Tissue substitute composition

Abstract

The invention relates to a tissue substitute composition, which enables postmortem examination of animal and human organs in a simple and reproducible manner. In particular, the tissue substitute composition, in combination with a contrast agent, enables, for example, improved computed tomography examination of blood vessels. The invention also relates to an imaging method, which comprises the use of the tissue substi- tute.

Description

[0001] Tissue substitute composition

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a tissue substitute composition, which enables postmortem examination of animal and human organs in a simple and reproducible manner. In particular, the tissue substitute composition, in combination with a contrast agent, enables, for example, improved computed tomography examination of blood vessels.

[0004] The invention also relates to an imaging method, which comprises the use of the tissue substitute.

[0005] BACKGROUND OF THE INVENTION

[0006] During ex vivo CT (computed tomography) angiography (CTA) examinations, blood vessels must be filled with a blood substitute material comprising a contrast agent. The requirement for the tissue substitute material substituting blood is that it must be able to reproduce the image quality obtained in in vivo examinations, i.e. its radiation absorption must be the same as that of the corresponding tissue, it must be compatible with the radiation-absorbing contrast agents used during the procedure (e.g., iopamidol, barium sulfate, gadolinium-based contrast agents), it must not damage the blood vessels and the formations, such as sclerotic plaques, occurring in the vessels, it must not exit the vascular system, it must not block blood vessels with a smaller diameter, and it must be able to fill blood vessels with a smaller diameter without bubbles.

[0007] Grabherr et al. summarized the procedures used during postmortem angiography and the preparations that can be administered into the blood vessels in their article. (Grabherr et al. Postmortem Angiography: Review of Former and Current Methods. AJR Am J Roentgenol. 2007 Mar;188(3):832-8. doi: 10.2214 / AJR.06.0787. PMID: 17312075.)

[0008] W02008034270A2 discloses an essentially oil-based material with a viscosity of 30-100 mPas and an essentially anhydrous contrast agent comprising hexadecane or tetradecane.

[0009] PCT / EP2018 / 055463 describes a contrast agent for use in microangiography, comprising polyurethane, a curing agent, a iodinated esterified oil and a ketone.

[0010] CN114010804A discloses a contrast agent comprising iohexol, sorbitol and water.

[0011] CN111840580A discloses a contrast agent for use in postmortem angiographic examinations, which comprises a compound containing an iodobenzene ring and a fat- soluble solvent of a given viscosity.

[0012] Becker et al. (Ex vivo coronary atherosclerotic plaque characterization with multi-detector-row CT. Eur Radiol. 2003 Sep;13(9):2094-8. doi: 10.1007 / s00330-003- 1889-5. Epub 2003 Apr 12. PMID: 12692681.) injected a mixture of iodinated contrast agent and methylcellulose into the coronary artery system postmortem. It was also reported that the mixture was unable to fill vessels with severe coronary artery lumen stenosis, as filling with the mixture was unsuccessful in cases of stenosis greater than 50% caused by plaque.

[0013] There is therefore a need for a tissue substitute that, in combination with a contrast agent, can fill constricted blood vessels with a small diameter, while meeting the requirements described above.

[0014] SHORT DESCRIPTION OF THE INVENTION

[0015] The invention provides the use of a cellulose ether for the substitution of a tissue ex vivo. Preferably the viscosity of the cellulose ether (2 m / m% aqueos solution, measured at 20°C) is 5- 35 mPas, particularly preferably 10-25 mPas or 12-18 rnPas, particularly preferably 15 mPas.

[0016] A method for the production of a tissue substitute composition is provided, comprising or consisting of: adding a low viscosity cellulose ether to warm water under stirring, cooling the mixture of the cellulose ether and the warm water by adding cold water, under stirring; if necessary further stirring at low temperature until a homogeneous solution is obtained, to obtain the tissue substitute composition.

[0017] A tissue substitute composition comprising cellulose ether is provided, wherein the viscosity of the cellulose ether (2 m / m% aqueos solution, measured at 20°C) is 5-35 mPas, particularly preferably 10-25 mPas or 12-18 mPas, particularly preferably 15 mPas.

