阅读说明：本技术 用于医疗用途的动物组织的保存和储存 (Preservation and storage of animal tissue for medical use ) 是由 塔拉·施瓦山卡·库普姆巴提 于 2020-01-15 设计创作，主要内容包括：本发明提供了一种用于处理组织以形成干燥组织部分的方法,所述干燥组织部分易于再水合并且在植入人体之前不需要冲洗。所述方法包括预处理和固定步骤,所述预处理和固定步骤包括具有柔性主链和至少一个极性基团的渗透剂分子；所述步骤还包括阳离子和阴离子以增强渗透剂分子整合到组织部分结构分子中并与其键合,以提供在弯曲期间所述干燥组织部分的抗破裂性。(The present invention provides a method for treating tissue to form a dry tissue portion that is easily rehydrated and does not require rinsing prior to implantation in the human body. The method comprises a pre-treatment and immobilization step comprising penetrant molecules having a flexible backbone and at least one polar group; the steps further include cations and anions to enhance the integration and bonding of penetrant molecules into the tissue portion structural molecules to provide resistance to cracking of the dry tissue portion during bending.)

1. A method of preparing a tissue portion, the method comprising:

A. pretreating the tissue portion with a pretreatment solution comprising osmolyte molecules having a flexible hydrocarbon portion capable of increasing the flexibility of the tissue portion, the osmolyte molecules further having a polar portion, the pretreatment solution further containing a counter ion, the counter ion consisting of an anion and a cation, the osmolyte molecules and the counter ion being capable of being physically integrated into a molecular structure containing structural molecules of the tissue portion, the counter ion providing an attractive force to the polar portion of the osmolyte molecules and to the structural molecules to maintain the osmolyte molecules in contact with the molecules of the structural molecules,

B. removing the tissue portion from the pre-treatment solution and placing the tissue portion in a fixation solution consisting of the penetrant molecules, the counterions and cross-linker molecules, the cross-linker molecules being capable of forming cross-links with the structural molecules of the tissue portion and with the penetrant molecules already capable of integrating into the molecular structure of the tissue portion, the molecular contact increasing the formation of the cross-links, the cross-links providing fixation of the tissue portion,

C. drying the tissue portion by removing the tissue portion from the fixation solution and placing the tissue portion in a drying solution comprising a drying agent capable of absorbing water molecules present in the molecular structure of the tissue portion,

D. removing the tissue portion from the drying solution and removing water molecules from the tissue portion, thereby forming a dried tissue portion,

E. whereby the flexible hydrocarbon portion of the osmotic agent molecule provides flexibility to the dry tissue portion against rupture due to bending of the tissue portion.

2. The method of claim 1, wherein:

A. the dry solution further comprises the penetrant molecules and the counter ions,

B. the counter ions present in the drying solution prevent the counter ions that have been able to integrate into the molecular structure of the tissue portion from diffusing out of the tissue portion, and the osmotic agent molecules present in the drying solution prevent the osmotic agent molecules that have been able to integrate into the molecular structure of the tissue portion from diffusing out of the tissue portion.

3. The method of claim 1, wherein the osmotic agent molecules within the fixation solution prevent diffusion of the osmotic agent molecules that have been able to integrate into the molecular structure of the tissue portion out of the tissue portion.

4. The method of claim 1, wherein the counter ions within the fixation solution prevent diffusion of the counter ions that have been capable of being integrated into the molecular structure of the tissue portion out of the tissue portion.

5. The method of claim 1, wherein the tissue portion is placed directly into the fixation solution from the pretreatment solution without rinsing the tissue portion to retain the osmotic agent molecules within the tissue portion that have been able to integrate into the molecular structure of the tissue portion.

6. The method of claim 1, wherein the osmotic agent is selected from the group consisting of polyethylene glycol, glycerol, fatty acids, vitamins, sugars, and polysaccharides.

7. The method of claim 1, wherein the fixative is selected from the group consisting of glutaraldehyde, formaldehyde, and other aldehydes.

8. The method of claim 1, wherein the drying agent is selected from the group consisting of isopropanol, ethanol, propanol, other polyols, and acetone.

9. The method of claim 1, wherein the counter ion is selected from the group consisting of sodium, calcium, ferrous, iron, potassium, chloride, hydroxide, sulfate, and phosphate.

10. The method of claim 1, wherein the concentration of the osmotic agent within the pretreatment solution is at least one hundred millimolar.

11. The method of claim 1, wherein the concentration of the osmotic agent in the fixing solution is at least one hundred millimolar.

12. The method of claim 2, wherein the concentration of the osmotic agent in the dry solution is at least one hundred millimolar.

13. The method of claim 1, wherein the concentration of the counter ion within the pretreatment solution is at least three hundred millimolar.

14. The method of claim 1, wherein the concentration of the counter ion within the fixation solution is at least three hundred millimolar.

15. The method of claim 2, wherein the concentration of the counter ion in the dry solution is at least three hundred millimolar.

16. The method of claim 1, wherein the concentration of the cross-linking agent in the fixation solution is at least one-half percent.

17. The method of claim 1, wherein the pre-treatment of the tissue portion is performed for five minutes to twenty-four hours and the fixation of the tissue portion is performed for twelve hours to thirty days.

18. A method of preparing a tissue portion, the method comprising:

A. pretreating the tissue portion with a pretreatment solution comprising osmolyte molecules having a flexible hydrocarbon portion capable of increasing the flexibility of the tissue portion, the osmolyte molecules further having a polar portion, the pretreatment solution further containing a counter ion, the counter ion consisting of an anion and a cation, the osmolyte molecules and the counter ion being capable of being physically integrated into a molecular structure containing structural molecules of the tissue portion, the counter ion providing an attractive force to the polar portion and the structural molecules of the osmolyte molecules to maintain the osmolyte molecules in contact with the molecules of the structural molecules,

B. removing the tissue portion from the pre-treatment solution and placing the tissue portion in a fixation solution consisting of the penetrant molecules, the counter-ions and cross-linker molecules capable of forming cross-links with the structural molecules of the tissue portion and with the penetrant molecules already capable of integrating into the molecular structure of the tissue portion to provide fixation of the tissue portion, the molecular contact increasing the formation of the cross-links.

