Abstract:
The present invention relates to a new method of preparing acellularized, biocompatible, implantation material starting from a biological material. In particular, the present invention relates to a method of preparing biological material for implantation comprising (a) fixation of the biological material with an aldehyde and (b) detoxification/anticalcification of the fixed biological material, wherein the detoxification/anticalcification step makes use of a novel class of substances of formula: 
       NH 2 —(CH 2 ) n —X   (I) 
     wherein
   X is COOH, SO 3 H, PO 3 H 2  and   n is an integer from 2 to 6.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a multistep, synergistic method of preparing acellularized, biocompatible implantation material starting from a biological material. 
       BACKGROUND OF THE INVENTION 
       [0002]    Biological materials of various origin to replace diseased or malfunctioning tissues have been in use for more than forty years. Such materials can be obtained from the patient (autologous tissue), from other individuals belonging to the same species (homologous tissue) or from other species (heterologous tissue). The source of the materials can be heart valves, pericardium, tendons, arteries, veins, dura mater, ligaments and bones. 
         [0003]    Since all tissues not obtained from the patient are subject to rejection, it was found necessary, from the very beginning, to develop chemical treatments capable of reducing the antigenic properties of these tissues and at the same time improving their mechanical properties and resistance to enzymatic degradation. 
         [0004]    The most commonly used substance for the purpose of fixing biological tissues is glutaraldehyde. Its success is most certainly due to its high crosslinking capability and to its mix of hydrophilic and hydrophobic properties, which allow the molecule to rapidly diffuse into the matrix. It can also mask antigenic determinants and thus suppress the immunological recognition of the tissues. Glutaraldehyde is also a powerful sterilizing agent. No other single compound exhibits such a range of useful properties. 
         [0005]    Nevertheless, the use of glutaraldehyde to fix biological tissues does induce several adverse effects, such as cytotoxicity and mineralization. Many authors have considered the presence of unreacted aldehyde groups as the main cause of cytotoxicity and of the development of inflammatory reactions. Aldehyde groups are also one of the contributory causes of tissue calcification. 
         [0006]    In order to detoxify biological materials treated with glutaraldehyde, molecules of various kind have been used. These detoxification methods generally rely on the formation of Schiff bases or imino groups between residual aldehyde groups and amino-containing substances. Thus, Girardot, M. N., et al., “Alpha-Aminoleic Acid, A New Compound Prevents Calcification of Bioprostheticheart Valves”, The 17th Annual Meeting of the Society for Biomaterials, May 1-5, 1991, p. 1141, describes the use of 2-aminooleic acid for this purpose. 
         [0007]    Other detoxification anticalcification approaches include the use of chondroitin sulfate, protamine, aminodifosfonates and chitosans. 
         [0008]    Especially effective methods for detoxifying biological materials treated with glutaraldehyde are based on use of aminodicarboxylic acids, e.g. glutamic acid, as disclosed in the U.S. Pat. No. 5,188,834 and aminocarboxylic acid, e.g. homocysteic acid, as disclosed in U.S. Pat. No. 5,873,812. 
         [0009]    The object of the present invention is to provide an alternative and more efficient method of preparing acellularized, biocompatible implantable materials. 
       SUMMARY OF THE INVENTION 
       [0010]    According to the present invention, that object is achieved thanks to a method having the characteristics referred to specifically in the ensuing claims. In particular, the object of the present invention is achieved by the use of a novel class of substances for the detoxification/anticalcification of biological tissues. These substances are, in fact, more reactive than the known substances used in the past and are more effective in penetrating biological structures and reduce disruption of the tissues. 
         [0011]    The claims form an integral part of the disclosure of the invention provided herein. 
     
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]    The invention will now be described, purely by way of non-limiting example, with reference to the annexed figures. 
           [0013]      FIG. 1  is a SEM image showing the pericardium after mechanical cleaning (step A). 
           [0014]      FIG. 2  shows a picture taken with ESEM of pericardium after treatment with glutaraldehyde followed by 72 hours of storage in paraben. 
           [0015]      FIG. 3  is a graph showing data obtained with the LDH test on bovine pericardium seeded with human fibroblasts from embryo pulmonary tissue (MRC5 line). 
           [0016]      FIG. 4  is a graph showing the results of Neutral Red Assay on bovine pericardium seeded with human fibroblasts from embryo pulmonary tissue (MRC5 line). 