[0018] Preferably the tissue substitute composition is an aqueous solution of a cellulose ether.

[0019] Use of a low viscosity cellulose ether for the production of a tissue substitute composition is provided.

[0020] Preferably the cellulose ether or the tissue substitute composition is used in a post mortem or ex vivo angiography examination, preferably in a CTA method, particularly preferably in the examination of coronary blood vessels. Preferably the cellulose ether or the tissue substitute composition is used in a post mortem or ex vivo imaging method.

[0021] Preferably the tissue for which the cellulose ether or tissue substitute material is used is blood. Preferably, the tissue substitute composition further comprises a contrast agent (radiation absorbing agent) suitable for imaging in a CTA method.

[0022] Preferably the cellulose ether is methylcellulose (MC; CAS: 9004-67-5) or hydroxypropyl methylcellulose (HPMC; CAS: 9004-65-3), particularly preferably methylcellulose. Preferably the viscosity of the cellulose ether (2 m / m% aqueos solution, measured at 20°C) is 5-35 mPas, particularly preferably 10-25 rnPas or 12-18 mPas, particularly preferably 15 mPas. Preferably the methoxy content of the MC is 20-40% or 25-35%, particularly preferably 27- 32% or 27,5-31,5%. Preferably the methoxy content of the HPMC is 20-40% or 25-35%, particularly preferably 28-30%, the hydroxylpropyl content of the HPMC is 2-20% or 5-17%, particularly preferably 7-12%.

[0023] Preferably the MC is A15 LV or METHOCEL A15 LV or METHOCEL A15 Premium LV. Preferably the HPMC is E15 LV or METHOCEL E15 LV or METHOCEL E15 Premium LV. Preferably the MC is an MC having the same physical (e.g. molecular weight, viscosity, friction) and chemical (e.g. methoxy content, number of glucose moieties) properties as the following MC: (retrieved on 13. 02.

[0024] 2024). Preferably the MC is an MC having the same physical and chemical properties as the following MC: Methocel A15 LV (64605- 100G-F, Sigma-Aldrich; 27,5-31,5% methoxyl basis) CAS Number: 9004-67-5, batch number: 3Hl / Xl,6 / S / 22 / 0 / ; BAM12359-M3; PCode: 102550267, Source: BCCH5199).

[0025] Preferably the temperature of the warm water is about 60-100 °C, preferably about 70-100 °C, preferably about 80-100 °C, particularly preferably about 75 °C or about 70-80 °C. Preferably the warm water is not boiling water. Preferably the temperature of the warm water is higher than the lower critical mixing temperature of methylcellulose (at normal pressure, relative to water). Preferably the temperature of the warm water is higher than 45°C or higher than 55°C. Preferably, the warm water is at a temperature that ensures wetting and / or (even) dispersion of MC particles in water (i.e. ensures that the effective - wettable - surface area of the MC added to the warm water is large). Preferably, the speed of mixing during the addition of the MC to the warm water ensures wetting and (even) dispersion of MC particles in water.

[0026] Preferably, the temperature of the cold water is such that when added to the mixture of warm water and MC, it ensures complete dissolution of the MC. Preferably, the temperature of the cold water is such that when added to the mixture of warm water and MC, the temperature of the resulting mixture is below the lower critical mixing temperature of MC (at normal pressure, relative to water). Preferably, the temperature of the cold water is such that when added to the mixture of warm water and MC, the temperature of the resulting mixture is lower than 45 °C or lower than 55 °C. Particularly preferably, the temperature of the cold water is 4-10 °C.

[0027] Preferably stirring is continued after the addition of cold water, until complete mixing is achieved (the MC is completely dissolved, a homogeneous solution is obtained). Preferably, after the addition of cold water, the resulting mixture is stirred at low temperature, preferably above 0 °C and below 55 °C, preferably above 0 °C and below 45 °C, preferably above 0 °C and below 25 °C, preferably between 2 and 20 °C, preferably between 4 and 10 °C, particularly preferably at a temperature of about 4 °C, until a homogeneous solution is obtained.