19. The method of claim 18, further comprising the steps of:

A. drying the tissue portion by removing the tissue portion from the fixation solution and placing the tissue portion in a drying solution comprising a drying agent capable of absorbing water molecules present in the molecular structure of the tissue portion,

B. removing the tissue portion from the drying solution and removing water molecules from the tissue portion, thereby forming a dried tissue portion,

C. whereby the flexible hydrocarbon portion of the osmotic agent molecule provides flexibility to the desiccated tissue portion against rupture during flexing of the desiccated tissue portion.

20. A method of preparing a natural tissue portion, the method comprising:

A. pretreating the tissue portion with a pretreatment solution comprising pretreatment solution penetrant molecules having a pretreatment solution flexible hydrocarbon portion and a pretreatment solution polar portion, the pretreatment solution comprising positive and negative pretreatment solution counterions, the pretreatment solution counterions and the pretreatment solution penetrant molecules capable of being physically integrated into a molecular structure comprising structural molecules of the tissue portion,

B. the pretreatment solution counter ions provide an attractive force between the pretreatment solution polar moiety and the structural molecules, thereby providing molecular contact between the pretreatment solution permeating molecules and the structural molecules,

C. removing the tissue portion from the pretreatment solution and placing the tissue portion in a fixation solution while retaining within the molecular structure the pretreatment solution penetrant molecules that have been able to integrate into the molecular structure of the tissue portion, the fixation solution being composed of cross-linker molecules, fixation solution penetrant molecules, and fixation solution counter ions, the fixation solution penetrant molecules having a fixation solution flexible hydrocarbon portion and a fixation solution polar portion,

D. providing fixation of the tissue portion by allowing the cross-linker molecules to form cross-links with the pre-treatment solution penetrant molecules already capable of integrating into the molecular structure of the tissue portion and with the structural molecules of the tissue portion, the cross-links increasing due to the molecular contact,

E. drying the tissue portion by removing the tissue portion from the fixation solution and placing the tissue portion in a drying solution comprising a drying agent capable of absorbing water molecules present in the molecular structure of the tissue portion,

F. removing the tissue portion from the drying solution and removing water molecules from the tissue portion, thereby forming a dried tissue portion,

G. whereby the pre-treatment solution flexible portion of the pre-treatment solution infiltrant molecules provides the dry tissue portion with increased flexibility to resist rupture due to bending of the dry tissue portion.

Background art:

tissues obtained from various animal sources, including human tissues, have been used as tissue parts in a variety of medical devices for implantation into the human body. Such tissue portions may be obtained from pericardial tissue, heart valve tissue, vascular tissue, or other tissue of animal origin. Alternatively, the tissue portion may be an engineered tissue formed by cells grown in an incubator and form a tissue portion composed of structural molecules including collagen, elastin, lipids and other molecules present in the molecular matrix of the tissue portion. The tissue portion may be used, for example, as a heart valve leaflet, a vascular graft, a tissue patch, or other application for applying replacement tissue such as a tissue portion in vivo.

Typically, such tissue portions are cross-linked in glutaraldehyde, for example, to reduce the immune response that the body may have to the tissue portion, which may lead to rejection of the tissue portion and potential cellular and enzymatic degradation of the tissue portion in vivo. After glutaraldehyde crosslinking, the tissue portion is typically packaged as a wet tissue portion in an aqueous preservative. The tissue portion is transported to a hospital as a wet tissue portion and awaits implantation into a patient. Physicians and operating room staff are then required to flush the aqueous antiseptic solution from the wet tissue portions with saline for an extended period of time to ensure that all of the antiseptic solution has been removed from the tissue portions prior to implantation.

A number of development activities have been applied to the overall processing of tissue portions in an effort to remove water from the cross-linked tissue portions so that the tissue portions can be packaged and shipped as dry tissue portions, thereby eliminating the lost time associated with rinsing the preservative solution from the tissue portions. The dried tissue portion may then be rehydrated in the operating room by the physician and implanted directly into the patient. Due to the fragile nature of the dried tissue portions using current treatment methods, such dried tissue portions have suffered increased disruption and tissue damage; in addition, rehydration of such dried tissue portions in the operating room is also very slow. There is a need for a treatment method for tissue portions that allows the tissue portions to be packaged and shipped as dry tissue portions, maintains their flexibility to avoid breakage, and allows a physician to easily rehydrate the tissue portions for direct implantation without the need to rinse out the preservative solution from the tissue portions.

The invention content is as follows:

the present invention is a tissue processing system and method for processing tissue portions such that the tissue portions may be packaged and transported to a hospital as dry tissue portions that are flexible and do not form weakable structures and ruptures that cause damage to the tissue portions. The processing method comprises three steps: pretreating, fixing and drying. Each step contributes to the formation of a flexible dry tissue portion.

The tissue portion may be formed of xenogenic or allogeneic pericardial tissue, heart valve tissue, vascular catheter tissue, or other tissue taken from a living animal, including porcine tissue, bovine tissue, equine tissue, human cadaver tissue, or other tissue that has been used for implantation within the body. Moreover, the tissue portion may be engineered tissue grown in an incubator by cells, whereby such cells produce collagen, elastin, lipids, and other molecules found in animal and human tissues. The tissue portion may be used to form, for example, leaflets for heart valves, vascular grafts, patches for covering openings in the body, and for other purposes in which a tissue portion may be desired in the body.

