 44 or 45, wherein the wound includes a surgical wound.

[0328] 47. The method described in any one of paragraphs 44 to 46, wherein the surgical wound results from the removal of a tumor.

[0329] 48. The method according to any one of paragraphs 44 to 47, wherein the surgical wound results from the removal of a breast tumor.

[0330] 49. The method according to any one of items 44 to 48, wherein the oligomeric collagen solution comprises a tissue filler.

[0331] 50. The method according to any one of paragraphs 44 to 49, wherein filling the wound does not result in defect atrophy and scar tissue formation.

[0332] 51. The method according to any one of paragraphs 44-50, wherein filling the wound does not result in an inflammatory mediator, inflammatory response, or foreign body reaction.

[0333] 52. The method according to any one of paragraphs 44 to 51, wherein filling a wound results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0334] 53. The method according to any one of paragraphs 44 to 52, wherein filling a wound results in the formation of breast tissue, including adipose tissue, mammary gland tissue, or a combination thereof.

[0335] 54. The method according to any one of paragraphs 44 to 53, wherein the tissue implantation response to wound filling is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesions, and / or areas of high opacity.

[0336] 55. A method for restoring and regenerating skeletal muscle tissue in a void or defect in a patient's tissue, comprising: introducing a self-assembling biopolymer into the void or defect in the tissue; and polymerizing the self-assembling biopolymer to form a shape-retaining matrix.

[0337] 56. The method described in paragraph 55, wherein the tissue void or defect includes a wound.

[0338] 57. The method described in paragraph 55 or 56, wherein the tissue void or defect includes a surgical wound.

[0339] 58. The method according to any one of paragraphs 55 to 57, wherein tissue cavities or defects result from the removal of the tumor.

[0340] 59. The method according to any one of paragraphs 55 to 58, wherein the restoration and regeneration of skeletal muscle tissue in a tissue void or defect does not result in defect atrophy or scar tissue formation.

[0341] 60. The method according to any one of paragraphs 55-59, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects does not result in inflammatory mediators, inflammatory responses, or foreign body reactions.

[0342] 61. The method according to any one of paragraphs 55 to 60, wherein the restoration and regeneration of skeletal muscle tissue in a void or defect in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0343] 62. The method according to any one of paragraphs 55 to 61, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects results in the generation of skeletal muscle together with adipose tissue.

[0344] 63. The method according to any one of paragraphs 55 to 62, wherein the tissue implantation response to restore and regenerate skeletal muscle tissue is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesions, and / or areas of high opacity.

[0345] 64. A method for restoring and regenerating skeletal muscle tissue in a tissue void or defect, comprising: introducing a mixture comprising an oligomeric collagen solution and a buffer into the tissue void or defect; polymerizing the oligomeric collagen solution to form a collagen-fibrillary matrix; wherein the oligomeric collagen solution comprises lyophilized type I oligomeric collagen and an acid.

[0346] 65. The method described in paragraph 64, wherein the tissue void or defect includes a wound.

[0347] 66. The method described in paragraph 64 or 65, wherein the tissue void or defect includes a surgical wound.

[0348] 67. The method according to any one of paragraphs 64 to 66, wherein tissue voids or defects result from the removal of the tumor.

[0349] 68. The method according to any one of paragraphs 64 to 67, wherein the restoration and regeneration of skeletal muscle tissue in a tissue void or defect does not result in defect atrophy or scar tissue formation.

[0350] 69. The method according to any one of paragraphs 64-68, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects does not result in inflammatory mediators, inflammatory responses, or foreign body reactions.

[0351] 70. The method according to any one of paragraphs 64 to 69, wherein the restoration and regeneration of skeletal muscle tissue in a void or defect in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

[0352] 71. The method according to any one of paragraphs 64 to 70, wherein the restoration and regeneration of skeletal muscle tissue in tissue cavities or defects results in the generation of skeletal muscle together with adipose tissue.

[0353] 72. The method according to any one of paragraphs 64 to 71, wherein the tissue implantation response for the restoration and regeneration of skeletal muscle tissue is not adversely affected by irradiation, and as a result, one or more of the following are not observed: lipid cysts, microcalcifications, lesions, and / or areas of high opacity.

[0354] 73. A method for preparing a matrix in a tissue void or defect, comprising using a single mixing step to polymerize collagen, wherein the single mixing step comprises mixing a collagen composition with a buffer to form a collagen solution, and polymerizing the collagen in the collagen solution to form a matrix.

[0355] 74. The method according to claim 73, further comprising incubating the collagen solution at a temperature above approximately 25°C to promote the polymerization of collagen in the collagen solution.

[0356] 75. The method according to paragraph 73 or 74, further comprising incubating the collagen solution at a temperature of approximately 37°C to promote the polymerization of collagen in the collagen solution.

[0357] 76. The method according to any one of items 73 to 75, wherein the collagen comprises collagen oligomers.

[0358] 77. The method according to any one of items 73 to 76, wherein the collagen comprises collagen molecules.

[0359] 78. The method according to any one of items 73 to 77, wherein the collagen consists of collagen oligomers.

[0360] 79. The method according to any one of paragraphs 73 to 78, wherein the collagen consists of intermolecularly cross-linked collagen molecules.

[0361] 80. The method according to any one of items 73 to 79, wherein the collagen substantially consists of intermolecularly cross-linked collagen molecules.

[0362] 81. The method according to any one of items 73 to 80, wherein the collagen further comprises telocollagen.

[0363] 82. The method according to any one of items 73 to 81, wherein the collagen further comprises atelocollagen.

[0364] 83. The method according to any one of claims 73 to 82, wherein collagen containing collagen oligomers is obtained from tissue containing collagen oligomers, from cells that produce collagen oligomers, or by chemically crosslinking collagen to obtain collagen oligomers.

[0365] 84. The method according to any one of items 73 to 83, wherein the collagen is derived from porcine skin tissue.

[0366] 85. The method according to any one of claims 73 to 84, wherein the collagen composition further comprises an acid.

[0367] 86. The method according to any one of claims 73 to 85, wherein the acid is selected from the group consisting of hydrochloric acid, acetic acid, lactic acid, formic acid, citric acid, sulfuric acid, and phosphoric acid.

[0368] 87. The method according to any one of headings 73 to 86, wherein the acid is hydrochloric acid.

[0369] 88. The method according to any one of the items 73 to 87, wherein the hydrochloric acid is hydrochloric acid of about 0.005 N to about 0.1 N.

[0370] 89. The method according to any one of the items 73 to 88, wherein the hydrochloric acid is approximately 0.01 N hydrochloric acid.

[0371] 90. The method according to any one of items 73 to 89, wherein the concentration of collagen in the collagen solution is approximately 0.1 mg / ml to approximately 40 mg / ml.

[0372] 91. The method according to any one of items 73 to 90, wherein the concentration of collagen in the collagen solution is approximately 7 mg / mL to approximately 8 mg / mL.

[0373] 92. The method according to any one of items 73 to 91, wherein the concentration of collagen in the mixture of collagen solution and buffer is approximately 6.3 to approximately 7.2 mg / mL.

[0374] 93. The method according to any one of items 73 to 92, wherein the collagen composition is sterilized.

[0375] 94. The method according to any one of claims 73 to 93, wherein a collagen composition, collagen solution, or collagen matrix is ​​sterilized by a method selected from the group consisting of exposure to chloroform, viral filtration, sterile filtration, gamma irradiation, ultraviolet irradiation, electron beam, and combinations thereof.

[0376] 95. The method according to any one of items 73 to 94, wherein the collagen composition is sterilized by filtration.

[0377] 96. The method according to any one of items 73 to 95, wherein the buffer solution contains approximately 0.03 mM to approximately 0.2 mM MgCl2.

[0378] 97. The method according to any one of items 73 to 96, wherein the buffer solution contains approximately 0.002 mM to approximately 0.02 mM MgCl2.

[0379] 98. The method according to any one of paragraphs 73 to 97, wherein the buffer solution contains less than approximately 0.02 mM MgCl2.

[0380] 99. The method according to any one of items 73 to 98, wherein the buffer solution does not contain MgCl2.

[0381] 100. The method according to any one of paragraphs 73 to 99, wherein the buffer further comprises approximately 0.3 mM to approximately 3 mM KH2PO4.

[0382] 101. The method according to any one of items 73 to 100, wherein the buffer further comprises approximately 1 mM to approximately 10 M Na2HPO4.

[0383] 102. The method according to any one of items 73 to 101, wherein the buffer solution further comprises approximately 0.1 mM to approximately 4 mM KCl.

[0384] 103. The method according to any one of items 73 to 102, wherein the buffer further comprises approximately 0.02 M to approximately 0.3 M of NaCl.

[0385] 104. The method according to any one of items 73 to 103, wherein the buffer further comprises NaOH at a concentration of approximately 0.002 N to approximately 0.02 N.

[0386] 105. The method according to any one of items 73 to 104, wherein the buffer further comprises about 0.5 weight percent to about 5 weight percent glucose.

[0387] 106. The method according to any one of items 73 to 105, wherein the buffer solution contains glucose at a rate of approximately 0.5 weight percent or less.

[0388] 107. The method according to any one of items 73 to 106, wherein the buffer solution does not contain glucose.

[0389] 108. The method according to any one of claims 73 to 107, further comprising adding cells to a collagen solution.

[0390] 109. The method according to any one of items 73 to 108, wherein the matrix comprises collagen fibrils.

[0391] 110. The method according to any one of items 73 to 109, wherein the collagen is soluble collagen.

[0392] 111. The method according to any one of paragraphs 73 to 110, wherein a collagen composition, collagen solution, and / or matrix are sterilized using UVC irradiation.

[0393] 112. The method according to any one of claims 73 to 111, wherein a collagen composition, a collagen solution, and / or a matrix are sterilized using UVC irradiation and sterile filtration.

[0394] 113. The method according to any one of claims 73 to 112, wherein the matrix obtained from the polymerization of a collagen solution maintains polymerization properties compared to an unirradiated collagen composition or unirradiated freeze-dried collagen.

[0395] 114. The method according to any one of items 73 to 113, wherein the polymerization property is the shear storage coefficient.

[0396] 115. The radiation dose is approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 The method described in any one of paragraphs 73 to 114, which falls within the range of the specified paragraph.

[0397] 116. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2 The method described in any one of paragraphs 73 to 115, which falls within the scope of the above.

[0398] 117. The method described in any one of paragraphs 73 to 116, wherein sterilization inactivates the virus.

[0399] 118. A method for preparing a matrix in a tissue defect or void, comprising polymerizing collagen by mixing a collagen composition with a buffer to form a collagen solution, and polymerizing the collagen in the collagen solution to form a matrix, wherein the buffer does not contain magnesium ions or manganese ions.

[0400] 119. The method according to item 118, further comprising incubating the collagen solution at a temperature above approximately 25°C to promote the polymerization of collagen in the collagen solution.

[0401] 120. The method according to paragraph 118 or 119, further comprising incubating the collagen solution at a temperature of approximately 37°C to promote the polymerization of collagen in the collagen solution.

[0402] 121. The method according to any one of items 118 to 120, wherein the collagen comprises collagen oligomers.

[0403] 122. The method according to any one of items 118 to 121, wherein the collagen comprises collagen molecules.

[0404] 123. The method according to any one of items 118 to 122, wherein the collagen consists of collagen oligomers.

[0405] 124. The method according to any one of items 118 to 123, wherein the collagen consists of intermolecularly cross-linked collagen molecules.