[0028] Preferably, such an amount of MC is dissolved that the viscosity of the final solution (tissue substitute composition) is the same as the viscosity of blood (under physiological conditions). Preferably, the amount of MC is such that 5-8 g, preferably 5.5-7.5 g, preferably 6-7 g, particularly preferably 6.6 g of MC are added to a total of 100 ml of water (warm water + cold water). Preferably, the concentration of MC is 50-80 g / dm3; preferably 55-75 g / dm3, preferably 60-70 g / dm3, particularly preferably 62-68 g / dm3or 66 g / dm3.

[0029] Preferably, the ratio of warm water to cold water is such that 70 ml of cold water is added to 30 ml of warm water.

[0030] Preferably, a contrast agent (radioabsorbing agent) suitable for CTA imaging is added to the tissue substitute composition. Preferably, the amount of contrast agent added is such that the radiodensity of the combination of tissue substitute and contrast agent is at least 250 Hounsfield units (HU).

[0031] Preferably, the added contrast agent is a iodine-containing contrast agent, for example and preferably iopamidol, iomeron, iomerol, preferably a gadolinium-containing contrast agent, for example gadoteric acid (dotarem, dotagraf), gadobutrol, gadoteridol, dotarem, preferably a barium sulfate-containing contrast agent, particularly preferably a solution contrast agent containing 400 mg / ml iodine, particularly preferably Iomeron 400.

[0032] SHORT DESCRIPTION OF THE FIGURES

[0033] Figure 1 Prepared heart

[0034] Figure 2 Fixing the cannula in the blood vessel.. A stitch is made under the blood vessel with a suture and fixed with a knot from the outside, so that it can be easily removed later and neither the vessel nor the plaques are damaged.

[0035] Figure 3 Representative images obtained with the three different CT scanners showing coronary calcification. Helical acquisition mode was used in images A, B and C with slice thicknesses of 0.625 mm, 0.67 mm, 0.4 mm. Ultrahigh resolution helical scanning mode was used in image D with slice thickness of 0.2 mm. Histological processing of ex vivo hearts and coronary plaques.

[0036] Figure 4 Main steps of the preparation, scanning and histological evaluation of ex vivo hearts and coronary plaques. Figure 5 Representative histological and coronary CTA cross-sections of advanced atherosclerotic lesions in ex vivo heart. A: Coronary CTA (CCTA) cross-sectional image and B: corresponding co-registered histological cross-section; showing plaque causing severe lumen narrowing. C-E: Coronary CTA and D-F: Histological cross-sections of two predominantly calcified plaques with different plaque volumes. G: Coronary CTA and H: Histological cross-section of a drug-eluting stent implanted for high-risk plaque. K: Coronary CTA and L: Histological crosssection of the entire myocardial bridge.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] A composition is provided which enables the substitution of a (mammalian) tissue, in particular blood, in an ex vivo test, in particular in an ex vivo CT or other test using X-rays. By adding a contrast agent to the composition, it perfectly represents the image quality and tissue radiation absorption (density) of tests performed in vivo (live).

[0039] The material produced with the methodology developed may be combined with various contrast agents (e.g. iodine, gadolinium, barium) and is easily reproducible, making it useful for other human or animal organs in addition to examining blood vessels.

[0040] During the production of the contrast agent developed, it was found that at 4 °C methylcellulose dimerizes to a lower extent, which results in a less viscous consistency. Therefore, when dissolved in the appropriate proportions, the tissue substitute composition does not enter the microcirculation, while collaterals with small diameters can also be filled with the tissue substitute composition, so that plaques causing significant stenosis may also be visualized on images generated in an imaging process. Due to the physical properties of the composition, it displaces bubbles trapped in the lumen of the blood vessels and fills them completely. When mixed with a contrast agent in an appropriate proportion, its density reaches the minimum value ensuring image quality during in vivo examinations, of 250 HU. After imaging, the tissue substitute composition can be washed out of the blood vessels easily, and does not damage their walls in any way, enabling, for example, further intravascular imaging.

