Tissue engineering, for example whole organ engineering, could help to address the problems discussed in the background to the invention, since the tissue used is biological and there is no rejection potential. Additionally, there is the potential of tissue regeneration and remodeling. In order to carry out tissue engineering the classical triangle is needed: a scaffold, a large number of different autologous cells and a bioreactor.
U.S. Pat. No. 7,438,850 describes a four-step sterilization method for the production of implantable or transplantable biological material of animal or human origin.
It is an object of this invention to provide an improved method of detoxification and stabilization of implantable or transplantable biological material of human or animal origin.
This invention relates to method of detoxification and stabilization of implantable or transplantable biological material of human or animal origin, the method including the following steps:
The solution containing an organic acid surfactant bile acid (secondary) preferably contains:
The antibiotic solution may contain at least one, preferably all of the following antibiotics:
Preferably, the antibiotic solution contains no Ca or Mg.
The solution to remove the organic acid surfactant bile acid (secondary) contains a lipopeptide anticmicrobial agent such as Fengycin or Iturin A in an amount of 5-800, preferably 20 μg/ml.
After step 4), the material is introduced to a storage solution containing antibiotics, preferably selected from one or all of the following antibiotics:
The invention also relates to the solution containing an organic acid surfactant bile acid (secondary) for use in a method of detoxification and stabilization of implantable or transplantable biological material of human or animal origin, said solution containing:
In accordance with the method of the present invention, tissue of human or animal origin is treated in four successive steps:
The solution of Step 2) is a physiological solution containing an organic acid surfactant bile acid (secondary), contains a triple combination of:
Deoxycholic acid (DOA), is a bile acid (secondary) an organic surfactant. Alternatively a derivative thereof such as ursodeoxycholic acid can also be used. It also takes care of the lipid parts of the membrane, not only the protein part. Additionally it has an anti-inflammatory activity which is also important in the production of an extracellular matrix.
The preferred anionic surfactant is sodium dodecyl sulfate (SDS), alternative anionic surfactants are ammonium dodecyl sulfate and potassium lauryl sulfate, which are of the same group, however a little different. These are anionic surfactant (anorganic) or detergent surfactant which denaturate proteins, but also microbicide including enveloped and non-enveloped viruses will be destroyed.
The preferred non-ionic surfactant is Triton-x100 (a polyoxyethylene surfactant) which will not denaturate proteins but results in membrane distortion to prepare the tissue for sodium dodecyl sulfate and deoxycholic acid to destroy the tissue (denaturate).
This triple combination has a synergistic effect: i.e. the combination of all three components allows the concentration of the individual components to be reduced. A lower concentration of components means that, in use, there is less chance that the stability of the extracellular matrix/scaffold will be changed during the detoxification and stabilization of the scaffold.
After step 4), the tissue is rinsed to remove all debridement out of the extracellular matrix. Thereafter, the material is introduced to a storage solution containing antibiotics, preferably selected from one or all of the following antibiotics:
The method may be used in the preparation of a heart, heart valves, grafts, patch material etc. and also other organs or tissue such as omentum.
The invention is described in more detail with reference to the following Examples. The invention is not restricted to these Examples.
An Example of the invention is the preparation of a large size heart, which was decellularized to create a scaffold on which autologous cells can be transplanted and later on implanted.
Technique to modify tissue by detoxification and stabilization:
Step 1)—treatment of the tissue with an antibiotic solution
The antibiotic solution without Ca or Mg contains a cocktail of antibiotics and antimycotic medication with flow or without flow at a shaker for several hours and at room temperature or at 37° C.
Thereafter the tissue is treated with distilled or purified water also for a particular time 15 minutes to 1 hour at room temperature or 37° C.
Step 2)—treatment of the tissue in a solution containing an organic acid surfactant bile acid (secondary)
The tissue is treated with a combination of deoxycholic acid (DOA) (0.5% v/v), sodium dodecyl sulfate (SDS) (2% v/v), and Triton x-100 (0.5% v/v). These substances have been used in the past, however never all together since there is a synergic effect in case using them together. Therefore the concentration can be lower and there is less chance that the stability of the extracellular matrix/scaffold can be changed. The collagen can be destroyed and deterioration can be increased due to this. This step is carried out at a particular temperature (room or 37° C.) for several hours or days (long).
Step 3)—treatment of the tissue in a solution to remove the organic acid surfactant bile acid (secondary)
The tissue is treated with Fengycin 100 μg/ml in DMSO 5 mmol to sterilize and to remove the DOA/SDS and Triton out of the tissue. It is also possible to use Iturin A for several hours. These are lipopeptide antimicrobial agents that are also antifungal. It will stabilize the tissue more. Concentrations can be changed, depending on the time. Temperature can also be different (room temperature or 37° C. is optimal). A shaker or flow can be used.
Step 4)—treatment of the tissue in a primary alcohol
Ethanol or another alcohol should be used to stabilize the tissue but will also sterilize the tissue. Again with or without shaker or under flow conditions for different time (several hours and at different temperature (room temperature or 37° C. is optimal).
Extensive rising of the tissue to get all the debridement out of the extracellular matrix. It would also be possible to control this by measurement to minimize the debridement at a minimum on the end.
Final step is storage with a specific store solution:
The process of the invention described above was carried out on different tissues and comparative tests were conducted using a single solution containing either deoxycholic acid, or sodium dodecyl sulfate, or Triton-X100, and a double solution containing either deoxycholic acid with sodium dodecyl sulfate, or deoxycholic acid with Triton-X100, or sodium dodecyl sulfate with Triton-X100. Only in the triple deoxycholic acid solution of the invention containing an organic acid surfactant bile acid (secondary); an anionic surfactant, and a non-ionic surfactant results in a cell free tissue without destroying the extracellular structures.
Number | Date | Country | Kind |
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2015/00478 | Jan 2015 | ZA | national |
Filing Document | Filing Date | Country | Kind |
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PCT/IB2016/050320 | 1/22/2016 | WO | 00 |