[0406] 125. The method according to any one of items 118 to 124, wherein the collagen substantially consists of intermolecularly cross-linked collagen molecules.

[0407] 126. The method according to any one of items 118 to 125, wherein the collagen further comprises telocollagen.

[0408] 127. The method according to any one of items 118 to 126, wherein the collagen further comprises atelocollagen.

[0409] 128. The method according to any one of claims 118 to 127, wherein collagen containing collagen oligomers is obtained from tissue containing collagen oligomers, from cells that produce collagen oligomers, or by chemically crosslinking collagen to obtain collagen oligomers.

[0410] 129. The method according to any one of items 118-128, wherein the collagen is derived from porcine skin tissue.

[0411] 130. The method according to any one of claims 118 to 129, wherein the collagen composition further comprises an acid.

[0412] 131. The method according to any one of claims 118 to 130, wherein the acid is selected from the group consisting of hydrochloric acid, acetic acid, lactic acid, formic acid, citric acid, sulfuric acid, and phosphoric acid.

[0413] 132. The method according to any one of headings 118 to 131, wherein the acid is hydrochloric acid.

[0414] 133. The method according to any one of the items 118 to 132, wherein the hydrochloric acid is hydrochloric acid of about 0.005 N to about 0.1 N.

[0415] 134. The method according to any one of the items 118 to 133, wherein the hydrochloric acid is approximately 0.01 N hydrochloric acid.

[0416] 135. The method according to any one of items 118 to 134, wherein the concentration of collagen in the collagen solution is approximately 0.1 mg / ml to approximately 40 mg / ml.

[0417] 136. The method according to any one of items 118 to 135, wherein the concentration of collagen in the collagen solution is approximately 7 mg / mL to approximately 8 mg / mL.

[0418] 137. The method according to any one of items 118 to 136, wherein the concentration of collagen in the mixture of collagen solution and buffer is approximately 6.3 to approximately 7.2 mg / mL.

[0419] 138. The method according to any one of the items 118 to 137, wherein the collagen composition is sterilized.

[0420] 139. The method according to any one of claims 118 to 138, wherein a collagen composition, collagen solution, or collagen matrix is ​​sterilized by a method selected from the group consisting of exposure to chloroform, viral filtration, sterile filtration, gamma irradiation, ultraviolet irradiation, electron beam, and combinations thereof.

[0421] 140. The method according to any one of items 118 to 139, wherein the collagen composition is sterilized by filtration.

[0422] 141. The method according to any one of items 118 to 140, wherein the buffer solution contains approximately 0.03 mM to approximately 0.2 mM MgCl2.

[0423] 142. The method according to paragraph 141, wherein the buffer solution contains approximately 0.002 mM to approximately 0.02 mM MgCl2.

[0424] 143. The method according to any one of items 118 to 142, wherein the buffer solution contains less than approximately 0.02 mM MgCl2.

[0425] 144. The method according to any one of paragraphs 118 to 143, wherein the buffer solution does not contain MgCl2.

[0426] 145. The method according to any one of paragraphs 118 to 144, wherein the buffer further comprises approximately 0.3 mM to approximately 3 mM KH2PO4.

[0427] 146. The method according to any one of items 118 to 145, wherein the buffer further comprises approximately 1 mM to approximately 10 M Na2HPO4.

[0428] 147. The method according to any one of items 118 to 146, wherein the buffer solution further comprises approximately 0.1 mM to approximately 4 mM KCl.

[0429] 148. The method according to any one of items 118 to 147, wherein the buffer further comprises approximately 0.02 M to approximately 0.3 M of NaCl.

[0430] 149. The method according to any one of items 118 to 148, wherein the buffer further comprises NaOH at a concentration of approximately 0.002 N to approximately 0.02 N.

[0431] 150. The method according to any one of items 118 to 149, wherein the buffer further comprises about 0.5 weight percent to about 5 weight percent glucose.

[0432] 151. The method according to any one of items 118 to 150, wherein the buffer solution contains glucose at a rate of approximately 0.5 weight percent or less.

[0433] 152. The method according to any one of items 118 to 151, wherein the buffer solution does not contain glucose.

[0434] 153. The method according to any one of items 118 to 152, further comprising adding cells to a collagen solution.

[0435] 154. The method according to any one of items 118 to 153, wherein the matrix comprises collagen fibrils.

[0436] 155. The method according to any one of items 118 to 154, wherein the collagen is soluble collagen.

[0437] 156. The method according to any one of paragraphs 118 to 155, wherein a collagen composition, a collagen solution, and / or a collagen matrix are sterilized using ultraviolet irradiation.

[0438] 157. The method according to any one of the items 118 to 156, wherein a collagen composition, a collagen solution, and / or a matrix are sterilized using UVC irradiation and sterile filtration.

[0439] 158. The method according to any one of claims 118 to 157, wherein the matrix obtained from the polymerization of a collagen solution maintains polymerization properties compared to an unirradiated collagen composition or unirradiated freeze-dried collagen.

[0440] 159. The method according to any one of items 118 to 158, wherein the polymerization property is the shear storage coefficient.

[0441] 160. The radiation dose is approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 The method described in any one of paragraphs 118 to 159, which falls within the scope of the specified range.

[0442] 161. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2 The method described in any one of paragraphs 118 to 160, which falls within the scope of the specified range.

[0443] 162. The method described in any one of paragraphs 118 to 161, wherein sterilization inactivates the virus.

[0444] 163. A collagen matrix prepared according to the method described in any one of paragraphs 1 to 162.

[0445] 164. A collagen matrix as described in paragraph 163, which is a medical graft.

[0446] 165. The collagen matrix according to paragraph 163 or 164, wherein the medical graft has a use selected from the group consisting of tissue graft material, injectable graft material, wound dressing material, hemostatic dressing material, delivery medium for therapeutic cells, and delivery medium for therapeutic agents.

[0447] 166. A collagen matrix as described in any one of paragraphs 163-165, for use in research.

[0448] 167. A collagen matrix as described in any one of the paragraphs 163 to 166, used for drug toxicity testing or drug development.

[0449] 168. A collagen matrix as described in any one of paragraphs 163 to 167, sterilized using ultraviolet irradiation.

[0450] 169. A collagen matrix according to any one of paragraphs 163 to 168, which maintains polymerization properties compared to an unirradiated collagen matrix.

[0451] 170. A collagen matrix according to any one of paragraphs 163 to 169, wherein the polymerization property is the shear storage coefficient.

[0452] 171. The radiation dose was approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 A collagen matrix as described in any one of paragraphs 163 to 170, which falls within the range of the specified paragraphs.

[0453] 172. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2A collagen matrix as described in any one of paragraphs 163 to 171, which falls within the range of the specified paragraphs.

[0454] 173. A collagen matrix as described in any one of paragraphs 163-172, wherein sterilization has inactivated the virus.

[0455] 174. A collagen matrix as described in any one of paragraphs 163 to 173, sterilized using UVC irradiation.

[0456] 175. A collagen matrix as described in any one of paragraphs 163 to 174, sterilized using UVC irradiation and sterile filtration.

[0457] 176. A collagen matrix prepared by introducing a self-assembling biopolymer into a void or defect in a tissue and polymerizing the self-assembling biopolymer to form a shape-retaining matrix, wherein the pH of the self-assembling biopolymer is in the range of about 5.5 to about 8.5, the self-assembly time of the self-assembling biopolymer is in the range of about 0.2 minutes to about 1.5 minutes, the shear storage coefficient (G') of the collagen matrix is ​​in the range of about 2.0 kPa to about 4.0 kPa, the shear loss coefficient (G”) of the collagen matrix is ​​in the range of about 0.1 kPa to about 0.7 kPa, and the compressibility coefficient of the collagen matrix is ​​in the range of about 5.0 kPa to about 10.0 kPa.

[0458] 177. The collagen matrix described in paragraph 176, wherein the pH of the self-assembling biopolymer is approximately 7.25 ± approximately 0.25, the self-assembly time of the self-assembling biopolymer is approximately 0.8 min ± approximately 0.3 min, the shear storage coefficient (G') of the collagen matrix is ​​approximately 3.1 kPa ± approximately 0.4 kPa, the shear loss coefficient (G”) of the collagen matrix is ​​approximately 0.4 kPa ± approximately 0.1 kPa, and the compressibility coefficient of the collagen matrix is ​​approximately 7.7 kPa ± approximately 1.9 kPa.

[0459] 178. A collagen matrix as described in paragraph 176 or 177, which is a medical graft.

[0460] 179. A collagen matrix according to any one of the claims 176 to 178, wherein the medical graft has a use selected from the group consisting of tissue graft material, injectable graft material, wound dressing material, hemostatic dressing material, delivery medium for therapeutic cells, and delivery medium for therapeutic agents.

[0461] 180. A collagen matrix as described in any one of paragraphs 176-179, for use in research.

[0462] 181. A collagen matrix as described in any one of the paragraphs 176-180, used for drug toxicity testing or drug development.

[0463] 182. A collagen matrix as described in any one of paragraphs 176-181, sterilized using ultraviolet irradiation.

[0464] 183. A collagen matrix according to any one of paragraphs 176 to 182, which maintains polymerization properties compared to an unirradiated collagen matrix.

[0465] 184. A collagen matrix according to any one of paragraphs 176 to 183, wherein the polymerization property is the shear storage coefficient.

[0466] 185. The radiation dose is approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 A collagen matrix as described in any one of paragraphs 176 to 184, which falls within the range of the specified paragraphs.

[0467] 186. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2 A collagen matrix as described in any one of paragraphs 176 to 185, which falls within the range of the specified paragraphs.

[0468] 187. A collagen matrix as described in any one of paragraphs 176-186, wherein sterilization has inactivated the virus.

[0469] 188. A collagen matrix as described in any one of paragraphs 176-187, sterilized using UVC irradiation.

[0470] 189. A collagen matrix as described in any one of paragraphs 176-188, sterilized using UVC irradiation and sterile filtration.

[0471] 190. A kit for restoring and regenerating tissue in tissue voids or defects, comprising an in-situ polymerizable collagen composition and a buffer.

[0472] 191. The kit according to item 190, wherein the in situ polymerizable collagen composition comprises liquid type I collagen.

[0473] 192. A kit according to paragraph 190 or 191, wherein the in-situ polymerizable collagen composition comprises type I oligomeric collagen derived from porcine dermis.

[0474] 193. A kit according to any one of items 190 to 192, wherein the in-situ polymerizable collagen composition comprises a solution containing freeze-dried oligomeric collagen and an acid.

[0475] 194. A kit according to any one of the following paragraphs, 190 to 193, comprising an in-situ polymerizable collagen composition containing a solution comprising lyophilized type I oligomeric collagen and an acid.

[0476] 195. A kit according to any one of claims 190 to 194, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid.

[0477] 196. A kit according to any one of items 190 to 195, wherein the ratio of in situ polymerizable collagen composition to buffer solution is approximately 9:1.

[0478] 197. A kit according to any one of items 190 to 196, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid, and the collagen concentration in the solution of the in-situ polymerizable collagen composition is approximately 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen.

[0479] 198. A kit according to any one of claims 190 to 197, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, and the solution of the in-situ polymerizable collagen composition is clarified using ultracentrifugation.

[0480] 199. The kit according to any one of the following paragraphs, 190 to 198, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution of the in-situ polymerizable collagen composition being clarified using ultracentrifugation and then filtered through a sterile membrane filter.