The pretreatment step of the method of the present invention comprises the steps of forming a pretreatment aqueous solution and using the pretreatment solution as a tissue portion. The pretreatment solution consists of a penetrant and a counter ion. The counter ion may be a cation or an anion added to the pretreatment solution. The osmotic agent is composed of molecules having a flexible backbone, wherein each osmotic agent molecule has at least one polar group or polar moiety attached to the backbone, which is capable of forming an attractive force with either a positive or negative ion; the polar moiety may be a functional group having a positive or negative charge or a molecule having a dipole capable of attracting positive or negative ions. Osmotic agents include molecules such as glycerol, polyethylene glycol, fatty acids, vitamins, sugars, and other molecules of similar structure. Adding counterions of both positive and negative charges to the pretreatment solution; such counterions include sodium (Na +), calcium (Ca + +), potassium (K +), ferric (Fe + + +), ferrous (Fe + +; chloride (Cl-), hydroxide (OH-), sulfate (SO 4-), phosphate (PO 4-), and other ions commonly found in the human body. Counterions are cations and anions that dissociate in aqueous solution and position themselves in the vicinity of polar or charged groups present on collagen molecules, lipid molecules, elastin molecules, or other molecules present in a tissue portion. The counter-ions help to allow the osmotic agent to penetrate into the intermolecular voids of the tissue portion by providing an attractive force between the polar groups of the osmotic agent and the charged or polar groups present on the structural molecules, including collagen, elastin, and lipid molecules, present in the molecular structure of the tissue portion. Counterions, such as sodium and hydroxide, can help convert hydrophobic carboxylic acids to react and form water soluble salts; the water-soluble salt can then penetrate into the molecular structure of the tissue portion and can be retained by, for example, attraction to polar groups of the collagen molecules.

The pretreatment step begins by placing the tissue portion in a pretreatment solution that allows the flexible osmotic agent to integrate into the molecular structure of the tissue portion and placing the osmotic agent molecules in proximity to the collagen molecules and other structural molecules of the tissue portion such that subsequent contact with the crosslinker molecules will create chemical bonds, including covalent bonds, between these molecules. The location of the osmotic agent molecules between each adjacent collagen molecule provides enhanced flexibility to the molecular structure of the tissue portion due to the flexible portion associated with the osmotic agent molecules.

The present invention also includes the formation of a fixation solution and the use of the fixation solution as a fixation step to crosslink molecules initially present in the native tissue portion and molecules that have diffused from the pretreatment solution and the fixation solution into the molecular structure of the tissue portion. The fixing solution consists of a cross-linking agent, a counter ion and a penetrating agent. The cross-linking agent may be glutaraldehyde, formaldehyde, other aldehydes, or a combination of various aldehydes and other molecules capable of reacting and forming chemical bonds between molecules present within the tissue portion and molecules present in the fixation solution. The presence of the counterion and the osmotic agent molecules in the fixation solution allows for a greater integration of the crosslinker molecules into the molecular structure of the tissue portion and prevents diffusion of the counterion and osmotic agent molecules that have been integrated into the molecular structure of the tissue portion from the pretreatment solution out of the tissue portion.

The counter ion can convert the non-aqueous fatty acid, for example, into a water-soluble salt that can penetrate into the molecular structure of the tissue portion and be held in close proximity to structural molecules, such as collagen molecules, for example, by ionic attraction, and be cross-linked to the collagen molecules by a cross-linking agent. The presence of the osmolyte molecules in the fixation solution further enhances the retention of the flexible osmolyte molecules at a location within the tissue portion between two existing structural molecules, such as collagen molecules. The presence of the counter ion and osmotic agent molecules in the fixation solution prevents the counter ion and osmotic agent molecules (from the pretreatment solution) that have been integrated into the molecular structure of the tissue portion from diffusing out of the tissue portion and toward the fixation solution.

The fixation step was performed as follows: the tissue portion is removed from its initial position within the pretreatment solution and placed in a fixation solution to cause cross-linking between structural molecules of the tissue portion, such as collagen molecules, and between structural molecules and osmotic agent molecules. The tissue portion is not rinsed after the pre-treatment step and before the fixation step such that the osmotic agent molecules and lipid molecules present in the tissue portion are retained and subsequently cross-linked into the molecular structure of the tissue portion by the cross-linking agent. In addition, the lipid and elastin molecules present in the molecular structure of the tissue portion are also cross-linked to each other, to other structural molecules, and to osmotic agent molecules. The presence of osmolyte molecules, the flexible backbone of which is integrated and cross-linked between corresponding or adjacent structural molecules of the tissue portion, such as collagen molecules, resulting in an increase in the intermolecular distance between the structural molecules from the native state to the distance present during the pre-treatment or fixation step, and providing the tissue portion with improved flexibility and the ability of the tissue portion to bend without breaking; this flexibility is maintained during subsequent processing steps.

The invention also includes the formation of a desiccating solution and the use of a desiccating solution to form a desiccated tissue portion that can be stored and transported as a desiccated tissue portion. The dry solution consists of a hygroscopic agent, a counter ion and a penetrant. The hygroscopic agent may be isopropanol, ethanol, propanol, acetone, other polyols, or other molecules currently used to absorb one or more water molecules from a solution and removable by air drying or lyophilization. The osmotic agent may include polyethylene glycol, glycerol, or other molecules having a flexible backbone and containing at least one polar group. The counter ion including the positive ion and the negative ion as previously described in the present specification helps to allow the moisture absorbent to be integrated into the molecular structure of the tissue portion and to allow water to be uniformly removed from the tissue portion. The counter ions form attractive forces that help to enhance and stabilize the moisture absorbing and penetrating agents in the vicinity of the molecules present in the tissue portion. The presence of the counter-ions and the penetrant molecules in the drying solution also ensures that the counter-ions and penetrant molecules that have penetrated into the tissue portion during the pre-treatment or fixation step cannot diffuse out of the tissue portion into the drying solution.

To initiate the drying step, the tissue portion is removed from the fixation solution and placed in a drying solution as described herein. The penetrant molecules are held in place within the molecular structure of the tissue portions by cross-linker molecular bonds and by electrostatic attraction assisted by counter-ions. Due to the presence of the counter-ion and penetrant molecules in the drying solution, the intermolecular distance between the individual molecules of the tissue portion is maintained during this portion of the drying step.