[0041] With the post-mortem methodology developed in conjunction with the tissue substitute composition, we are able to image the entire organ, with image quality comparable to or better than in vivo imaging. The bubble-free filling of the blood vessels, without compression of the shape / structure of the plaque, with a contrast agent with a blood viscosity of at least 250 HU absorption is a significant novelty. In connection with the method, we have developed a carrier medium, which makes the ex vivo organ suitable for transport and approximately represents the radiation absorption of the human chest. After post-mortem imaging, a histological cross-sectional series can provide the gold standard validation.

[0042] The developed composition is closely related to and fits into the methodology of post-mortem coronary CT angiography (CCTA) examinations. The developed tissue substitute composition and the post-mortem coronary CTA methodology enable the optimization of the examination parameters of clinical devices, improving the imaging focusing on the composition of plaques, however, the developed model can also be applied to other clinical imaging devices.

[0043] The matching of histological sections and images taken with the imaging procedure (coregistration) is essential for the validation and development of imaging devices and software, which cannot be achieved without the use of post-mortem examination methodology and tissue substitute composition.

[0044] Histological validation of CT slice images for carotid plaques has already been achieved with the HistoMatch and ElucidVivo (Elucid) software. The main limitation of the above softwares is that they are only suitable for the analysis of carotid plaques. The main reason for this is that carotid plaques are now routinely removed during surgery under anesthesia, so these plaques are easily accessible. The same cannot be done in coronary arteries in a living human body, and ex vivo is almost impossible due to the extremely limiting pathological regulations worldwide. The tissue substitute composition hereby provided and the application of the post-mortem examination methodology can also assist in the development of new imaging tools (e.g. micro- CT, phase contrast CT) for human and animal blood vessels, heart and other organs, and can also be used for the validation of other imaging tools (e.g. cardiac MRI, IVUS, OCT, NIRS). Another advantage of the method is that a post-mortem examination model can be prepared in a short time (approximately 1 hour), thus shortening the ischemic time. The prepared contrast agent can be stored refrigerated.

[0045] According to an aspect of the invention, production of an organ preparation suitable for an ex vivo examination is provided, comprising the following steps: providing an organ ex vivo, inserting a cannula into the organ and fixing it, preferably with sutures, inserting a valve into the cannula and fixing it, filling the container containing the preparation with a lipid-based material whose radiation absorption is identical to that of the tissue surrounding the organ in vivo, optionally fixing the organ to the container, optionally inserting a balloon into the organ, wherein the balloon is equipped with a cannula and a valve, optionally filling the balloon with liquid, preferably bubble-free, or / and then with the tissue substitute composition bubble-free through the valve so that the organ maintains its shape corresponding to the in vivo state, optionally introducing liquid into the organ through the valve, filling the organ with the tissue substitute composition without bubbles through the valve.

[0046] Preferably the organ is a hollow organ. Preferably the organ is an artery, particularly preferably a heart. Particularly preferably the organ is a heart and coronary arteries.

[0047] Preferably, the organ is filled with the fluid and the tissue substitute composition via different valves and / or different cannulas. Preferably, the valve is three-pronged to allow separate introduction of the fluid and the tissue substitute composition into the cannula. Preferably, the cannula is, for example, a dedicated coronary cannula, preferably, for example, an ANDOCOR VC3 - 3 mm, free blunt tip type cannula (According to the ANDOCOR n. v. Cardiac catheters and cannulae catalogue 2021; https: / / www.andocor.com / andocor-cardiac-catheters-cannulae; downloaded 2024. 02. 13.).

[0048] Preferably, the liquid is distilled water or physiological saline.