[0481] 200. The kit according to any one of the following paragraphs, 190 to 199, wherein the in-situ polymerizable collagen composition comprises a solution containing lyophilized type I oligomeric collagen and hydrochloric acid, the solution of the in-situ polymerizable collagen composition being clarified using ultracentrifugation, irradiated with ultraviolet light, and then filtered through a sterile membrane filter.

[0482] 201. The in-situ polymerizable collagen composition comprises a solution containing freeze-dried type I oligomeric collagen and hydrochloric acid, and the solution of the in-situ polymerizable collagen composition is clarified using ultracentrifugation to 500 mJ / cm³. 2 A kit as described in any one of paragraphs 190 to 200, which is irradiated with ultraviolet light and then filtered through a sterile membrane filter.

[0483] 202. The kit according to any one of claims 190 to 201, further comprising a syringe configured for delivering a mixture of an in-situ polymerizable collagen composition and a buffer to a void or defect in tissue.

[0484] 203. A kit as described in any one of sections 190 to 202, wherein the buffer solution contains approximately 0.03 mM to approximately 0.2 mM MgCl2.

[0485] 204. A kit as described in any one of items 190 to 203, wherein the buffer solution contains approximately 0.002 mM to approximately 0.02 mM MgCl2.

[0486] 205. A kit described in any one of items 190-204, containing a buffer solution of less than approximately 0.02 mM MgCl2.

[0487] 206. A kit described in any one of sections 190-205, wherein the buffer solution does not contain MgCl2.

[0488] 207. A kit as described in any one of sections 190 to 206, wherein the buffer further contains approximately 0.003 M to approximately 0.03 M of KH2PO4.

[0489] 208. A kit as described in any one of sections 190 to 207, wherein the buffer solution further contains approximately 0.01 M to approximately 0.1 M of Na2HPO4.

[0490] 209. A kit as described in any one of sections 190 to 208, wherein the buffer solution further contains approximately 0.001 M to approximately 0.04 M of KCl.

[0491] 210. A kit as described in any one of sections 190 to 209, wherein the buffer solution further contains approximately 0.2 M to approximately 3.0 M of NaCl.

[0492] 211. A kit as described in any one of sections 190 to 210, wherein the buffer solution further contains NaOH at a concentration of approximately 0.02 N to approximately 0.2 N.

[0493] 212. A kit according to any one of items 190 to 211, wherein the buffer further comprises approximately 0.2 to 5 weight percent glucose.

[0494] 213. A kit according to any one of items 190-212, wherein the buffer solution contains glucose at a rate of approximately 0.5 weight percent or less.

[0495] 214. A kit described in any one of sections 190-213, wherein the buffer solution does not contain glucose.

[0496] 215. A kit according to any one of items 190 to 214, wherein the concentration of collagen in the in-situ polymerizable collagen composition is approximately 0.1 mg / ml to approximately 40 mg / ml.

[0497] 216. A kit according to any one of items 190 to 215, wherein the concentration of collagen in the in situ polymerizable collagen composition is approximately 7 mg / mL to approximately 8 mg / mL.

[0498] 217. A kit according to any one of items 190 to 216, wherein the concentration of collagen in a neutralized collagen filler comprising an in-situ polymerizable collagen composition and a buffer is approximately 6.3 to approximately 7.2 mg / mL.

[0499] 218. A kit according to any one of items 190 to 217, wherein the collagen solution contains hydrochloric acid ranging from approximately 0.005 N to approximately 0.1 N.

[0500] 219. The kit according to any one of claims 190 to 218, wherein the buffer is configured to polymerize the in situ polymerizable collagen composition in a single mixing step comprising mixing the in situ polymerizable collagen composition with the buffer.

[0501] 220. A kit according to any one of items 190 to 219, wherein the in situ polymerizable collagen composition and buffer are contained in separate containers.

[0502] 221. A kit as described in any one of the items 190 to 220, wherein each individual container contains a sterile vial.

[0503] 222. A kit as described in any one of the headings 190 to 221, wherein each individual container contains a separate compartment for a dual-barrel syringe.

[0504] 223. A kit according to any one of the following sections, 190 to 222, comprising a dual-barrel syringe containing a mixing element.

[0505] 224. A kit described in any one of sections 190-223, wherein the dual-barrel syringe is sterile.

[0506] 225. The kit described in any one of sections 190 to 224, further including instructions for using the components of the kit.

[0507] 226. A kit according to any one of the paragraphs 190 to 225, further comprising at least one therapeutic agent configured for local delivery to a tissue void or defect.

[0508] 227. A kit according to any one of the paragraphs 190 to 226, wherein at least one therapeutic agent comprises a chemotherapeutic agent, an anti-inflammatory agent, an antibiotic, an analgesic, or a combination thereof.

[0509] 228. A kit according to any one of the paragraphs 190 to 227, wherein the tissue void or defect includes a wound.

[0510] 229. A kit according to any one of the paragraphs 190 to 228, wherein the tissue void or defect includes a surgical wound.

[0511] 230. A kit according to any one of paragraphs 190-229, wherein tissue voids or defects result from tumor removal.

[0512] 231. A kit according to any one of paragraphs 190-230, wherein tissue voids or defects result from the removal of a breast tumor.

[0513] 232. A kit for regenerating tissue after breast-conserving surgery, as described in any one of paragraphs 190 to 231.

[0514] 233. A kit for preparing a matrix in a tissue void or defect, as described in any one of the paragraphs 190 to 232.

[0515] 234. A kit according to any one of the following paragraphs, 190 to 233, wherein the in-situ polymerizable collagen composition or freeze-dried oligomeric collagen is sterilized using ultraviolet irradiation.

[0516] 235. The kit according to any one of items 190 to 234, wherein the collagen matrix obtained from the polymerization of an in-situ polymerizable collagen composition maintains its polymerization properties compared to an unirradiated collagen composition or unirradiated lyophilized collagen.

[0517] 236. A kit described in any one of sections 190 to 235, wherein the polymerization property is the shear storage coefficient.

[0518] 237. The radiation dose was approximately 5 mJ / cm². 2 From approximately 800 mJ / cm² 2 A kit described in any one of paragraphs 190 to 236, which falls within the range of the specified items.

[0519] 238. The radiation dose was approximately 30 mJ / cm². 2 From approximately 300 mJ / cm² 2 A kit as described in any one of paragraphs 190 to 237, which falls within the range of [the specified area].

[0520] 239. A kit described in any one of paragraphs 190-238, wherein sterilization has inactivated the virus.

[0521] 240. A kit according to any one of the following paragraphs, 190 to 239, wherein the in-situ polymerizable collagen composition or lyophilized oligomeric collagen is sterilized using UVC irradiation.

[0522] 241. A kit according to any one of the paragraphs 190 to 240, wherein the collagen composition or freeze-dried oligomeric collagen is sterilized using UVC irradiation and sterile filtration.

[0523] The purified fibrillating liquid type I collagen derived from porcine dermis for use in this instruction is described in the applicant's concurrently pending U.S. Patent Application No. 16 / 482,465, filed July 31, 2019, and International Publication No. 2018 / 144496A1. The entire contents of both documents are incorporated herein by reference.

[0524] With regard to collagen preparations for use in the methods and compositions described herein, any method known in the art for preparing collagen may be used. In exemplary embodiments, collagen may be prepared by the methods described in Bailey JL, Critser PJ, Whittington C, Kuske JL, Yoder MC, Voytik-Harbin SL; Collagen oligomers modulate physical and biological properties of three-dimensional self-assembled matrices, Biopolymers (2011) 95(2):77-93, Kreger ST, Bell BJ, Bailey J, Stites E, Kuske J, Waisner B, Voytik-Harbin SL; Polymerization and matrix physical properties as important design considerations for soluble collagen formulations, Biopolymers (2010) 93(8):690-707, U.S. Patent Application Publication No. 20080268052, or U.S. Patent Application Publication No. 20120027732, which are incorporated herein by reference, respectively.

[0525] In various exemplary embodiments, the collagen for use in the methods and compositions described herein may be obtained from any suitable raw material of collagen known in the art, provided that at least a portion of the collagen comprises polymerizable oligomeric collagen. Exemplary collagen sources generally include submucosal tissue (U.S. Patents 4,902,508, 5,281,422, and 5,275,826), pericardial tissue, bladder submucosa, gastric submucosa, liver basement membrane tissue, placental tissue, ovarian tissue, animal tail tissue, skin tissue [e.g., Gallop, et al., Preparation and Properties of Soluble Collagens, Meth. Enzymol. 6: 635-641 (1963), incorporated herein by reference], and extracellular matrix-containing tissues. In various embodiments, the type of collagen to be used in the methods and compositions described herein may be any suitable type of collagen, but is not limited to, type I collagen, type II collagen, type III collagen, or type IV collagen, or a combination thereof.

[0526] In some embodiments, collagen oligomer-rich tissue (e.g., porcine skin tissue) may also be used to obtain collagen for use in the methods and compositions described herein, or the collagen may be obtained from cells that produce collagen oligomers (e.g., cells modified by recombinant technology to express collagen oligomers), or by chemically crosslinking collagen to obtain collagen oligomers (e.g., using crosslinking agents known in the art). In some embodiments, the collagen for use in the methods and compositions described herein may contain oligomers or consist of oligomers. In some embodiments, the collagen may include oligomers and other forms of collagen, such as monomers, telocollagen, and / or atelocollagen.

[0527] In another embodiment, the collagen may be soluble collagen or solubilized collagen. In embodiments where the collagen is soluble collagen or solubilized collagen, the collagen is substantially free of insoluble collagen, but may contain some insoluble collagen. In another embodiment, the collagen consists of soluble collagen or solubilized collagen.

[0528] In various exemplary embodiments, collagen, collagen compositions, collagen matrices, collagen solutions, lyophilized collagen, and / or buffers (also referred herein as neutralizing buffers or self-assembling reagents) may be sterilized using sterilization techniques known in the art, including, but not limited to, propylene oxide or ethylene oxide treatment, gas plasma sterilization, gamma irradiation (e.g., 0.1–10 Mrad), ultraviolet irradiation (e.g., UVC irradiation), electron beam, viral filtration, sterile filtration (e.g., using a 0.22 μm filter), chloroform exposure, and / or peracetic acid sterilization, and combinations thereof. In this embodiment, the sterilization procedure should not adversely affect the structure of the sterilized collagen, the polymerization properties of the collagen, or the biological properties of the collagen. In various embodiments, the collagen may be sterilized before or after lyophilization (lyophilization procedures are described below).