The drying step of the present invention further comprises removing the tissue portion from the drying solution and placing it on a flat surface to allow air drying or drying by lyophilization to form a dried tissue portion. During this part of the drying step, the permeating molecules are located between, e.g. adjacent, corresponding structural molecules of the tissue portion, such as collagen molecules, e.g. located adjacent to each other. Other structural molecules such as lipids and elastin are also bonded to the osmotic agent molecules through cross-linkers and through ionic bonds formed with counter ions. Due to the presence of the permeant molecules, the intermolecular distance between corresponding structural molecules, such as collagen molecules, located adjacent to each other within the molecular structure is increased to a greater intermolecular distance than when the permeant molecules and counter ions are absent. The resulting dried tissue portions are able to flex and bend without creating a rupture that can weaken the tissue portions and cause damage to the tissue portions. The presence of the counter ions and polar portions of the osmotic agent molecules present within the molecular structure of the dried tissue portion enhances the ability of the dried tissue portion of the present invention to be rehydrated more rapidly by a physician in an operating room than if the polar groups of such counter ions and osmotic agent molecules were not present within the molecular structure.

Description of the drawings:

fig. 1 is a process flow diagram showing the pretreatment step, the fixation step, and the drying step, as well as the types of molecules and counter ions present in the solution used in each step.

FIG. 2 is a view of a tissue portion in a pretreatment solution and a close-up view of the molecular structure of the tissue portion, wherein molecules of the pretreatment solution penetrate into the molecular structure.

Fig. 3 is a view of a tissue portion within a fixation solution and shows a close-up of the molecular structure of the tissue portion, wherein cross-linking occurs between permeating molecules and structural molecules of the tissue structure.

Fig. 4 is a view of a tissue portion disposed within a drying solution and a close-up view of the molecular structure of the tissue portion showing the interaction of moisture absorbent molecules with water molecules of the drying solution.

FIG. 5 is a close-up view of the molecular structure of a dried tissue portion after removal of a substantial number of water molecules by air drying or lyophilization; the molecular structure shows that the permeant molecules and counter-ions remain within the molecular structure.

The specific implementation mode is as follows:

figures 1 to 5 show a process flow diagram and describe three steps in the method of the invention for treating a tissue portion. The three steps shown in fig. 1 include pretreatment in a solution containing a penetrant, cations and anions (counterions) (5); fixation (10) in a solution containing a penetrant, a fixative, cations, and anions; and drying (15) in a solution containing a hygroscopic agent, a penetrant, a cation and an anion, and then drying, for example, by air drying or by lyophilization. The cations or anions (i.e., charged groups or charged species) held in proximity to the charged groups or polar groups (or polar moieties) of the molecules having opposite charge to the cations or anions are referred to as counterions or cations. The tissue portions may be obtained from xenogeneic sources, homologous sources, or from engineered tissues formed from cells grown in an incubator to produce structural molecules such as collagen, elastin, and lipid molecules, similar to those present in body tissues. The tissue portions formed by the present methods may be used in a variety of medical applications, including leaflets for heart valves, vascular grafts, patches for attaching tissue, preventing fluid leakage, and other applications. In the description that exists in this specification, applanation tissue portions from natural tissue sources will be described, however, it should be understood that the same processes and methods may be used to treat tissue obtained from other sources and other shaped tissue portions that may be tubular, have a 3-D shape, or formed, for example, as tissue portions used as separate components of medical device assemblies, such as the heart leaflets of a heart valve.

The pre-treatment step (5) comprises taking clean fresh native tissue and forming a tissue portion (20) having a tissue portion thickness (25), as shown in fig. 2. The tissue portion thickness (25) depends on several factors, including the source of the tissue material and the application in which the tissue portion is used. During the pretreatment step (5), the tissue portion is placed in a pretreatment aqueous solution (30) containing a pretreatment solution penetrant (35) (i.e., penetrant molecules present in the pretreatment solution (30)) and a combination of cations and anions or pretreatment solution counterions (40) (i.e., counterions added to and thus located in the pretreatment solution (30)). The pretreatment solution penetrant (35) may be a molecule having a flexible backbone (45) and at least one polar group or polar moiety (50) positioned along the chain, the polar group or polar moiety being capable of forming an attractive force (55) or a polar attraction (55) with a positive or negative ion or counter ion within the tissue portion. The polar moiety (50) may be a functional group located along the backbone of the permeant molecule, for example, having a dipole moment capable of attracting positive or negative ions. For example, a polar group or portion (50) may form an ionic or attractive force (55) by forming a salt that is attracted to an oppositely charged counterion. The pretreatment solution penetrant molecules (35) may be polyethylene glycol, glycerol, fatty acids, vitamins, sugars, polysaccharides, or other molecules having a flexible backbone (45) and at least one polar group (50) attached to the backbone. Cations and anions include, but are not limited to: sodium (Na +), calcium (Ca + +), potassium (K +), ferric (Fe + + +), ferrous (Fe + +); chloride (Cl-), hydroxide (OH-), sulfate (SO 4-), and phosphate (PO 4-).

The anions and cations help to allow the pretreatment solution penetrant molecules (35) to leave the pretreatment solution (30) and diffuse or migrate into the molecular structure (60) of the tissue portion (20), as shown in fig. 2, and become penetrant molecules (62) located within the molecular structure of the tissue portion (20). The counter ions (68) (i.e., anions and cations) that have migrated into the molecular structure (60) of the tissue portion (20) may form carboxyl groups of the water insoluble fatty acid, e.g., form soluble salts that may form ionic bonds with polar and charged groups extending from the backbone of the structural molecules (65) of the native tissue portion (20), including, for example, collagen molecules or other structural molecules (65) located in the molecular structure (60) of the tissue portion (20). The pretreatment solution counterions (40) leave the pretreatment solution (30) and diffuse or migrate into the molecular structure (60) of the tissue portion (20) to become (due to their location) counterions (68) of the tissue portion (20). The counterions (i.e., cation and anion) both help to increase penetration of the penetrant molecules into the molecular structure (60) of the tissue portion (20) and also help to retain the penetrant molecules (62) within the molecular structure (60), e.g., adjacent to structural molecules (65), such as collagen molecules, present in the tissue portion (20), by attractive forces (55) associated with the ionic charges of the counterions (68) (cation or anion). The counter ions provide increased molecular contact (72) between the penetrant molecules and the structural molecules such that subsequent exposure of the tissue portions to the cross-linking agent will result in increased cross-linking between the penetrant molecules and the structural molecules.