[0049] Preferably, the lipid-based material is solid at room temperature, preferably below 20°C, preferably below 10°C. Preferably, the lipid-based material is liquid at temperatures above 10°C or above 20°C, preferably above room temperature. Preferably, the lipid-based material changes from solid to liquid state at temperatures between 20-80°C, preferably between 25-80°C, preferably between 30-80°C. Preferably, the lipid-based material is free of bubbles (homogeneous) when poured into the container. Preferably, the lipid-based material is essentially lard, gelatine, coconut fat, margarine or shea butter or any mixture thereof, particularly preferably lard. Preferably, the lipid-based material can be removed from the organ without mechanical damage to the organ.

[0050] Preferably, the container is made of a material that transmits the rays used during an angiographic examination, preferably a CT procedure, thus enabling an angiographic, preferably a CT examination, of the organ placed in the container.

[0051] According to one aspect of the invention, the use of a lipid-based material in the preparation of an organ preparation suitable for ex vivo examination is provided, wherein the lipid-based material is solid at room temperature, preferably below 20°C, preferably below 10°C. Preferably, the lipid-based material is liquid at a temperature above 10°C or above 20°C, preferably above room temperature. Preferably, the lipid-based material changes from a solid to a liquid state at a temperature between 20-80°C, preferably between 25-80°C, preferably between 30-80°C. Preferably, the lipid-based material is free of bubbles (homogeneous) when poured into the container. Preferably, the lipid-based material is essentially lard, gelatin, coconut fat, margarine or shea butter or any mixture thereof, particularly preferably lard. Preferably, the lipid-based material can be removed from the organ without mechanical damage to the organ.

[0052] 1. A few applicable lipid-based materials

[0053] In one aspect of the invention, the invention provides an imaging method using the product or method of the invention.

[0054] EXAMPLES

[0055] Production of the tissue substitute composition

[0056] 1. Cool 140 ml distilled water

[0057] 2. Warm 60 ml of distilled water until it is (just) not yet boiling (to remain free of air bubbles)

[0058] 3. Measure out 13.2 g methylcellulose (A 15 LV or A15 Premium LV or METHOCEL A15 LV or METHOCEL A15 Premium LV)

[0059] 4. Stir the heated water on a magnetic stirrer at medium speed

[0060] 5. Add the methylcellulose to the stirred warm water (warm water promotes the (even) dispersion of the methylcellulose particles)

[0061] 6. Add the cooled water to the methylcellulose+warm water mixture, stir at low temperature (e.g., below room temperature but above 0 °C) until a homogeneous solution is obtained.

[0062] Production of a tissue substitute composition comprising a contrast agent

[0063] 1. Cool 140 ml distilled water (in a refrigerator)

[0064] 2. Heat 60 ml of distilled water in a 250 ml Erlenmeyer flask (almost to boiling point, but do not boil, as boiling will prevent water from being air free)

[0065] 3. Measure 13.2 g methylcellulose (A 15 Premium LV, Dow Chemicals)

[0066] 4. Place the flask containing heated water on a magnetic stirrer 5. Place a magnetic bar into the flask containing the heated water with a forceps

[0067] 6. Start stirring at a medium setting

[0068] 7. Add the methylcellulose carefully to the flask (high temperature enables the dispersion of methylcellulose)

[0069] 8. Add the cooled water to the content of the flask (methylcellulose dissolves at low temperature and dimerizes less)

[0070] 9. Add 7 ml of iopamidol to the content of the flask

[0071] 10. Close the opening of the flask with a film

[0072] 11. Stir the flask for at least 4 h in a cold environment (e.g. by placing the flask in a container containing ice)

[0073] 12. After 4 hours check whether the solution is homogenous

[0074] 13. Remove the magnetic bar with a forceps

[0075] Ex vivo CT scan of the heart

[0076] During ex vivo organ preparation, the coronary arteries were filled with methylcellulose-based iodinated contrast composition using dedicated cannulas. A water-inflated balloon was placed in the left ventricle to maintain the shape of the organ. A lipid-based material was used to stabilize the prepared hearts, which mimics human body fat during CCTA aquisition.