[0529] In embodiments including ultraviolet irradiation (e.g., UVC irradiation), the collagen matrix obtained from collagen polymerization can maintain polymerization properties compared to unirradiated collagen, unirradiated collagen composition, unirradiated collagen matrix, unirradiated collagen solution, or unirradiated lyophilized collagen. In such embodiments, polymerization properties may be selected from shear storage coefficient, elastic modulus (Young's modulus), tensile modulus, compressive coefficient, fibrillary structure, proteolysis, cellular signaling, and combinations thereof. In various embodiments, the amount of ultraviolet irradiation (e.g., UVC irradiation) is about 5 mJ / cm². 2 From approximately 800 mJ / cm²2 , about 5mJ / cm 2 From approximately 700 mJ / cm² 2 , about 5mJ / cm 2 From approximately 600 mJ / cm² 2 , about 5mJ / cm 2 From approximately 500 mJ / cm² 2 , about 5mJ / cm 2 From approximately 400 mJ / cm² 2 , about 5mJ / cm 2 From approximately 300 mJ / cm² 2 5 mJ / cm 2 From approximately 200 mJ / cm² 2 5 mJ / cm 2 From approximately 100 mJ / cm² 2 5 mJ / cm 2 From approximately 50 mJ / cm² 2 , about 30mJ / cm 2 From approximately 800 mJ / cm² 2 , about 30mJ / cm 2 From approximately 700 mJ / cm² 2 , about 30mJ / cm 2 From approximately 600 mJ / cm² 2 , about 30mJ / cm 2 From approximately 500 mJ / cm² 2 , about 30mJ / cm 2 From approximately 400 mJ / cm² 2 , about 30mJ / cm 2 From approximately 300 mJ / cm² 2 , about 30mJ / cm 2 From approximately 200 mJ / cm² 2 , about 30mJ / cm 2 From approximately 100 mJ / cm² 2 , about 30mJ / cm 2 From approximately 50 mJ / cm² 2 , about 200mJ / cm 2 From approximately 800 mJ / cm² 2 , about 300mJ / cm 2 From approximately 800 mJ / cm² 2 , about 400mJ / cm 2 From approximately 800 mJ / cm² 2 , about 500mJ / cm 2 From approximately 800 mJ / cm² 2 , about 600mJ / cm 2From about 800 mJ / cm 2 to about 50 mJ / cm 2 from about 300 mJ / cm 2 to about 100 mJ / cm 2 from about 300 mJ / cm 2 to about 200 mJ / cm 2 from about 300 mJ / cm 2 or may be in the range of. In all embodiments of the ultraviolet irradiation described herein (e.g., UVC irradiation), sterilization inactivates the virus. In this embodiment, "inactivating the virus" means inactivating all viruses regardless of whether they are infected or not, reducing the number of infectious viruses, or inhibiting the activity of the virus regardless of whether they are infected or not.

[0530] In one aspect, the collagen for use in the methods and compositions described herein may be purified by methods known in the art of collagen purification. As used herein, "purified" means, but is not limited to, removal of contaminants including cell contaminants, nucleotide contaminants, and endotoxins. In various embodiments, the collagen may be purified to remove contaminants such that it has a purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5%. In other embodiments, the collagen may be isolated. As used herein, "isolated" means, but is not limited to, substantially free of contaminants including cell contaminants, nucleotide contaminants, and endotoxins.

[0531] In some embodiments, collagen for use in the methods and compositions described herein may be freeze-dried and then reconstituted to form a collagen composition which is then mixed with the buffers described herein. In such embodiments, the reconstitution of freeze-dried collagen is not a mixing step for polymerizing collagen. As used herein, the term “freeze-drying” means, for example, that water is removed from a protein, compound, or composition by freeze-drying under vacuum. Any freeze-drying method known to those skilled in the art may be used. In some embodiments, collagen may be freeze-dried in an acid, such as acetic acid, hydrochloric acid, formic acid, lactic acid, citric acid, sulfuric acid, or phosphoric acid. In other embodiments, collagen may be freeze-dried in water. In further embodiments, a freeze-protecting agent or a freeze-drying protectant, or a combination thereof, may be used during freeze-drying.

[0532] In some embodiments, lyophilized collagen may be reconstituted to form a collagen composition as described herein and mixed with a buffer to polymerize the collagen. In some embodiments, the collagen may be reconstituted in an acidic solution or in water. In some embodiments, the acidic solution may contain acetic acid, hydrochloric acid, formic acid, lactic acid, citric acid, sulfuric acid, or phosphoric acid. In some embodiments, the acidic solution for reconstitution may have an acid concentration of about 0.005 N to about 0.1 N, about 0.005 N to about 0.08 N, about 0.005 N to about 0.06 N, about 0.005 N to about 0.04 N, about 0.005 N to about 0.02 N, about 0.005 N to about 0.01 N, or about 0.01 N. In some embodiments, the acid may be hydrochloric acid, and the hydrochloric acid may be hydrochloric acid of about 0.005 N to about 0.1 N. In other embodiments, the acid may be hydrochloric acid, and the hydrochloric acid may be hydrochloric acid of about 0.01 N.

[0533] In some embodiments, the collagen concentration in the collagen composition or collagen solution may be about 0.1 mg / ml to about 40 mg / ml, about 0.1 mg / ml to about 5 mg / ml, or about 0.5 mg / ml to about 4 mg / ml. In other embodiments, the collagen concentration in the collagen composition or collagen solution may be about 0.05 to about 5.0 mg / ml, about 1.0 mg / ml to about 3.0 mg / ml, about 0.05 mg / ml to about 10 mg / ml, about 0.05 to about 20 mg / ml, about 0.05 to about 30 mg / ml, about 0.05 to about 40 mg / ml, about 0.05 to about 50 mg / ml, about 0.05 to about 60 mg / ml, about 0.05 to about 80 mg / ml, about 5 mg / ml to 10 mg / ml, about 5 mg / ml to 20 mg / ml, about 5 mg / ml to about 40 mg / ml, about 5 mg / ml to about 60 mg / ml, about 5 mg / ml to about 100 mg / ml, about 20 mg / ml to about 40 mg / ml, about 20 mg / ml to about 60 mg / ml, or about 20 mg / ml to about 100 mg / ml.

[0534] In some embodiments, the collagen composition is mixed with a buffer in a single step to polymerize the collagen. In other embodiments, the collagen composition is mixed with a buffer in the absence of magnesium or manganese ions to polymerize the collagen. In some embodiments, the collagen composition is mixed with a buffer to form a collagen solution, which is incubated at a temperature above about 25°C to promote the polymerization of collagen in the collagen solution. In other embodiments, the collagen solution may be incubated at about 37°C to promote the polymerization of collagen in the collagen solution. In some embodiments, the collagen solution may be incubated at about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 38°C, 39°C, or 40°C to promote the polymerization of collagen in the collagen solution. In other embodiments, the collagen solution may be incubated from about 25°C to about 40°C to promote the polymerization of collagen in the collagen solution. In other embodiments, polymerization may be carried out at a temperature above 20°C or at a temperature selected from the range of about 20°C to about 40°C. In such embodiments, collagen may be polymerized to form proficient fibers similar to those found in the body.

[0535] In some embodiments, the buffer that will be mixed with the collagen composition to form a collagen solution may contain, or not contain, about 0.03 mM to about 0.2 mM MgCl2, about 0.002 mM to about 0.02 mM MgCl2, or less than about 0.02 mM MgCl2. In other embodiments, the buffer that will be mixed with the collagen composition to form a collagen solution may contain, about 0.3 mM to about 3 mM KH2PO4, about 1 mM to about 10 M Na2HPO4, about 0.1 mM to about 4 mM KCl, about 0.02 M to about 0.3 M NaCl, and about 0.002 N to about 0.02 N NaOH. In other embodiments, the buffer that will be mixed with the collagen composition to form a collagen solution may contain, or not contain, about 0.5% to about 5% by weight glucose, or less than about 0.5% by weight glucose.

[0536] In some embodiments, the buffer may be diluted 10-fold, 5-fold, 2-fold, or from any suitable starting concentration to prepare a 1-fold buffer having any of the component concentrations in the preceding section. In some embodiments, the kit according to this teaching may contain a buffer having a 10-fold, 5-fold, or 2-fold concentration, or any suitable starting concentration, for dilution to prepare a 1-fold buffer. In some embodiments, the 10-fold buffer may contain the following components at the following concentrations: 1.37M NaCl 0.027M KCl 0.081M Na2HPO4 0.015M KH2PO4 0.1N NaOH 55.5 mM glucose as needed

[0537] In other embodiments, the 1x buffer may contain the following components at the following concentrations: 0.137M NaCl 0.0027M KCl 0.0081M Na2HPO4 0.0015M KH2PO4 0.01N NaOH 5.55 mM glucose as needed

[0538] In these embodiments, NaOH is present in the buffer. In conventional known methods for polymerizing collagen, NaOH was added separately as an additional mixing step in the method for polymerizing collagen. In some embodiments, calcium chloride may be present in the buffer at a concentration of about 0.4 mM to about 2.0 mM.

[0539] In some embodiments, the buffer in the buffer solution may be selected from the group consisting of phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-n,n'-bis(2-ethanesulfonic acid) (PIPES), [n-(2-acetamide)]-2-aminoethanesulfonic acid (ACES), N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), and 1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis Trispropane). In some embodiments, the buffer is PBS.

[0540] In some embodiments, the pH of the collagen solution for polymerizing collagen is selected from the range of about 5.0 to about 11, about 6.0 to about 9.0, and about 6.5 to about 8.5, and in some embodiments, the pH is about 7.3 to about 7.4.

[0541] In some embodiments, nutrients including minerals, amino acids, sugars, peptides, proteins, vitamins, or glycoproteins that promote cell proliferation, such as laminin and fibronectin, hyaluronic acid, or growth factors, such as epidermal growth factor, platelet-derived growth factor, transforming growth factor β, or fibroblast growth factor, as well as glucocorticoids, such as dexamethasone, may be added to the collagen solution before, during, or after the completion of collagen polymerization. In other embodiments, cells may be added to the collagen solution before, during, or after the completion of collagen polymerization. In some embodiments, cells may be selected from the group consisting of epithelial cells, endothelial cells, mesoderm-derived cells, mesothelial cells, synovial cells, nerve cells, glial cells, osteoblasts, fibroblasts, chondrocytes, tendinocytes, smooth muscle cells, skeletal muscle cells, cardiomyocytes, pluripotent primordial cells (e.g., myelocytes, induced pluripotent stem cells, and other stem cells), adipocytes, osteogenic cells, and specific cell derivatives from pluripotent stem cells.

[0542] In some embodiments, collagen matrices prepared according to any of the methods described herein are provided. In some embodiments, the collagen matrices may be medical grafts. In some embodiments, the medical grafts have uses selected from the group consisting of tissue graft materials, injectable graft materials, wound dressings, hemostatic dressings, delivery media for therapeutic cells, and delivery media for therapeutic agents. In other embodiments, the methods described herein may be used to produce bioink formulations for printing tissues or organs. In other embodiments, the collagen matrices are used for research purposes, e.g., drug toxicity testing or drug development. In some embodiments, matrices prepared by the methods described herein may serve as a substrate for the regrowth (e.g., remodeling) of endogenous tissue at the implantation site, and the matrices may have the characteristics of injured or pathological tissue that is replaced at the implantation or injection site.

[0543] In some embodiments, the matrix described herein has a fibril surface fraction (defined as a percentage area of ​​the total area occupied by fibrils in the cross-sectional surface of the matrix) or fibril volume fraction (fibrils in three dimensions) of approximately 0.1% to approximately 100%, approximately 0.5% to approximately 100%, approximately 0.5% to approximately 26%, approximately 1% to approximately 100%, approximately 1% to approximately 26%, approximately 1% to approximately 7%, approximately 1% to approximately 15%, approximately 7% to approximately 26%, approximately 20% to approximately 30%, approximately 20% to approximately 50%, approximately 20% to approximately 70%, approximately 20% to approximately 100%, approximately 30% to approximately 50%, approximately 30% to approximately 70%, or approximately 30% to approximately 100%. The material may include a fraction of the total area occupied by the fibers, and / or a coefficient (e.g., elastic or linear coefficient defined by the slope of the linear region of the stress-strain curve obtained using a conventional mechanical testing protocol; i.e., stiffness), compressibility, or shear storage coefficient in the ranges of approximately 0.5 kPa to approximately 40 kPa, approximately 30 kPa to approximately 100 kPa, approximately 30 kPa to approximately 1000 kPa, approximately 30 kPa to approximately 10000 kPa, approximately 30 kPa to approximately 70000 kPa, approximately 100 kPa to approximately 10000 kPa, or approximately 100 kPa to approximately 70000 kPa.