The pre-treatment (5) (i.e., the pre-treatment step (5)) includes placing the tissue portion (20) in a pre-treatment solution (30) for 10 hours (ranging from 5 minutes to 24 hours) to allow the osmotic agent molecules (62) to penetrate into the molecular structure (60) of the tissue portion (20). The tissue portion (20) has an initial tissue portion thickness (25) that is dependent upon its application as a heart valve leaflet, such as a patch, vascular graft, or other application of the tissue portion (20). The thickness of the natural tissue portion prior to pretreatment (5) may be in the range of about 0.002 inches to 0.020 inches. Due to the presence of the penetrant molecules (62), the intermolecular distance (70) between structural molecules (65), such as collagen molecules, present in the molecular structure (60) of the pre-treated tissue portion (20) is increased over the intermolecular distance (70) present in the native tissue portion. For example, the presence of flexible osmotic agent molecules (62) is located between separate and adjacent collagen molecules and is maintained by ionic attractive forces (55) (or polar forces) provided by counter ions (68) that diffuse into the molecular structure and are located between structural molecules of the tissue portion. The counter ions are derived from the pretreatment solution counter ions (40) which are added to the pretreatment solution (30) as part of the present invention to provide flexibility to the tissue portion (20); this flexibility will remain unchanged even after the tissue portion (20) has been exposed to subsequent fixation (10) and drying steps (15). For the pretreatment solution (30), a lower concentration limit of 100 millimolar (mM) of the pretreatment solution osmotic agent (35) is necessary to provide the tissue portion (20) with the desired flexibility to prevent rupture during bending of the final dried tissue portion, as described below. The lower concentration limit of the pretreatment solution counter ions (40) requires 300mM to provide the attractive forces (55) required to retain the penetrant molecules (62) within the molecular structure (60) and subsequently crosslink the structural molecules (65) bonded to the tissue portion (20). The molar ratio of pretreatment solution counter ion (40) to pretreatment solution penetrant (35) in pretreatment solution (30) is, for example, 3: 1; the present invention is not limited by a particular molar ratio.

The pretreatment solution (30) of the present invention may employ one or more penetrants at concentrations ranging from 1% to 95% by weight. Furthermore, the pretreatment solution (30) may incorporate one or more counter ions at a concentration of 1% to 20% by weight. Examples of pre-treatment solution (30) formulations included in the present invention are discussed herein; the pretreatment solution examples are not intended to limit the scope of pretreatment solution selection, but rather to provide specific formulations that have been tested and shown to produce treated dried tissue portions that can be successfully dried and will remain flexible without rupture during bending.

In pre-treatment solution example a (as described herein), the pre-treatment solution (30) is formed from an aqueous solution having a pre-treatment solution penetrant (35) concentration of 50% by weight (range 1% to 95%); the pretreatment solution penetrant (35) is polyethylene glycol. The pretreatment solution (30) contains pretreatment solution anions and pretreatment solution cations (monovalent, divalent, or trivalent ions) at a concentration of 10% by weight (ranging from 1% to 20%).

Pretreatment solution example B had a glycerol pretreatment solution penetrant (35) at a concentration of 50% by weight (ranging from 1% to 95%). The pretreatment solution (30) contains pretreatment solution anions and pretreatment solution cations (monovalent, divalent, or trivalent ions) at a concentration of 10% by weight (ranging from 1% to 20%).

In pretreatment solution example C, the pretreatment solution penetrant (35) is polyethylene glycol at a concentration of 50% by weight (range 1% to 95%) and glycerol at a concentration of 50% by weight (range 1% to 95%). The pretreatment solution (30) contains pretreatment solution anions and pretreatment solution cations (monovalent, divalent, or trivalent ions) at a concentration of 10% by weight (ranging from 1% to 20%).

It should be understood that the pre-treatment solution (30) may be comprised of one or more permeant molecules. Furthermore, it should be understood that separate permeation molecules may be used in a separate pretreatment step (5) to form a pretreatment (5) of the tissue portion (20). For example, a combined pretreatment step as described for pretreatment solution example a (5) followed by pretreatment solution example B to form a pretreated tissue portion (20) may be applied.

Initiating a fixation step (10) (or fixation of a tissue portion (10)) after removal of the tissue portion (20) from the pretreatment solution (30); as shown in fig. 3, fixation (10) is initiated by placing the tissue portion (20) in a fixation solution (75); the fixing solution contains fixing solution penetrant molecules (80) (i.e., penetrant molecules located in the fixing solution (75)), cross-linker molecules (85), and fixing counterions (90) (i.e., cations and anions in the fixing solution (75)). The tissue portion (20) is not rinsed after the pre-treatment step (5) such that the osmotic agent molecules (62) and the lipid molecules of the tissue portion (20) remain within the molecular structure (60) of the tissue portion (20). The osmotic agent molecules (62), together with the lipid molecules present in the tissue portion (20), provide the tissue portion with flexible molecules that will remain within the molecular structure (60) and provide the tissue portion (20) with flexibility even after the tissue portion is dried to form a dried tissue portion (130) as shown in fig. 5.

The fixation solution penetrant molecules (80) include molecules having a flexible backbone (45) and at least one polar group (50) attached to the backbone, including, for example, the pretreatment solution penetrant molecules (35) present in the pretreatment solution (30) described previously. It is necessary that the fixative solution osmotic agent molecules (80) be present in a sufficient minimum concentration within the fixative solution (75) to ensure that diffusion of the osmotic agent molecules (62) that have migrated into the tissue portion (20) during the pre-treatment step (5) from the tissue portion (20) into the fixative solution (75) is prevented during the fixation step (10).

Cross-linker molecules (85) include glutaraldehyde, formaldehyde, other aldehydes, and other cross-linkers (85) currently used to form cross-links (95) between structural molecules (65), including collagen, lipids, elastin, and other molecules found in native tissue portions (20). Glutaraldehyde is often used as the cross-linking agent (85) because its bifunctional two carbonyl groups is capable of chemically bonding to the amino groups present on the protein molecules of collagen. Glutaraldehyde molecules may also be bonded to other functional groups present on structural molecules (65) present in proteins, lipids and elastin, including to thiol, phenolic and imidazole groups.