[0077] CT imaging of ex vivo hearts was performed using micro-CT and three different clinical scanners, including a photon counting detector (PCD) CT scanner from Siemens Healthineers and two 128-slice conventional clinical scanners (energy integrating detector (EID-CT)) from General Electric and Philips Healthcare (Figure 3).

[0078] Coronary artery histological sections were prepared and stained within 24 hours after CCTA scanning. Coronary artery specimens were cut into 2 mm long segments and embedded in paraffin for histological staining. To ensure accurate image registration between CT and histology, coronary artery tissue blocks were continuously sliced at 5-10 pm and the distance between each slice and the coronary orifice was recorded. Hematoxylin-eosin (HE) staining was performed.

[0079] For further studies, micro-CT aquisition of coronary tissue blocks was performed. Coronary vessels were recorded and imaged with a Pannoramic-X micro-CT machine (3DHISTECH Kft, Budapest) with a spatial resolution of 20 pm. The main steps are shown in Figure 4.

[0080] The main steps of the work are summarized in Figure 4. Before histopathological processing, the location of each coronary plaque was precisely determined and its distance from the origin of the given artery was measured. The hearts, arteries and all plaques were assigned specific identification numbers.

Claims

CLAIMS1. Ex vivo use of a cellulose ether for the substitution of a tissue, wherein the viscosity of a 2% by weight solution of the cellulose ether measured at 20 °C is 5-35 mPas and wherein the cellulose ether is used in the form of an aqueous solution.

2. The use according to claim 1, wherein the cellulose ether is a methylcellulose with a methoxy content of 20-40%.

3. The use according to claim 1 or 2, wherein the cellulose ether is METHOCEL A15 LV or A15 Premium LV methylcellulose.

4. The use according to any one of the previous claims, wherein the concentration of the cellulose ether in the solution is about 5-8 g cellulose ether / 100 ml.

5. The use according to any one of the previous claims, wherein the tissue is blood.

6. The use according to any one of the previous claims, wherein the cellulose ether is used for the substitution of a tissue in a test using X-ray and wherein a radiation absorbing agent suitable for imaging in a method using X-ray is added to an aqueous solution of the cellulose ether such, that the radiodensity of the solution comprising the radiation absorbing agent corresponds to the in vivo radiodensity of the tissue comprising the same radiocontrast agent.

7. The use according to any one of the previous claims, wherein the cellulose ether is used in angiography, preferably in computer tomography angiography.

8. The use according to any one of the previous claims, wherein the cellulose ether is used in testing coronary arteries.

9. The use according to any one of the previous claims, wherein the cellulose ether is METHOCEL A15 LV or A15 Premium LV methylcellulose in the form of an aqueous solution, wherein the concentration of the aqueous solution corresponds to a solution prepared by the following steps: adding 13.2 g METHOCEL A15 LV or A 15 Premium LV methylcellulose to60 ml distilled water with a temperature of 80-90 °C to ensure the dispesion of the methylcellulose particles, then cooling the obtained mixture to a temperature of about 4-10 °C by addig 160 ml cold distilled water while stirring and stirring until a homogenous solution is obtained.

10. A method for obtaining an organ preparation suitable for ex vivo examination, comprising the following steps: providing an organ ex vivo, introducing a cannula into the organ and fixing it preferably with surgical suture, inserting a valve into the cannula and fixing it, filling the container containing the preparation with a lipid-based material, whose radiation absorbance is the same as that of the tissue surrounding the organ in vivo, and wherein the lipid-based material is solid at room temperature and liquid at temperatures above room temperature, optionally securing the organ to the container, optionally introducing a balloon into the organ, wherein the balloon is equipped with a cannula and valve, optionally filling the balloon with a liquid, preferably without bubbles or / then with an aqueous solution of a cellulose ether defined in any one of claims 1-9 without bubbles via the valve such that the organ preserves its form as in its in vivo state, optionally introducing a liquid into the organ via the valve, filling the organ with an aqueous solution of a cellulose ether defined in any one of claims 1-9 without bubbles via the valve.