[0544] In some embodiments, a kit is provided comprising lyophilized collagen, a hydrochloric acid solution, and a buffer. In other embodiments, a kit is provided comprising a collagen composition and a buffer. In these kit embodiments, the buffer may contain or not contain MgCl2 in amounts ranging from about 0.03 mM to about 0.2 mM, from about 0.002 mM to about 0.02 mM, or less than about 0.02 mM. In various embodiments, the buffer further comprises about 0.003 M to about 0.03 M KH2PO4, about 0.01 M to about 0.1 M Na2HPO4, about 0.001 M to about 0.04 M KCl, about 0.2 M to about 3.0 M NaCl, and about 0.02 N to about 0.2 N NaOH. In other embodiments, the buffer may contain or not contain glucose in amounts ranging from about 0.2 wt percent to about 5 wt percent, or less than about 0.5 wt percent glucose.

[0545] In some embodiments of a kit containing a hydrochloric acid solution, the hydrochloric acid solution may contain hydrochloric acid ranging from about 0.005 N to about 0.1 N. In embodiments of a kit containing lyophilized collagen, hydrochloric acid solution, and buffer, the lyophilized collagen, hydrochloric acid solution, and buffer may be provided in separate containers. In embodiments of a kit containing a collagen composition and buffer, the collagen in the collagen composition may be at a concentration of about 0.1 mg / ml to about 40 mg / ml or about 0.1 mg / ml to about 10 mg / ml. In some embodiments, the collagen composition has a concentration between about 7 mg / mL and about 8 mg / mL. In some embodiments, the concentration of collagen in the mixture of collagen solution and buffer is about 6.3 to about 7.2 mg / mL. In some embodiments, upon neutralization, formulation yields polymerizable collagen having one or more of the following characteristics: final collagen concentration: 6.3–7.2 mg / mL; polymerization time: 0.5–1.1 mins; shear storage coefficient: 2.7–3.5 kPa; shear loss coefficient: 0.3–0.5 kPa; and / or compressibility coefficient: 5.8–9.6 kPa. In such embodiments, the collagen composition and buffer may be provided in separate containers, for example, in sterile vials or in separate compartments of a dual syringe containing a mixing element. In any embodiment of the kit described herein, the kit may further include instructions for using the components of the kit. In any embodiment of the kit described herein, the buffer may be capable of polymerizing collagen using a single mixing step comprising mixing the buffer with lyophilized collagen reconstituted in hydrochloric acid solution or with the collagen composition.

[0546] In some embodiments, the kit comprises collagen provided in a lyophilized form, a buffer as described herein, and a solution of an acid for reconstituting the lyophilized collagen, such as acetic acid, or another dilute acid including, for example, hydrochloric acid, formic acid, lactic acid, citric acid, sulfuric acid, or phosphoric acid.

[0547] The following examples illustrate specific embodiments in greater detail. These examples are provided for illustrative purposes only and should in no way be construed as limiting the present invention.

Example

[0548] [Example 1]

[0549] Methods and Materials Two collagen tissue filler formulations with a single trademark and different manufacturing processes were prepared and evaluated by GeniPhys; since no differences in performance were observed, the results were combined and presented as a single formulation. For both formulations, lyophilized type I oligomeric collagen was dissolved in 0.01 N hydrochloric acid to obtain a solution that was presumably 8 mg / ml (based on the dry weight of the lyophilized material). After solubilization, the solution was clarified using ultracentrifugation (142,400 × g). Then, Formulation 1 was filtered through a sterile 0.2 μm membrane filter (SterliTech, Kent, WA) and subjected to the quality control tests described below. Formulation 2 was irradiated with ultraviolet (UV) radiation of 500 mJ / cm 2 at a wavelength of 254 nm parallel beam, then filtered through the same type of membrane filter and subjected to quality control tests. A neutralizing solution with a trademark was prepared according to GeniPhys's standard procedure and filter sterilized through a 0.2 μm filter. To prepare a kit for use in surgery, syringes were aseptically filled with Formulation 1, Formulation 2, and the neutralizing solution. The volume ratio of the oligomeric collagen solution used to the neutralizing solution was 9:1.

[0550] The material properties of collagen formulations were defined, and quality control was performed based on the evaluation of molecular purity, self-assembly dynamics, and viscoelastic mechanical properties. Self-assembly dynamics and viscoelastic properties were measured for four independent collagen prototype batches (N=4) for 6 to 8 repetitions (n=6-8). To evaluate collagen purity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on collagen samples and molecular weight standards using 4-20% and 6% gels (Invitrogen) (Novex SeeBlue Plus2, Invitrogen, Carlsbad, CA), and stained with Coomassie Blue (Sigma-Aldrich, St. Louis, MO) according to established methods. Collagen concentration was determined using the Sirius Red (Direct Red80, Sigma-Aldrich) assay. Time-dependent vibratory shear fluid measurements were performed to determine the self-assembly dynamics and shear storage (G') and loss (G'') coefficients. Briefly, neutralized oligomeric collagen samples were tested on an AR2000 rheometer (TA instruments, New Castle, DE) using a 40 mm parallel plate outline and solvent trap. The Peltier plate was maintained at 4°C before loading the sample and for the first two minutes of the test. Vibratory shear measurements were performed at a 1% strain for these first two minutes and continued for 10 minutes after the temperature rose to 37°C. After the vibratory shear test, the sample was subjected to uniaxial compression at a strain rate of 20 μm / s. To define the dynamics of matrix formation, a plot of the shear storage coefficient over time was prepared, and the time at which collagen reached its maximum stiffness (G') was defined as the polymerization time. Using this point, the matrix G' and G'' values ​​were also defined. To obtain the compressibility factor, stress-strain curves were constructed from uniaxial compression data, and the slope was calculated in a specified low-strain region (20-40% strain) corresponding to low stress / strain coefficients reported in the literature for soft tissue (e.g., human breast). Four independent batches of prototype collagen were tested, with each batch repeated 6 to 8 times (N=4 batches; repeated 6-8 times per batch). [Example 2]

[0551] Pig simulation model for mammary gland tumor removal This study tests novel strategies to improve functional and cosmetic outcomes after breast surgery procedures, including mammary tumor resection and mastectomy, using the mammary glands of miniature pigs. Simulated mammary tumor resections were performed on female Yucatan miniature pigs weighing between 45 and 65 kg, using a protocol approved by the Purdue Animal Care and Use Committee. A total of eight pigs were used in two studies: six for the longitudinal study and two for the radiographic study. In both studies, the mammary glands were randomly assigned to the experimental and control groups, with unfilled and no-surgery groups serving as negative and positive controls, respectively. Postoperative evaluations for the long-term study were performed at 1, 4, and 16 weeks (two animals per time point), with 12 repeats for the collagen filler groups (n=12 for each collagen filler formulation; n=6 for each) and 6 repeats for the unfilled and no-surgery groups (n=6). For the radiation study, postoperative evaluations were performed 4 and 16 weeks after surgery. The collagen filler group underwent 6 repetitions (n=6), the non-filler group 3 repetitions (n=3), and the no-surgery group 1 repetition (n=1). The pair of mammary glands closest to the tail was used as the non-irradiated, no-surgery control. Outcomes from irradiated animals were compared with those from unirradiated animals in the long-term study. Postoperative evaluations at each time point included semi-quantitative scoring of macroscopic appearance and homogeneity / concentration of the mammary gland / surgical site, ultrasound, radiography, macroscopic evaluation of the explant, and histopathological analysis. Both semi-quantitative scoring and histopathological analysis were performed in a blinded manner.

[0552] The animals were anesthetized, intubated, and placed in a supine position. For each simulated mammary tumor excision, a 3 cm skin incision was made using a surgical scalpel, orienting the incision laterally and just outside the areola and nipple of each mammary gland. Approximately one-quarter of the mammary tissue was excised using an electrocautery device, and its volume was measured using a standard volume substitution method. Uniaxial compression tests were performed on a portion of the excised normal mammary tissue to characterize its mechanical properties (strain rate: 1 mm / min, compression coefficient determined in the linear region of 20-40% strain). Titanium marker clips (Ethicon Small LigaClips, West CMR, Clearwater, FL) were placed in a portion of the animal to facilitate the identification of margins in the surgically treated areas with collagen and without filler. In areas treated with collagen filler, the postoperative space was filled with neutralized liquid collagen. Negative control areas were not filled (untreated). A portion of the pig mammary gland that did not undergo surgery was used as a positive control. All surgical sites were closed with reabsorbable sutures and bandaged with non-adhesive pads (McKesson, San Francisco, CA) and Tegaderm (3M, St. Paul, MN) dressings. The animals' health was monitored daily based on appetite, behavior, movement, and elimination. [Example 3]

[0553] Postoperative radiation therapy after adjuvant breast tumor removal To address the question of how radiotherapy affects the tissue response to collagen tissue fillers, two animals that had undergone simulated mammary tumor excision and treatment were treated with radiation. Pig udders were again randomly assigned to the treatment group, with unfilled and unsurgery-free udders serving as negative and positive controls, respectively. Two weeks after surgery, a total dose of 20 Gy was delivered to five pairs of cranial mammary glands in 5 consecutive fractions using CT-based 3D-CRT technology with a 6MV Varian EX clinical linear accelerator (Varian, Palo Alto, California) equipped with a 120-leaf multi-leaf collimator. Pairs of mammary glands near the tail were excluded as non-irradiated controls. [Example 4]

[0554] Postoperative procedures and evaluation At the design stage, the animals were anesthetized, and each mammary gland was evaluated using a semi-quantitative scoring system for macroscopic mammary gland / surgical site appearance, including erythema / crusting and edema formation, as well as mammary gland uniformity / concentration scoring, as shown in Figure 10. Furthermore, ultrasound imaging of each mammary gland was performed using a Mindray M7 ultrasound instrument (Mindray North America, Mahwah, NJ) and a linear 4-7 MHz transducer. After euthanasia, mastectomy was performed on each mammary gland, preserving all surgical sites, any implants, and surrounding tissues. Each mammary gland was placed in 10% buffered formalin and radiographed using an Inno Vet Select Radiograph unit (Summit, Niles, IL) with a Genesis Vet DR plate installed using Genesis VxVue acquisition software (Genesis Digital Imaging, Los Angeles, CA), followed by processing for histopathological analysis. [Example 5]

[0555] histopathology Formalin-fixed explant tissues were bisected, imaged, paraffin-embedded, and then cut. The fragments were stained with hematoxylin and eosin (H&E). To detect epithelial cells, the fragments were stained with pancytokeratin (ab9377, Abcam, Cambridge, MA) at a 1:100 dilution, followed by treatment with 6 μg / mL of secondary DyLight 488 goat anti-rabbit (DI-1488, Vector Labs, Burlingame, CA). The nuclei were counterstained with DAPI (4',6-diamidino-2'-phenylindole, dihydrochloride; EN62248, Pierce Biotechnology, Rockford, IL). Images were obtained using an Aperio VERSA 8-hole slide scanner (Leica Biosystems, Buffalo Grove, IL). [Example 6]

[0556] Liquid collagen transforms into a stable fibril matrix that conforms to the external shape and possesses properties similar to soft tissue. We evaluated collagen tissue filler formulations specifically designed to act as regenerative and regenerative fillers for damaged or defective tissues created by BCS, such as tissue cavities. Collagen tissue filler formulations were defined based on their compositional and mechanical properties, including molecular purity, collagen content, polymerization (self-assembly) time, and viscoelastic properties under vibrational shear and uniaxial compression loading. To evaluate the biocompatibility and efficacy of the tissue fillers, a simulated mammary tumor excision procedure was performed on pig mammary glands (breasts) by fellowship-trained breast surgeons. Prototype formulations were then used to fill portions of mammary cavities, and surgical outcomes were compared to untreated defects (unfilled; negative control) representing standard treatment for BCS. Normal mammary glands that had not undergone breast surgery served as positive controls. Long-term studies were conducted at 1, 4, and 16 weeks to define tissue response schedules and gain insights into the mechanism of action of the tissue fillers (tissue implantation response). At one week, a small number of sites were used to determine whether the material impaired or interfered with the surgical re-excision procedure. A second study was then conducted to evaluate how the tissue filler and its associated tissue response were affected by irradiation, which is often used as a postoperative treatment to prevent local cancer recurrence. Outcome measurements included visual and palpation of the entire breast and surgical site, and semi-quantitative assessment of erythema, scabbing, edema, and breast homogeneity / concentration. Furthermore, the entire breast was imaged using ultrasound and radiography. Finally, after euthanasia, the breast exgrafts were collected and subjected to macroscopic and histological analysis. Combining these data provides support for improved healing outcomes after the use of tissue fillers during breast-conserving surgery.