           [0017]      FIG. 5  is a graph showing measurement of alkaline phosphatase (ALP) activity on bovine pericardium seeded with human fibroblasts from embryo pulmonary tissue (MRC5 line). 
           [0018]      FIG. 6  is a SEM image of bovine pericardium after treatment with the method of the present invention seeded with human fibroblasts from embryo pulmonary tissue (MRC5 line) taken 72 hours after seeding. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0019]    All patents, published applications and other publications and references cited herein are hereby incorporated by reference in their entirety into the present disclosure. 
         [0020]    The present invention makes use of a novel class of substances for the detoxification anticalcification of biological tissues. It was found that compounds having the general formula: 
         [0000]      NH 2 —(CH 2 ) n —X  (I) 
         [0000]    wherein 
       X is COOH, SO 3 H or PO 3 H 2  and 
       [0021]    n is an integer from 2 to 6
 
are very effective for neutralizing excess aldehyde groups and at same time in preventing tissue calcification.
 
         [0022]    Compounds represented by the general formula (I)—useful according to the present invention—are:
   3-aminopropanoic acid also known as β-alanine;   4-aminobutanoic acid also known as γ-aminobutyric acid (GABA);   5-aminopentanoic acid;   6-aminohexanoic acid;   2-aminoethanesulfonic acid, also known as taurine,   3-aminopropane-1-sulfonic acid;   4-aminobutane-1-sulfonic acid;   5-aminopentane-1-sulfonic acid   6-aminohexane-1-sulfonic acid;   2-amino ethanephosphonic acid;   3-aminopropane-1-phosphonic acid;   4-aminobutane-1-phosphonic acid;   5-aminopentane-1-phosphonic acid;   6-aminohexane-1-phosphonic acid.   
 
         [0037]    Among these compounds, especially effective were found γ-aminobutyric acid (GABA) and 3-aminopropane-1-sulfonic acid. 
         [0038]    The main difference between such compounds and those described in U.S. Pat. No. 5,188,834 and U.S. Pat. No. 5,873,812 is the absence of the carboxyl group bound to the same carbon atom as the amino functionality. The basicity and nucleophilicity and hence the reactivity of primary amines in α-amino acids are reduced by the inductive effect caused by the carboxyl group in close proximity. Thus, the pK 5  of glycine is 9.60, compared to 10.65 of a primary amine, such as metilamine; pK 2  for glutamic acid is 9.76 vs. 10.56 for γ-aminobutyric. 
         [0039]    Moreover, compounds described by the general formula (I) have no net charge in a wide pH interval, and especially at physiological pH, while α-amino acids with acidic side chains as those previously described, and in particular glutamic acid and homocysteic acid have an excess of negative charge at pH values higher than about pH 4.2 and 2.2, respectively. Neutral species are more effective in penetrating biological structures and reduce disruption of the tissues. 
         [0040]    Additionally, compounds such as γ-aminobutyric are much more water soluble than glutamic acid and, therefore, the solution prepared with it are more concentrated and thus more effective than those containing glutamic acid as detoxifier/anticalcifier. 
         [0041]    Treatment with the compounds described by formula (I) is carried out with acidic aqueous solution having pH within the range from 3.0 to 5.0 and, preferably, between 3.0 and 3.5. The reaction medium is a buffered solution, preferably belonging to the group of sodium citrate/HCl, potassium hydrogen phthalate/HCl, citric acid/phosphate, and citrate-phosphate-borate/HCL. The concentration of the acid solutions is generally within the range from 10 mmM to saturated solutions, more preferably from 25 to 100 mM. 
         [0042]    In order to fully exploit the advantages of the novel reagents, it was found useful to develop a complete protocol, including steps before the fixation with glutaraldehyde and post-treatment steps after the detoxification anticalcification procedure. More specifically, according to the present invention the biological material is subjected to a carefully designed sequence of treatments, namely cleaning of the sample, osmotic shock, extraction of cellular debris with detergents, fixation with an aldehyde, detoxification anticalcification with an α, ω amino acid, reconditioning and storage. 
         [0043]    Thus, a complete treatment of biological material for implantation consists of the following steps, or procedures (a) thorough mechanical cleaning of the tissue; (b) osmotic shock; (c) decellularization; (d) fixation; (e) detoxification/anticalcification; (f) reconditioning and storage in buffer. 