As shown in fig. 3, the cross-linker molecules (85) are able to penetrate into the voids of the molecular structure (60) of the tissue portion (20) and form cross-links (95) with structural molecules (65) present in the tissue portion (20), such as collagen molecules, lipid molecules, elastin molecules and other long chain molecules. The cross-linker (85) creates cross-links (95), such as covalent bonds, between the cross-linker molecules (85) and the structural molecules (65), such as collagen molecules, and also creates cross-links (95) with adjacent structural molecules (65), for example, to cross-link adjacent structural molecules (65) to each other. The cross-links may be covalent bonds. In addition, the cross-linker molecules (85) form cross-links (95) with penetrant molecules (62) of the molecular structure (60) that have been previously delivered to the tissue portion (20), thereby cross-linking the penetrant molecules (62) with adjacent penetrant molecules (62), or cross-linking the penetrant molecules (62) with adjacent collagen molecules or other structural molecules of the tissue portion (20). The presence of the counter-ion within the molecular structure of the tissue portion increases molecular contact (72) between the penetrant molecules and the structural molecules of the tissue portion, resulting in increased formation of cross-links between these molecules upon exposure to the cross-linking agent as compared to when the molecular contact (72) is not formed.

The presence of cations and anions (i.e., fixation solution counterions (90)) in the fixation solution (75) helps to provide an attractive force (55) that enhances penetration of the crosslinker molecules (85) and the osmotic agent molecules (62) into the molecular structure (60) of the tissue portion (20) and ensures that the counterions (68) that have diffused into the tissue portion (20) cannot diffuse out of the tissue portion (20) and toward the fixation solution (75). The counter ions (68) maintain the pretreatment solution penetrant molecules (35) located within the molecular structure (60) of the tissue portion (20) in close proximity to the structural molecules (65) of the tissue portion (20) by the attractive force (55) to provide improved cross-linking between these molecules and the cross-linker molecules (85) as compared to if such pretreatment solution counter ions (40) were not added to the pretreatment solution (30) and the fixation solution (75). The cations and anions convert polar carboxyl groups present on the protein molecules of the tissue portion into water soluble salts, thereby allowing better integration of the cross-linker molecules (85) and the osmotic agent molecules (62) into the molecular structure (60) of the tissue portion (20).

During the fixation step (10), the tissue portion (20) is placed in a fixation solution (75) for a period of 7 days (ranging from 12 hours to 30 days) to allow the cross-linking agent (85) to penetrate into the molecular structure (60) of the tissue portion (20) and achieve the desired cross-linking between the structural molecules (65) and the penetrant molecules (62) located within the molecular structure (60) of the tissue portion (20). The penetrant molecules have been positioned between and in contact with the structural molecules of the tissue portion and are held in place by the attractive forces provided by the counter-ions prior to exposure to the cross-linker molecules. Due to the presence of the penetrant molecules (62) located within the molecular structure (60) of the tissue portion (20), the intermolecular distance (70) between structural molecules (65), such as collagen molecules, present in the tissue portion (20) is increased over that present in the native state. For example, the presence of the flexible backbone (45) of the osmotic agent molecules (62) located between the separated structural molecules (65) and held by the ionic attraction (55) of the polar groups (50) and cross-linked to the structural molecules (65) by the cross-linker molecules (85) provides flexibility to the tissue portion (20), which will remain in place even after the tissue portion (20) has been exposed to a subsequent drying step (15).

For the fixation solution (75), the lower concentration limit of the fixation solution osmotic agent (80) is 100 millimolar (mM) to ensure that osmotic agent molecules (62) located within the molecular structure (60) of the tissue portion (20) cannot diffuse out of the fixation solution (75). The lower limit of the concentration of the counter ions (90) of the fixation solution is 300mM to prevent diffusion of the counter ions (68) located within the molecular structure (60) of the tissue portion (20) from the tissue portion (20) into the fixation solution (75). For example, the molar ratio of the fixing solution counterions (90) to the fixing solution penetrants (80) in the fixing solution (75) is about 3:1, although the invention is not limited to a particular ratio of counterions to penetrant molecules.

The fixing solution (75) of the present invention may use one or more penetrants at a concentration ranging from 1% to 85% by weight. Furthermore, the fixing solution (75) may incorporate one or more counter ions at a concentration of 1% to 20% by weight. The fixing solution (75) may utilize one or more crosslinker molecules (85) at a concentration of 0.1% to 10% by weight. Examples of fixative solution (75) formulations included in the present invention are discussed herein; the fixation solution examples are not intended to limit the scope of fixation solution selection, but rather provide a specific formulation that has been tested and demonstrated to produce a treated tissue portion that can be successfully dried and will remain flexible without rupture during bending.

In fixing solution example a (as described herein), the fixing solution (75) is formed from an aqueous solution having a concentration of 40% by weight (ranging from 1% to 85%) of a cross-linking solution penetrant (i.e., the penetrant used in the fixing solution (75) during the cross-linking step); the fixing solution penetrant (80) is polyethylene glycol. The crosslinking solution contains a concentration of crosslinking solution anions (i.e., anions used in the crosslinking solution during the crosslinking step) and crosslinking solution cations (i.e., cations used in the crosslinking solution); the anion and cation may be monovalent, divalent or trivalent ions at a concentration of 10% (ranging from 1% to 20%) by weight. The crosslinking solution contains a crosslinking agent (85) of glutaraldehyde, formaldehyde or other crosslinking agent (85) at a concentration of 5% (ranging from 0.1% to 10%).

In fixing solution example B, the cross-linking solution penetrant is glycerol at a concentration of 50% by weight (range 1% to 95%). The crosslinking solution contains crosslinking solution anions and crosslinking solution cations (mono-, di-or trivalent ions) in a concentration of 10% by weight (range 1% to 20%). The crosslinking solution contains a crosslinking agent (85) of glutaraldehyde, formaldehyde or other crosslinking agent (85) at a concentration of 5% by weight (in the range of 0.1% to 10%).