[0557] Collagen tissue filler formulations were obtained as kits from GeniPhys (Zionsville, Indiana). As shown in Figure 1A, the kit consisted of a syringe containing sterile type I oligomeric collagen in dilute acid (0.01 N hydrochloric acid), a syringe containing sterile neutralizing solution (buffer), a sterile Luer lock adapter, and a sterile applicator tip. The oligomeric collagen components of these kits were prepared, and quality control was performed from closed-group pig skins according to ASTM F3089-14 guidelines for polymerizable collagen. Immediately before use, the two syringes were connected using the Luer lock adapter (Figure 1B), and the collagen and neutralizing reagent were mixed in a 9:1 ratio to bring the collagen solution to physiological pH and ionic strength. After mixing, the viscous liquid could be injected into various forms, if suitable for the shape, and then transferred to a physically stable fibrous collagen matrix (Figure 1B). To demonstrate collagen purity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 4–20% and 6% gels. The gels revealed band patterns characteristic of oligomeric collagen, and no contamination of non-collagen proteins or other types of collagen was detected (Figure 1C). Other functional performance parameters, including polymerization time of the neutralized collagen tissue filler solution and viscoelastic properties of the matrix formed by the tissue filler, were measured, and a summary is provided in Figure 1D. In particular, the concentration of oligomeric collagen before neutralization was approximately 7.7 mg / mL. During neutralization, the matrix formation reaction took just under 1 minute on average when measured rheometrically at 37°C. When analyzed under vibrational shear and uniaxial compression, the formed matrix exhibited solid-like behavior, with shear storage (G') and loss (G'') coefficients of 3.16 ± 0.16 kPa and 0.40 ± 0.02 kPa, respectively, and a compressibility coefficient of 7.67 ± 0.42 kPa.While oligomeric collagen can be used to produce a wide variety of polymer materials with tunable combinations of compositional and mechanical properties (i.e., elastic modulus and strength values), the specific tissue filler formulations developed and tested here were designed using a particular combination of material properties to exhibit viscoelastic mechanical properties similar to those of soft tissue. [Example 7]

[0558] The collagen tissue-filling matrix maintains volume, induces angiogenesis and breast tissue formation, and does not cause the inflammatory response typically observed during healing or the foreign body reaction typically observed in the tissue implantation response. To evaluate the effectiveness of tissue fillers in improving the appearance, structure, and function of soft tissue defects, a long-term study was conducted involving simulated mammary tumor excision procedures on normal udders of healthy Yucatan miniature pigs (Figure 2). Female miniature pigs represent a preferred large animal model for such translational studies based on their size and anatomical and physiological similarities to humans. Furthermore, since pigs generally have 12 mammary glands (udders), and each udder can serve as either an experimental or control group, the total number of animals required for the study is reduced. Prior to surgery, pig udders were randomly assigned to the treatment group, with unfilled and unsurgery-free udders serving as negative and positive controls, respectively. All surgical procedures and udder evaluations were performed by fellowship-trained breast surgeons. Approximately one-quarter of the udder tissue volume was excised (Figure 2E), ranging from 2 to 5.5 mL of tissue (average approximately 3 mL) depending on the individual udder size (Figure 2A). For breast tissue fillers, if the shape was complex, liquid collagen was mixed and immediately injected into the tissue cavity, then transferred to a fibrous collagen matrix within 5 minutes under these conditions (Figures 2B-D). Breast surgeons used their discretion when filling each defect, and the applied collagen volume varied depending on the defect size and shape. Postoperative cavities were filled with collagen of at least the same volume as the removed tissue, with most being given a collagen volume exceeding 1-2 mL. Negative control sites were left untreated (unfilled), which was consistent with the standard care BCS procedure. All incisions were closed with reabsorbable sutures and bandaged (Figure 2F). All animals maintained their body weight (±5 kg), the surgical sites remained closed, and no procedural complications occurred throughout the study period (Figure 2G).

[0559] Consistent with observations between males and females, the mammary glands of pigs were found to vary in volume, consistency, and composition both within and between individual animals. At a microscopic level (Figure 9), the mammary glands consisted of multiple lobes, each composed of a system of small secretory lobules and tubules (channels) organized as tufts, ultimately exiting into the skin via the nipple. The lobules and tubules were supported by intralobular stroma, primarily composed of fibrous type I collagen. Furthermore, collagenous connective tissue was found between the lobes (interlobular stroma), supporting the mammary gland and determining its shape. Adipose tissue initially determined the mammary gland size and filled the space between the glands and fibrous connective tissue. When evaluated under uniaxial compression, the mammary glands located cranially (towards the head) were relatively firm, with an average compressibility of 19.0 ± 12.9 kPa. Moving towards the tail (towards the tail), the mammary glands became softer with an increasing lipid composition, and the average compressibility was 6.56 ± 2.51 kPa for the mammary glands closest to the tail.

[0560] To evaluate the biocompatibility and tissue response of collagen fillers, animals were anesthetized at the designed time points of 1, 4, and 16 weeks. All mammary glands were visually examined, palpated, and semi-quantitatively scored in a blinded manner according to the criteria in Figure 10. Collagen-treated mammary glands and unfilled control mammary glands showed no evidence of erythema (redness) or crusting (crusting, dead tissue) at any of the time points. Mild edema was evident at 1 week in surgically treated mammary glands; however, the degree of swelling was similar for both the collagen and unfilled groups and subsided quickly thereafter. The homogeneity / concentration score for collagen-treated mammary glands was similar to that of the unfilled control at all three time points, decreasing from approximately 1.2 at 1 week to 0.25 at 16 weeks (Figure 3A). Such findings are important to demonstrate that collagen fillers do not result in mammary inconsistencies that may be clinically interpreted as the cause of sequelae or patient discomfort. All normal mammary glands were given a score of 0. Furthermore, when breast surgeons performed simulated surgical re-excisions on collagen-treated breasts, the filler material did not impair or interfere with the procedure.

[0561] The biocompatibility and tissue response of collagen fillers were further defined based on macroscopic and histological examinations of transverse fragments of breast exgrafts, compared with unfilled and normal breast controls. These analyses clearly demonstrated high biocompatibility, with collagen fillers maintaining their volume (minimal defect atrophy), and exhibiting a tissue response in the absence of inflammatory responses typically seen in the healing of untreated tissue cavities, or foreign body reactions typically observed in tissue implantation responses. When cells infiltrated the tissue filler matrix and new breast tissue was generated, it presented a tissue-like appearance that was difficult to macroscopically distinguish from the surrounding normal tissue (Figure 3B). In this case, surgical clips were useful as markers of the original defect margins (Figures 3B, 4). Upon histological analysis at one week, collagen fillers were evident within the tissue cavities, appearing as a homogeneous, bright pink (eosinophilic) staining material (Figure 4). Much of what surrounded the fillers consisted of hemorrhage, fibrin, and some leukocyte bands, which were attributable to the surgical manipulation of the tissue (Figure 4A). At the filler-host tissue boundary, there was a localized, extensive area of ​​proliferative fibroblasts (mesenchymal cells), and a few small-diameter vessels infiltrating the matrix margin were observed. The surrounding breast tissue appeared mostly normal, along with the remodeling area adjacent to the surgical site. These areas contained aggregates of remodeling epithelial cells, some of which appeared to be tubules, while others were more irregular in shape, suggesting underdeveloped lobules (Figure 4A). Note that there was no evidence of inflammation-mediated xenobiotic reaction or active biodegradation, which are characteristic of conventional implantable materials. At 4 weeks, fibroblasts had branched into deeper parts of the collagen-filled matrix along with newly formed vascular systems, and infiltrating cells were most abundant at the periphery, further decreasing towards the center (Figure 4A). Multifocal aggregates of epithelial cells were observed, which were also consistent with precursors of glandular structures (Figure 4A). By 16 weeks, the matrix was fully cellularized, matured, and emerged as remodeled collagen fibers and bundles, with some areas showing small, identifiable regions of acellular eosinophilic filler material.Small-diameter blood vessels were scattered and uniformly distributed throughout the matrix (Figure 4A). Within the vascularized collagen matrix, newly formed lobules and tubules, positively stained for cytokeratin, and adipose tissue were particularly present in the periphery (Figures 11A,B, 4A). The glandular morphology was well-developed and mature, and no significant pathological signs were observed.

[0562] In contrast, at week 1, hematoma formation was evident in both macroscopic and histological evaluations of the unfilled breast explant (Figures 3B, 4B). Hemorrhage, fibrin clots, and leukocytes, including neutrophils and macrophages, were evident within the mammary tumor excision site. Mixed within the hemorrhagic area were proliferative fibroblasts, with a few small-diameter vessels, consistent with fibrovascular scar tissue associated with repair and wound healing. Scattered areas of necrosis with active inflammation also clearly surrounded the defect. By week 4, these tissue defects had atrophied, as evidenced by macroscopically significant clip migration and a histologically stellate, atrophic appearance (Figure 4B). Vascular connective tissue scar tissue was prominent within the defect, with multiple small areas of necrosis and inflammation observed throughout and near the defect boundary (Figure 4B). Active remodeling of glandular and adipose tissue was observed in the tissue areas surrounding the defect (Figure 4B). By 16 weeks, the fibrous scar tissue had increased in density and appeared as a vortex of parallel-aligned fibrous tissue densely packed with myofibroblasts, exhibiting a specific orientation. Lobules, tubules, and adipose tissue were identified around the defect, but multiple necrotic glands with nearly undeveloped morphological features were found within the periphery of the scar tissue, as evidenced by the presence of inflammatory mediators and residual low levels of diffused cytokeratin staining (Figures 4B, 11C, 11D). [Example 8]

[0563] The tissue-filled matrix does not impair the interpretation of sonograms and X-ray images. Mammography and ultrasound are commonly used as follow-up diagnostic procedures for BCS to monitor cancer recurrence. To ensure that the collagen filler does not impair or interfere with image interpretation, ultrasound was performed on all pig udders before euthanasia, and individual whole-breast X-rays were taken after mastectomy. Sonograms obtained over a 16-week study showed that the tissue filler matrix did not obscure or obstruct the matching of breast tissue and did not generate any unexpected echogenic areas (Figure 5A). At week 1, large, irregularly shaped hypoechoic areas with varying degrees of heterogeneous echoes were observed within the collagen-treated udders (Figure 5A). Such signals were not surprising considering that the filler microstructure represents a meshwork of randomly oriented collagen fibrils, measured to a diameter of approximately 400 μm. These areas appeared to maintain their volume over time, but gradually took on the appearance of normal tissue, confirming macroscopic cytosis and angiogenesis observed within the explant and histologically (Figure 5A). Unfilled spaces also showed irregularly shaped hypoechoic areas consistent with seroma and hemorrhage at week 1 (Figure 5A). By weeks 4 and 16, these areas decreased in size and produced heterogeneous signals consistent with atrophy and scar formation (Figure 5A).