         [0044]    Step (a) requires careful removal of connective and fat tissue, while keeping the sample at 0° C. 
         [0045]    Next, the tissue undergoes one or more treatments aimed at removing the cellular components. As is known to the art, it may be preferable to first subject the tissue to osmotic shock (b), by alternatively treating it with distilled water and saline buffers. This procedure destroys the integrity of cellular walls and removes part of the cellular debris. 
         [0046]    The remaining of this debris is more thoroughly removed by employing washing steps with detergents (c). Again, it is well known to the art the use of ionic detergents, such as sodium dodecyl sulfate (SDS), non-ionic detergents, such as Triton X-100, zwitterionic detergents, sodium deoxycholate. Mixtures of the aforesaid detergents are also often used, to increase their effectiveness. Additionally, enzymes such as DNAses and RNAses, as well as common chelators, such as EDTA are added to the detergent buffer. Certain solvent of medium polarity can also be used, alone or together with detergent to remove said cellular debris, especially acetone, lower alcohols, chlorinated hydrocarbons, such as dichloromethane, chloroform, alone or in mixtures. 
         [0047]    After rinsing off the excess detergent with a buffer solution, the sample is subjected to the fixation procedure (d), which is generally accomplished by immersing the sample in a dilute solution of glutaraldehyde, for several hours, up to a few days. 
         [0048]    The fixation is followed by detoxification/anticalcification (e) with the compounds of formula (I), and especially with γ-aminobutyric acid (GABA) and 3-aminopropane-1-sulfonic acid. 
         [0049]    It is advantageous to first treat the fixed tissues with a pH 3.3 citrate buffer, to help depolymerization of glutaraldehyde oligomers formed under the experimental condition. These polymerization reactions are, in fact, known to be reversible at lower pH values. As a results, masked aldehyde groups become exposed and can react with the amino groups of detoxifying agent in the next step. If prewashing with acidic buffer is not performed at this point, aldehyde groups masked by the aldolic self-condensation of glutaraldehyde would be i) slowly released spontaneously and ii) eventually responsible for a delayed increase in cytotoxicity of the implanted tissue. 
         [0050]    For similar reasons, the detoxification/anticalcification step with γ-aminobutyric acid (GABA) and 3-aminopropane-1-sulfonic acid, or other compounds described by the general formula (I), is performed at acidic pH and, in particular, at pH 3.3. 
         [0051]    The final step (f) requires reconditioning the sample at neutral pH and storage in a biologically compatible sterilizing solution, such as a paraben solution (0.02% n-propyl p-hydroxybenzoate and 0.18% methyl p-hydroxybenzoate). 
         [0052]    The following example illustrates the invention in a detailed manner. 
       EXAMPLE 
       [0053]    STEP A. Immediately after the explant, bovine pericardium from the local abattoir was placed in sterile physiological solution (0.9% NaCl) kept at 4° C. 
         [0054]    A sample (12×15 cm) was carefully cleaned by removal of connective tissue and fat residues, while working at 0° C. 
         [0055]    STEP B. Subsequently, the sample was subjected to osmotic shock, by washing it alternatively with distilled water and saline solution (with ice) at least five times. It is important to thoroughly rinse the sample and then store it for at least 20 minutes in distilled water, before the next immersion in saline solution. The purpose of this treatment is to break cellular walls and then remove cellular components as much as possible. 
         [0056]    STEP C. The sample is then washed with a detergent solution formulated as follows: 1% Triton X-100, 0.25 M sodium deoxycholate, 0.02% EDTA, 0.1% DNAase and RNAase under continuous stirring for 48 hours at 37° C. After the first 24 hours the detergent solution was replaced with fresh solution. The sample washed thoroughly (at least 20 minutes) with phosphate saline buffer (PBS) three times. 
         [0057]    STEP D. The sample was then fixed with 0.5% glutaraldehyde in pH 7.4 PBS buffer at 0° C. for 24 hours. At the end of this period the sample was subjected to two, 20 minutes washes with a pH 3.3, 0.16 M citrate buffer to remove glutaraldehyde in excess. 
         [0058]    STEP E. A detoxification/anticalcification step was performed by treating the sample, for a total of three times, with a 60 mM solution of γ-aminobutyric acid (GABA) in pH 3.3, 0.1 M citrate buffer. This first treatment lasted 24 hr, the second 36 hours, and the third, 24 hours. The pH of the solution was checked, and if necessary, adjusted to pH 3.3 with HCl or NaOH. 