In fixation solution example C, the cross-linking solution penetrant is polyethylene glycol at a concentration of 50% by weight (range 1% to 95%) and glycerol at a concentration of 50% by weight (range 1% to 95%). The crosslinking solution contains crosslinking solution anions and crosslinking solution cations (mono-, di-or trivalent ions) in a concentration of 10% by weight (range 1% to 20%). The crosslinking solution contains a crosslinking agent (85) of glutaraldehyde, formaldehyde or other crosslinking agent (85) at a concentration of 5% by weight (in the range of 0.1% to 10%).

It is to be understood that the crosslinking solution may be comprised of one or more penetrant molecules, and may contain one or more crosslinker molecules (85). Furthermore, it should be understood that individual permeant molecules can be used in a single pretreatment step (5) to form a pretreatment (5) of the tissue portion (20). Furthermore, it should be understood that individual crosslinker molecules may be used in a single crosslinking step to form crosslinks for the tissue portion (20). For example, the fixation step (10) may be performed using fixation solution example a, followed by fixation (10) using the fixation formulation present in fixation solution example B to form a combined cross-linking step for cross-linking the tissue portion (20).

Removing the tissue portion (20) from the cross-linking solution prior to forming the drying step (15); as shown in fig. 4, the drying step (15) begins by placing the tissue portion (20) in a dry aqueous solution (100) containing molecules of a moisture absorbent (105), counter ions of a drying solution (110) (i.e., cations and anions used in the drying step), and molecules of a drying solution penetrant (115). The dry solution penetrant molecules (115) include molecules having a flexible backbone (45) and at least one polar group attached to the backbone, including, for example, the aforementioned penetrant molecules. The dry solution desiccant molecules (105) include isopropyl alcohol, ethyl alcohol, propyl alcohol, acetone, other polyols, or other molecules currently used in the medical device industry to absorb water. The moisture absorbent molecules (105) providing one or more functional groups capable of bonding with water molecules (125) present within the interstices of the molecular structure (60) of the tissue portion (20); the hygroscopic molecules, as well as the water present in the tissue portion (20), are then removed as the tissue portion is air dried or lyophilized, as described below.

As shown in fig. 4, the presence of the drying solution counter ions (110) (i.e., cations and anions previously identified but used herein in the drying solution (100)) in the drying solution (100) help provide attractive forces (55) that enhance penetration of the hygroscopic agent molecules (120) and osmotic agent molecules (62) within the molecular structure (60) of the tissue portion (20). The cations and anions convert polar carboxyl groups present on the protein molecules of the tissue portion into water soluble salts, thereby integrating the hygroscopic agent molecules (120) and the osmotic agent molecules (62) into the molecular structure (60) of the tissue portion. It was found that the concentration of the dry solution counter ions (110) and the dry solution osmotic agent molecules (115) in the dry solution (100) would prevent the diffusion of the counter ions (68) from the molecular structure (60) of the tissue portion out of the tissue portion and prevent the diffusion of the osmotic agent molecules (62) from the molecular structure (60) of the tissue portion out of the tissue portion and into the dry solution (100). The intermolecular distance between corresponding adjacent structural molecules (65) has increased due to the penetration and bonding of the permeant molecules (62) located between the adjacent structural molecules (65) caused by the presence of the attractive forces (55) provided by the counter ions (68), i.e., cations and anions. The presence of flexible osmotic agent molecules (62) within the molecular structure (60) of the tissue portion (20) provides greater flexibility and resistance to rupture during bending of the tissue portion.

The tissue portion is placed in a drying solution (100) for 7 days (ranging from 30 minutes to 14 days) to allow the drying solution moisture absorber (105) and osmotic agent molecules (62) to penetrate into the molecular structure (60) of the tissue portion and become a moisture absorber (120) associated with the tissue portion. For example, the intermolecular distance between structural molecules (65), such as collagen molecules, present in the tissue portion is increased relative to the intermolecular distance (70) between structural molecules (65) present in the native state of the tissue portion. The presence of flexible permeant molecules (62), e.g., located between isolated collagen molecules, and maintained by ionic attraction (55) and covalent bonds associated with cross-links (95), e.g., between structural molecules (65) and permeant molecules (62), provides flexibility to the tissue portion, even after the tissue portion has been completely dried by air drying or freeze drying (by vacuum freeze drying) to remove water molecules (125) from the tissue portion as described below, the tissue portion will remain in place. When present in the drying solution (100), the intermolecular distance (70) between adjacent structural molecules (65) of the tissue portion remains similar to the intermolecular distance (70) present in the cross-linked tissue portion.

For the dry solution (100), the lower concentration limit of the dry solution osmotic agent is 100 millimolar (mM) to prevent migration of the osmotic agent molecules (62) out of the tissue portion (20) and into the dry solution (100). A lower concentration limit of 300mM of the dry solution counter ion (110) is required to prevent diffusion of the counter ion (68) from the molecular structure (60) of the tissue portion (20) into the dry solution (100). For example, the molar ratio of the dry solution counterion (110) to the dry solution penetrant in the dry solution (100) is 3:1, although this molar ratio can vary and still be suitable for the present invention.

The dry solution (100) of the present invention may employ one or more osmotic agents at a concentration ranging from 1% to 85% by weight. Furthermore, the dry solution (100) may incorporate one or more counter ions at a concentration of 1% to 20% by weight. The dry solution (100) further contains a dry solution moisture absorbent (105) at a concentration of 10 to 100% by weight. Examples of dry solution formulations included in the present invention are discussed herein; the drying solution examples are not intended to limit the scope of drying solution selection, but rather to provide specific formulations that have been tested and shown to produce treated tissue portions that can be successfully dried and will remain flexible without rupture during flexing.