[0564] The tissue-filling matrix did not interfere with the interpretation of radiographs, but rather showed opacities consistent with normal tissue throughout the study period (Figure 5B). Furthermore, radiographs provided further evidence that the collagen matrix maintained void volume with limited clip movement over time (Figure 5B). Most of the untreated (unfilled) postoperative voids also produced radiographs that appeared consistent with normal tissue at week 1, with a few sites showing distinct dark areas consistent with voids, seromas, or hematomas (Figure 5B). The gradual movement of surgical clips observed at weeks 4 and 16 provided further evidence of defect atrophy and scarring over time (Figure 5B). [Example 9]

[0565] Irradiation does not adversely affect collagen fillers or tissue implantation response. To determine whether collagen fillers were suitable for radiotherapy, a cohort of animals underwent abdominal irradiation two weeks after a simulated mammary tumor resection procedure. A computed tomography (CT)-based three-dimensional conformal treatment (3D-CRT) plan was created for each animal, and a total dose of 20 Gy was delivered in five consecutive fractions to the five cranial pairs of mammary glands using 6 MV X-rays from a Varian EX clinical linear accelerator. The pairs near the tail of the mammary glands were excluded as non-irradiated controls. Irradiated animals showed increased skin pigmentation over time, as evidenced by darkening of skin color (Figure 2G), which would be similarly expected in humans receiving therapeutic irradiation. At a microscopic level, moderate epidermal hyperplasia or thickening was evident, with increased melanin deposition, particularly in the basal epidermis (Figure 9). At 16 weeks, the mammary tissue was significantly hardened, which was also a common change observed in radiotherapy. Furthermore, clear signs of lipid necrosis and atypical hyperplasia of the tubules and glands were observed (Figure 9).

[0566] Aside from differences in skin pigmentation, all mammary glands and surgical sites healed well and appeared similar to those of unirradiated animals. Mean mammary uniformity / concentration scores for the collagen-filled and unfilled groups were slightly higher in irradiated animals compared to unirradiated animals at each time point, with the only exception being the collagen-filled group at 16 weeks, where the scores were similar (Figures 6A, 3A). Macroscopic explant and histological cross-sectional examinations revealed no apparent adverse effects of irradiation or associated tissue responses on the tissue filler matrix; subjectively, the overall healing schedule of the irradiated sites appeared somewhat delayed (Figures 6B, 6C). Throughout the 16-week study period, the collagen filler persisted within the surgical site, supporting gradual cellularization, angiogenesis, and mammary tissue formation progressing inward from the filler-host tissue boundary. As expected, the unfilled group showed atrophy and development of fibrous scar tissue (Figures 6B, 6D). The sonograms (Figure 7A) and X-ray images (Figure 7B) were nearly identical for both irradiated and unirradiated animals, confirming that the collagen filler was not adversely affected by irradiation and that no suspicious imaging abnormalities were generated. [Example 10]

[0567] Analysis of the results In this study, porcine mammary glands differed in size and tissue composition, resulting in differences in consistency, which were evident both qualitatively and quantitatively. The measured range of compressibility coefficients (approximately 6–19 kPa) was encompassed by mammary consistency observed in females, which was reported to range from 0.7–66 kPa depending on mammary composition (e.g., fibrous gland vs. lipid) and test parameters (e.g., strain rate, preconditioning). The healing response of untreated mammary defects was similar to that observed in females after BCS, yielding scar tissue structurally and functionally distinct from normal mammary tissue. A 16-week long-term study showed progression through the classic overlapping stages of repair wound healing, which led to scarring including congestion and inflammation, proliferation, and remodeling, as shown in Figure 8A. Substantial atrophy of the defects was facilitated by the initial fibrin clot and primary matrix, as evidenced by clip migration and stellate scar tissue morphology, which were mechanically weaker compared to normal mammary ECM. The process of scar formation and remodeling over time is perhaps the most unpredictable and problematic aspect of BCS, and all of these have a negative emotional and psychological impact on women, as they are known to contribute to pain, distortion of breast contour and firmness, and loss of sensation.

[0568] Filling defect volumes with a persistent fibrous collagen matrix that is naturally metabolized and remodeled rather than actively degraded resulted in a healing response with virtually no immune mediators, and the outcome was more regenerative than restorative (Figure 8B). Based on these results, the proposed regenerative healing response with respect to the collagen filler is illustrated in Figure 8B. The injectable in situ-forming matrix fills, conforms to the defect, and integrates effectively with the surrounding host tissue, thus re-establishing structural and mechanical continuity across the tissue, which is known to be important for scar-free healing and tissue morphogenesis. In particular, the compressibility coefficient of the collagen filler (7.67 ± 0.42 kPa) fell within the range of the mammary mechanical properties of both pigs and humans. The high-density microstructure and compressibility of the collagen filler effectively resisted the atrophic forces provided by the surrounding normal tissue and infiltrating cells. Furthermore, because the matrix mechanical properties were similar to those of soft tissue, none of the palpable mammary inconsistencies occurred. From a translational perspective, it is important to maintain patient satisfaction and comfort, as well as the ability to detect recurrent cancer through palpation.

[0569] Because the collagen fibrils formed by the tissue filler contain multiple functional cells and molecular binding domains, the matrix may be effectively involved in both biochemical and mechanochemical signaling, as performed by the tissue ECM. Unlike conventional implantable materials, the matrix was initially present with fibroblast-like mesenchymal cells along with angiogenic cells rather than inflammatory mediators. The rapid and robust neo-angiogenic response was consistent with other in vivo studies used for in vitro investigations of the fundamental mechanisms of angiogenesis, where oligomers are embedded in other microenvironments. Histogenesis continued as these apex cells advanced deeper toward the matrix center over time, forming adipose tissue, as well as mammary glands including secretory lobules and ducts. Interestingly, the newly formed lobules, particularly evident at 4 and 16 weeks, showed almost no macrophage infiltration, reminiscent of those found in nulliparous (pre-pregnancy) breasts. In summary, the regenerative tissue response observed with collagen filling shows many similarities to processes associated with tissue development and morphogenesis, including mammary gland tissue, highlighting the importance of maintaining interstitial collagen and its associated mechanobiological continuity.

[0570] As part of this study, it was also demonstrated that collagen fillers were not adversely affected by radiation therapy and did not impair the interpretation of diagnostic imaging procedures. In this study, irradiation was applied two weeks after simulated mammary tumor excision, which was within the range of adjuvant radiation administration after BCS. Tumors and tissues with rapid cellular turnover, such as the epidermis of the skin, are most sensitive to the effects of radiation, and the degree of damage depends on the total radiation dose and the time the radiation is delivered. Irradiation resulted in hyperpigmentation of the skin, as well as moderate levels of lipid necrosis and hyperplasia of glands and tubules, which are expected side effects similar to the response to photodermatitis or sunburn observed in humans. For both collagen-filled and non-filled treatment groups, healing progressed similarly to the individual non-irradiated groups; however, the healing rate appeared somewhat slower based on breast density scores and histopathological analysis. Such results were not surprising, as it is known that irradiation delays wound healing. Based on a combination of histopathological radiographs and ultrasound analysis, collagen fillers and their associated signaling capacity were determined to be largely unaffected by irradiation. X-ray and ultrasound examinations also showed no suspicious artifacts caused by the collagen filler. Extensive changes in breast tissue detected through these imaging techniques, ranging from seemingly benign lipid cysts to findings suggestive of malignancy, such as microcalcifications, lesions, and areas of suspected high opacity, were the main drawbacks of lipid grafting.

[0571] Given that this study represents an early principle illumination assessment, these studies are not without limitations. Initially, because breast size differs between pigs and humans, quadrantectomy was performed, removing approximately 25% of the volume of the pig breast. The defect volume ranged from 2 to 5.5 mL, with an average defect volume of approximately 3 mL. While quadrantectomy is rare, even in women, these complete defect volumes fell within the range of human clinical procedures. In particular, published human clinical reports indicate that 67% and 82% of breast tumors have a diameter (volume) of ≤1.9 cm (≤3.6 mL) and ≤2.9 cm (≤12.8 mL), respectively. Further research is needed to determine how defect size affects material performance, but adverse outcomes are not predicted based on the observed material mode of action and tissue implantation response. However, it is predicted that the time to complete cytogenesis and healing will be directly proportional to the defect volume. Secondly, since the longest time point evaluated was 16 weeks, additional animal and human clinical studies are needed to define long-term (i.e., more than 6 months) outcomes of collagen fillers. A third limitation of these large animal studies was that the pigs were not cancerous. Because the pigs used in this large animal study were not cancerous, the effects of collagen fillers on tumor promotion and recurrence could not be fully evaluated. For a number of reasons, it is not predicted that collagen fillers will pose a risk to oncological safety. Firstly, breast surgeons may gain more confidence in excising more tissue to achieve negative margins, as they will be able to maintain breast contour and density more predictably. Furthermore, we have shown that collagen fillers do not induce inflammatory or xenobiotic responses, which is particularly important because inflammation-related macrophage infiltration and other processes (e.g., cytokine release) are involved in tumor promotion. In addition, when tested in vitro with various cancer cell types, the high fibril density / rigidity of the oligomeric collagen matrix was found to limit the proliferation and migration of tumor cells.Finally, to further prevent tumor recurrence, chemotherapy or other anticancer agents could be easily added to the matrix-forming reaction to achieve targeted and local delivery. This dramatically reduces the amount of drug administered and minimizes the side effects associated with systemic administration.