         [0059]    STEP F. The final step, was performed by reconditioning the sample by rinsing three times for 20 minutes with saline solution (0.9% NaCl in water). It is important that the sample does not contain the amino acid employed in the detoxification/anticalcification step. It was stored in a paraben solution (0.02% n-propyl p-hydroxybenzoate and 0.18% methyl p-hydroxybenzoate). 
         [0060]    After each step the sample was observed by electron microscopy (SEM and ESEM). 
         [0061]      FIG. 1  (SEM) shows the sample after step A, mechanical cleaning. The sample surface shows the presence of cellular components. Collagen fibers on the surface are well covered with fat layers. 
         [0062]      FIG. 2  shows pictures taken with ESEM, in Low-Vacuum mode, after 72 hours of storage in paraben. The collagen structure appears to have been modified by the glutaraldehyde treatment but anatomically within the norm. 
         [0063]    Cytotoxicity and cytolysis were evaluated by lactate dehydrogenase (LDH) and neutral red assays. 
         [0064]    LDH is a stable cytoplasmatic enzyme found in most cells. It is released when the cytoplasmatic membrane is damaged. The assay is based on the conversion of yellow tetetrazolium salts into a red formazan dye (absorbance maximum at 500 nm). By measuring the activity of LDH released by damaged or dead cells it is possible to evaluate any cytotoxic or cytolytic effect due to the material onto which test cell are grown. Any increase in the number of damaged/dead cell is followed by a correspondent increase in LDH activity and is directly proportional to the amount of formazan produced. In a quantitative form, it can be stated as follows (LDH Cytotoxicity Detection Kit Manual, TAKARA Cat. MK 401): 
         [0000]      Cytotoxicity%=[(espectedvalue−control/(low))/(control(high)−control/(low))]×100  (II) 
         [0065]    Neutral red assay is a way of measuring the number of viable cells by absorption of the dye into the cells. Live cells absorb this dye and incorporate it into their liposomes. An increase/decrease in the number of cells and their physiological well-being correspond to an increase/decrease in the amount of neutral red incorporated by culture cells. The method was implemented by means of a SIGMA kit (No. TOX-4). Best results are obtained when cells are in logarithmic growth and at concentrations less than or equal to 10 6  cell/ml. 
         [0066]    A sample of bovine pericardium, treated as in the example, was seeded with human fibroblasts from embryo pulmonary tissue (MRC5 line). Data obtained with the LDH test, reported in  FIG. 3 , show complete lack of cytotoxicity. The control is polystyrene (empty well). Expected value for pericardium (72 hours after seeding) is 1.66±0.36. Expected value for control is 1.98±0.20.  FIG. 4  shows the results of neutral red assay. Cells seeded on pericardium after 72 hours are 1.66E+05, cells seeded on control 72 hours after seeding are 3.06+05. 
         [0067]    Since, in the absence of cytotoxicity, a decrease in the tendency to proliferate arises in the presence of an increased cellular activity, the samples were also tested for cellular activity. 
         [0068]    A test based on the quantitative measurement of alkaline phosphatase (ALP) was used. This enzyme, widely distributed in tissues, hydrolyses p-nitrophenol phosphate (colorless) to p-nitrophenol red at basic pH. The absorbance maximum of p-nitrophenol is at 405 nm. Therefore the absorbance increase at 405 nm is directly proportional to ALP activity. A Sigma kit (No. 245) was used for this test. Results are reported in  FIG. 5 . Cellular activity is twice as high for cells seeded on pericardium than for those seeded on polystyrene (control), thus compensating the decrease in proliferation. 
         [0069]    These results are consistent with SEM images taken 72 hours after seeding with MRC5 cells,  FIG. 6 . Good adhesion of the seeded cells to pericardium substrate is evident in the picture, numerous cells are adherent to the substrate and cell interconnections can also be observed. 
         [0070]    Those of skill in the art will promptly appreciate that all the numerical values provided herein are to be understood by taking into account the tolerances currently associated with determining/measuring such values. 
         [0071]    Of course, without prejudice to the principle of the invention, the details of fabrication and the embodiments may vary widely with respect to what is described and illustrated herein, without thereby departing from the scope of the present invention, as defined by the annexed claims.