In dry solution example a (as described herein), the dry solution (100) is formed from an aqueous solution having a dry solution penetrant (i.e., the penetrant used during the drying step (15)) concentration of 40% by weight (ranging from 1% to 85%); the dry solution penetrant is polyethylene glycol. The drying solution (100) contains a concentration of drying solution anions (i.e., anions used during the drying step (15)) and drying solution cations (i.e., cations used during the drying step (15)); the anion and cation may be monovalent, divalent or trivalent ions at a concentration of 10% (ranging from 1% to 20%) by weight. The dry solution (100) contains a hygroscopic or other hygroscopic agent of isopropanol or ethanol at a concentration of 50% (ranging from 10% to 100%).

In dry solution example B, the dry solution penetrant is glycerin with a concentration of 50% by weight (range 1% to 95%). The dry solution (100) contains a dry solution anion and a dry solution cation (monovalent, divalent or trivalent ion) at a concentration of 10% by weight (range 1% to 20%). The dry solution (100) contains a dry solution desiccant (105) of isopropanol, ethanol or other desiccant at a concentration of 50% (ranging from 10% to 100%).

In dry solution example C, the dry solution penetrant is polyethylene glycol at a concentration of 50% by weight (range 1% to 95%) and glycerol at a concentration of 50% by weight (range 1% to 95%). The dry solution (100) contains a dry solution anion and a dry solution cation (monovalent, divalent or trivalent) in a concentration of 10% (ranging from 1% to 20%).

It is to be understood that the dry solution (100) may be composed of one or more permeant molecules and may contain one or more dry solution desiccant molecules (105). Moreover, it should be understood that separate osmotic agent molecules may be used in a separate drying step to form a drying of the tissue portion (20). Furthermore, it should be understood that separate moisture absorbent molecules may be used in a separate drying step to form the drying of the tissue portion (20). For example, a drying step (15) involving drying solution example a followed by a drying step (15) incorporating drying solution example B of drying solution (100) may be applied to form a combined drying step for drying the tissue portion (20).

To complete the drying step (15), the tissue portion (20) is removed from the drying solution (100) and placed on a flat surface for air drying, or the tissue portion (20) may be exposed to a lyophilization process to complete the drying step (15) and remove water from the tissue portion (20). The moisture absorbent molecules have functional groups capable of bonding with water molecules (125) present within the tissue portion (20). Air drying or freeze drying of the tissue portion (20) will allow the moisture absorbent molecules (120) to evaporate, e.g. from the molecular structure (60) of the tissue portion, together with the water molecules (125), thereby forming a dried tissue portion (130) as shown in fig. 5; the dry tissue portion (130) has a majority (50% to 90%) of water molecules (125) removed from the molecular structure (60) of the dry tissue portion (130) with the moisture absorbent (120). The intermolecular distance (70) between the structural molecules (65) of the dried tissue portion (130) in a fully dried state is reduced relative to the intermolecular distance (70) present when the tissue portion is within the drying solution (100). The cross-linking of the osmotic agent molecules with the structural molecules (65) of the tissue portion (20) retains the osmotic agent molecules (62) and counter-ions (68) within the molecular structure (60) of the tissue portion (20) and provides enhanced flexibility to the tissue portion (20) in a fully dried state without rupture due to bending of the tissue portion (20).

The dry tissue portion (130) (after substantially all or most of the water has been removed) retains the osmotic agent molecules (62) in the dry tissue portion (130) and thus will remain more flexible than other methods that do not utilize the concentrations of counterions (i.e., cations and anions) required by the present invention to enhance permeation and ionic bonding to maintain the osmotic agent molecules adjacent to structural molecules (65), such as collagen molecules, and other native molecules adjacent to the tissue portion (20) during the pretreatment (5), fixation (10), and drying steps (15) of the present invention. Maintaining the osmotic agent molecules at the desired concentration for the present invention, and maintaining the lipid molecules within the molecular structure (60) of the tissue portion (20) by crosslinking via the crosslinking agent and the ionic attraction (55) formed by the counter ions (68) located within the molecular structure (60), will provide flexibility to the dry tissue portion (130) during bending without forming cracks in the absence of the osmotic agent molecules due to brittleness of the tissue portion (20). Due to the presence of the counter ions (68) within the molecular structure (60) of the dry tissue portion (130) at the concentrations provided by the invention, the dry tissue portion (130) can be rehydrated more rapidly by a physician in an operating room than other dry tissue portions that do not contain counter ions within the molecular structure (60) of the tissue portion (20).

Reference numerals used in the specification and found in any one of fig. 1 to 5 of the present invention are intended to denote similar structures having similar functions. Moreover, it is to be understood that aspects of one or more embodiments may be combined with other aspects from another embodiment to form yet other embodiments also included in the present invention.

For example, an alternative embodiment of the present invention may apply an initial pre-treatment-fixation step to the natural tissue portion using a pre-treatment-fixation solution having the same concentration of osmolyte molecules, counterions and cross-linker molecules as the fixation solution described previously. The counterions and penetrant molecules present in the pre-treatment-fixation solution will diffuse and integrate into the molecular structure of the native tissue portion, while the cross-linker molecules form cross-links with the penetrant molecules and the structural molecules of the tissue portion. In this embodiment, the penetrant molecules and counterions do not have the opportunity to become fully integrated into the molecular structure of the tissue portion prior to forming the cross-links, as would be the case if separate pretreatment and fixation steps were used. The consistency of the molecular structure forming the cross-links will be more variable and will depend on the timing of diffusion of the counter-ion and penetrant molecules into the tissue portion.

In another embodiment, a moisture absorbent may be added to the pre-treatment-fixing solution to form a pre-treatment-fixing-drying solution. The native tissue portion may be placed in a single pre-treatment-fixation-drying solution initially containing a small amount of hygroscopic agent, and the hygroscopic agent concentration may be increased from a small concentration to the same level as present in the previously identified drying step, within a time period similar to that described for the fixation step. The tissue portions may be removed from the solution and allowed to air dry completely or dried by lyophilization. This single-step treatment of the tissue portion is faster and easier than the three-step treatment described in the earlier embodiments of the invention, but does not provide control over each step, which can provide consistency to the characteristics of the tissue portion, including flexibility and resistance to bending rupture of the dry tissue portion.