[0572] Finally, a regenerative and regenerative tissue filler formed in situ and made from collagen polymers is described, which appears to address the demands of surgeons and overcome the major limitations associated with conventional implantable materials. This is the first report of a breast filler that is persistent, maintains its volume, and induces progressive breast tissue regeneration, including mammary glands, ducts, and adipose tissue. Furthermore, the findings have significant implications for regenerative medicine, suggesting that reducing inflammation and maintaining the structural and mechanical continuity of collagen shifts the healing balance from repair (scar formation) to regeneration. This study sets the stage for future preclinical and clinical research in which the translational potential of this tissue filler will be further validated in relation to BCS and other tissue recovery and reconstruction needs. [Example 11]

[0573] Skeletal muscle recovery and regeneration Yucatan miniature pigs were placed under general anesthesia. A defect (approximately 2 cm x 2 cm) was created within the skeletal muscle and adipose tissue area on the dorsal side of the neck, as shown in Figure 12. The tissue cavity was filled with a liquid collagen filler that conformed to the shape of the cavity. Within approximately 1 minute after application, the liquid collagen self-assembled (polymerized) in situ, forming a fibrous collagen matrix that restored the tissue's continuity and morphology. The site was then sutured closed. Eleven weeks after the creation of the tissue defect, the defect site and surrounding normal tissue were harvested and placed in 10% buffered formalin. The formalin-fixed explant tissue was bisected, embedded in paraffin, and thinned. The fragments were stained with hematoxylin and eosin (H&E) and Masson's trichrome. Figure 13 shows the newly formed skeletal muscle and adipose (lipid) tissue within the collagen matrix 11 weeks after implantation. In Figure 13C, the collagen matrix is ​​shown, F represents lipids, M represents skeletal muscle, and the arrows indicate the associated microvascular system. [Example 12]

[0574] Tissue filler kits and related performance characteristics The tissue filler comprises oligomeric collagen derived from porcine dermis and a neutralizing buffer. In some embodiments, the in-situ forming collagen device may be supplied as a disposable kit, as shown in Figure 14, comprising: a sterile glass vial containing collagen solution (10 mL) in dilute (0.01 N) hydrochloric acid; a sterile glass vial containing neutralizing buffer (self-assembling reagent; 2 mL); two sterile 10 mL syringes; two sterile needleless vial adapters; a sterile Luer lock connector; and a sterile applicator tip. In other embodiments, a pre-filled dual-barrel syringe with a static mixing tip may be provided. This dual-barrel product format may be used to support mixing of the collagen solution and neutralizing buffer during administration.

[0575] Table 1 provides an overview of the neutralizing buffer components and their roles in bringing the collagen solution to physiological pH, ionic strength, and osmolality to induce the matrix formation reaction.

[0576] Table 1. Overview of the components of the neutralizing buffer (self-assembly reagent). [Table 1]

[0577] As shown in Table 1, the neutralizing buffer represents 10-fold phosphate-buffered saline, which, when mixed with the collagen solution, brings it to physiological conditions including pH, osmolality, and ionic strength. Using a needleless vial adapter, 9 mL of collagen solution is prepared in one syringe and 1 mL of neutralizing buffer in the other. The user then connects the two syringes using a Luer lock connector and thoroughly mixes the two reagents. After mixing, the neutralized collagen solution may be injected to fill and fill tissue voids and defects, including those that are difficult to access and / or irregularly shaped. Upon application, the collagen undergoes an in-situ matrix formation reaction via molecular self-assembly. The tissue filler device achieves its intended use by providing a solid fibrous collagen matrix suitable for cellularization and angiogenesis (Figure 15A) and maintaining a supportive environment for tissue regeneration. In some embodiments, the neutralizing buffer component is glucose, as shown in Table 1. In other embodiments, the neutralizing buffer component does not contain glucose.

[0578] Table 2 summarizes the technical and performance characteristics of the neutralizing buffer (self-assembling reagent) components according to this instruction.

[0579] Table 2. Technical and performance characteristics of tissue fillers [Table 2]

[0580] The above-mentioned detailed description and accompanying drawings are provided for illustrative and illustrative purposes only and are not intended to limit the scope of the appended claims. Many of the variations in the preferred embodiments shown herein will be obvious to those skilled in the art and will remain within the scope of the appended claims and their equivalents.

[0581] It should be understood that the elements and features listed in the attached claims may be combined in various ways to generate novel claims that also fall within the scope of the present invention. Therefore, while the dependent claims attached below depend only on a single independent claim or dependent claim, these dependent claims may instead depend on substitutes from any prior claim, whether independent or dependent, and such novel combinations should be understood to form part of this specification.

Claims

1. A composition containing a solution of polymerizable oligomeric collagen for filling tissue voids or restoring tissue volume in a patient, The composition The oligomer collagen solution is used by a process that includes introducing a sufficient amount of the solution into the tissue voids to fill the tissue voids, The process includes polymerizing the polymerizable oligomeric collagen in situ to form a shape-retaining matrix that maintains the introduced volume over time. The composition.

2. The composition according to claim 1, wherein the shape-retaining matrix supports the recovery of functional tissue over time.

3. The composition according to claim 1, wherein the patient is a mammal.

4. The composition according to claim 1, wherein the patient is a human being.

5. The composition according to claim 1, wherein the oligomeric collagen contains type I collagen.

6. The composition according to claim 1, wherein the type I collagen is derived from porcine dermis.

7. The composition according to claim 1, wherein the polymerizable oligomeric collagen solution comprises a solution containing type I oligomeric collagen and an acid.

8. The composition according to claim 7, wherein the acid is hydrochloric acid.

9. The composition according to claim 1, wherein the polymerizable oligomeric collagen solution comprises a solution containing lyophilized type I oligomeric collagen and 0.01 N hydrochloric acid, the concentration of the solution being about 8 mg / mL based on the dry weight of the lyophilized type I oligomeric collagen.

10. The composition according to claim 1, wherein the solution is introduced into the tissue cavities via a syringe.

11. The composition according to claim 2, wherein tissue voids are present in the breast tissue, and a shape-retaining matrix supports the formation of breast tissue comprising adipose tissue, glandular tissue, or a combination thereof.

12. An oligomeric collagen solution for filling tissue cavities in breast tissue in a patient, wherein the tissue cavities are created by the breast tumor removal or mastectomy procedure. The oligomeric collagen solution is The process involves introducing a mixture containing oligomeric collagen solution and buffer into tissue cavities in an amount sufficient to fill the cavities. The process includes polymerizing the oligomeric collagen in situ to form a shape-retaining collagen-fibrillary matrix that maintains the introduced volume over time. The oligomeric collagen solution.

13. The oligomeric collagen solution according to claim 12, wherein the shape-retaining collagen-fibrillary matrix supports the recovery of functional tissue over time.

14. The oligomeric collagen solution according to claim 12, wherein the oligomeric collagen solution comprises type I oligomeric collagen and an acid.

15. The oligomeric collagen solution according to claim 12, wherein the ratio of oligomeric collagen solution to buffer solution is approximately 9:

1.

16. The oligomer collagen solution according to claim 14, wherein the acid contains 0.01 N hydrochloric acid, and the concentration of the oligomer collagen solution is about 8 mg / mL based on the dry weight of freeze-dried type I oligomer collagen.

17. An oligomeric collagen solution for filling tissue cavities in breast tissue in a patient, wherein the tissue cavities are created by the breast tumor removal or mastectomy procedure. The oligomeric collagen solution is The process involves introducing a mixture containing oligomeric collagen solution and buffer into tissue cavities in an amount sufficient to fill the cavities. The process includes polymerizing the oligomeric collagen in situ to form a shape-retaining collagen-fibrillary matrix that restores or supports the shape, consistency, and function of the tissue over time. The oligomeric collagen solution contains type I oligomeric collagen and 0.01 N hydrochloric acid; The concentration of the oligomeric collagen solution is approximately 8 mg / mL based on the dry weight of type I oligomeric collagen; The ratio of oligomeric collagen solution to buffer solution is approximately 9:

1. The oligomeric collagen solution.

18. The oligomeric collagen solution according to claim 17, wherein the tissue voids include surgical wounds.

19. The oligomeric collagen solution according to claim 17, wherein the tissue voids are due to the removal of the tumor.

20. The oligomeric collagen solution according to claim 17, wherein the tissue voids are due to the removal of a breast tumor.

21. The oligomeric collagen solution according to claim 17, wherein filling the voids in the tissue results in a compressibility coefficient or range of compressibility substantially the same as that of natural tissue.

22. The oligomeric collagen solution according to claim 17, wherein filling the voids in the tissue results in the formation of breast tissue, which includes adipose tissue, mammary gland tissue, or a combination thereof.

23. A kit for supporting organizations that exist in organizational gaps, This kit is, In-situ polymerizable collagen solution and buffer solution; A device for mixing the oligomer collagen solution and the buffer solution; An applicator, which is attached to the device and configured to deliver the mixture of the oligomeric collagen solution and the buffer solution into the voids of the tissue, including, The kit.

24. The kit according to claim 23, wherein the in-situ polymerizable oligomeric collagen solution contains type I oligomeric collagen.

25. The kit according to claim 23, wherein the in-situ polymerizable oligomeric collagen solution contains type I oligomeric collagen derived from porcine dermis.

26. The kit according to claim 23 or 24, wherein the in-situ polymerizable oligomeric collagen solution comprises oligomeric collagen and an acid.

27. The kit according to claim 23, wherein the in-situ polymerizable oligomeric collagen solution comprises type I oligomeric collagen and hydrochloric acid.

28. The kit according to claim 23, wherein the ratio of in-situ polymerizable collagen solution to buffer solution is approximately 9:

1.

29. The kit according to claim 23, wherein the in-situ polymerizable collagen solution comprises type I oligomeric collagen and 0.01 N hydrochloric acid, and the concentration of type I oligomeric collagen is about 8 mg / mL based on the dry weight of type I oligomeric collagen.

30. The buffer solution is approximately 0.03 mM to approximately 0.2 mM MgCl ２ The kit according to claim 23, including the following:

31. The buffer solution is approximately 0.002 mM to approximately 0.02 mM MgCl ２ The kit according to claim 23, including the following:

32. The buffer solution contains less than approximately 0.02 mM MgCl ２ The kit according to claim 23, including the following:

33. Buffer is MgCl ２ The kit according to claim 23, which does not include the above.

34. The buffer solution contains approximately 0.003 M to 0.03 M KH ２ PO ４ The kit according to claim 33, further comprising:

35. The buffer solution contains approximately 0.01 M to 0.1 M Na ２ HPO ４ The kit according to claim 34, further comprising:

36. The kit according to claim 35, wherein the buffer further comprises about 0.001 M to about 0.04 M of KCl.

37. The kit according to claim 36, wherein the buffer further comprises about 0.2 M to about 3.0 M of NaCl.

38. The kit according to claim 36, wherein the buffer further comprises NaOH at a concentration of approximately 0.02 N to approximately 0.2 N.

39. The kit according to claim 37, wherein the buffer further comprises about 0.2 weight percent to about 5 weight percent glucose.

40. The kit according to claim 38, wherein the buffer solution contains glucose at a concentration of approximately 0.5 weight percent or less.

41. The kit according to claim 38, wherein the buffer solution does not contain glucose.

42. The kit according to claim 23, wherein the concentration of collagen in the in-situ polymerizable collagen solution is approximately 0.1 mg / ml to approximately 40 mg / ml.

43. The kit according to claim 23, wherein the concentration of collagen in the in-situ polymerizable collagen solution is approximately 7 mg / mL to approximately 8 mg / mL.

44. The kit according to claim 23, wherein the collagen solution contains approximately 0.005 N hydrochloric acid to approximately 0.1 N hydrochloric acid.

45. The kit according to claim 23, wherein the in-situ polymerizable collagen composition and buffer are present in separate containers.

46. The kit according to claim 45, wherein each container includes a separate compartment for a dual-barrel syringe.

47. The kit according to claim 46, wherein the dual-barrel syringe includes a mixing element.

48. The kit according to claim 23, further comprising at least one therapeutic agent configured for local delivery into tissue cavities.

49. The kit according to claim 48, wherein at least one therapeutic agent comprises a chemotherapeutic agent, an anti-inflammatory agent, an antibiotic, an analgesic, or a combination thereof.

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