• 专利附录
  • 专利说明
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    • 专利摘要:
    The invention provides an improved method of producing a natural, acellular matrix scaffold for subsequent use in tissue-engineered replacement of tissues such as the 5 bladder. Decellularisation is carried out on an expanded or distended bladder and the product retains the strength and compliance of natural material, The invention also provides use of the matrix scaffolds as wound healing material and to investigate tissue structure and function in vitro.
    公开号:AU2013200822A1
    申请号:U2013200822
    申请日:2013-02-14
    公开日:2013-03-07
    发明作者:Fiona Bolland；Elleen Ingham；Sotirios Korossis；Jennifer Southgate
    申请人:TISSUE REGENIX Ltd；
    IPC主号:A61L27-36
    专利说明:
    1 IMPROVEMENTS RELATING TO DECELLULARISATION OF TISSUE MATRICES FOR BLADDER IMPLANTATION 5 The present invention relates to a method of producing a natural, acellular matrix scaffold for subsequent use in tissue-engineered replacement of tissue. The invention provides a biomaterial that is suitable for tissue replacement and/or repair particularly but not exclusively in the fields of gastroenterology, urology, cosmetic surgery, wound repair and reconstructive surgery. The invention also provides use of the matrix 10 scaffolds to investigate tissue structure and function in vitro. BACKGROUND At present, natural biomaterials are predominantly used in reconstructive surgery, mainly 15 in the fields of gastroenteroogy, urology and wound-healing, but with increasing applications in cardiovascular and cosmetic surgery. One such biomaterial, porcine small intestinal submucosa (SIS), has been used in gastroenterology, urology and wound-healing applications as it is easily incorporated into host tissue and remodelled. However, recent work to investigate the adherence and viability of human cells seeded 20 onto SIS demonstrated that commercially-available SS specimens contained porcine nuclear residues and was cytotoxic in vitro. In addition, clinical use of SIS has resulted in localised inflammation, suggesting the material can cause an immunological response in vivo. An alternative natural biomaterial is decellularised porcine dermis (PermacolTM) which has a wide range of uses in medical and cosmetic procedures and has been 25 implanted in over 8500 patients in over 70 different surgical procedures since being licensed for use in humans in 1998. However, PermacolTM a disadvantage associated with this material is that not only is it only partially resorbable but when it is used in an animal model of bladder augmentation it has been shown to cause micro-calcification and irregular detrusor regeneration. Furthermore, PermacolTM is unable to support in 30 vitro recellularisation thus preventing its use as a biomaterial that could be seeded with cells and functionalised prior to implantation. An improved natural biomaterial that is immunologically inert and able to support recellularisation would offer an immediate advantage in the art. 35 It is estimated that over 400 million people worldwide suffer from some form of bladder dysfunction. A variety of diverse congenital and acquired conditions result in bladder 2 dysfunction, for example cancer, congenital abnormalities, nerve damage or trauma. Currently, the major surgical solution is surgical reconstruction. It is known in the prior art to repair or augment or replace the bladder during these procedures with vascularised segments of the patient's own tissue derived from their stomach or more 5 commonly their intestine. However, this latter procedure ('enterocystoplasty') is associated with significant clinical complications that arise due to the exposure of the epithelial lining of the intestine to urine. it has been found that the use of intestine results in significant complications, such as infection and development of bladder stones, as the intestine is lined by an absorptive and mucus-secreting epithelium that is 10 incompatible with long-term exposure to urine. Consequently, a number of alternative approaches have been proposed to find a practical and functional substitute for native bladder tissue. One of the alternative solutions is 'composite enterocystoplasty', where the de-epithelialized intestine wall is lined with bladder epithelial cells that have been propagated in vitro, to augmenting the urinary system with natural or synthetic 15 biomaterials that may incorporate in vitro-propagated cells. However even this modified form of enterocystoplasty has been associated with adverse side effects. The lack of an entirely satisfactory clinical procedure has led researchers to investigate alternative strategies. 20 Attempts have been made to develop suitable biomaterials for urological tissue engineering using both synthetic materials, for example polyglycolic acid and poly L lactic acid, and naturally derived materials including SIS, PermacolTM and porcine bladder matrix. However none of these materials have been found to be totally successful vis a vis immunogenicity and rejection and recellularisation. 25 Tissue matrices prior to implantation undergo a process of decellularisation in order to reduce their immunogenicity once implanted. This process involves removing the donor cells, whilst ideally retaining the biomechanical structure and function of the matrix. As regards the bladder, it is especially desirable to maintain the normal mechanical 30 properties and its elasticity. A problem associated with the use of a bladder matrix is that bladder tissue is relatively thick (1-5 mm when not distended). This in turn means that it is difficult to decellularise bladder tissue to provide an immunologically inert scaffold matrix using routine methods 35 known in the art. Attempts to decellularise dissected segments of full thickness porcine 3 bladder have resulted in incomplete decellularisation. Histological analysis of these bladder have resulted in incomplete decellularisation. Histological analysis of these samples indicated that cells had not been removed from the muscular bladder wall. In its retrieved form, the porcine bladder was too thick to allow efficient penetration of the 5 solutions used throughout the decellularisation process. In order to overcome this problem segments of bladder tissue of reduced thickness could be used to enable successful decellularisation. An improved method of decellularisation that could be used to decellularise whole 10 bladders or other membranous sacs of full thickness, whilst retaining the biomechanical properties of the tissue, would offer immediate advantage in the field of particularly but not exclusively, urological tissue engineering. BRIEF SUMMARY OF THE DISCLOSURE 15 According to a first aspect of the invention there is provided a method of decellularisation of a tissue comprising a distensible membranous sac, the method comprising: (i) immersing the distensible membranous sac in a buffer solution at a mild alkaline pH which includes active amounts of a proteolytic inhibitor; (ii) distending the distensible membranous sac by introducing a sufficient 20 volume of the same buffer solution into the interior cavity of the sac and; (iii) continuing decellularisation of the sac by replacing and introducing fresh solutions both around the exterior surface of the sac and into the sac interior itself so as to maintain distension of the sac during decellularisation. 25 It will be appreciated that step (ii) may be performed prior to step (i) and that the essence of these two steps is to ensure that both the inside and outside of the distensible membranous sac is in contact with the buffer for effective decellularisation to take place. Whether the sac is fluid filled with the buffer and then immersed in the buffer 30 or whether it is immersed in the buffer and then filled with the buffer is not material to the method providing that the sac is in contact with the buffer on both its inner and outer membranous surfaces and that it remains in a distended form whilst decellularisation occurs. 35 It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.
    4 Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. 5 Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. 10 Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. 15 Reference herein to a distensible membranous sac is intended to include a tissue or sac or balloon of tissue that has an elastic or flexible membrane. That is to say tissue when in vivo has the ability to expand and to return to a normal resting state without rupture or disruption of its mechanical properties. 20 Reference herein to decellularisation is intended to include the removal of cellular membranes, nucleic acids, lipids, cytosolic components and retaining an ECM having as major components collagens and elastins. 25 Preferably, the distensible membranous sac is clamped before and after each step of fluid replacement so as to prevent fluid loss and to maintain distension of the sac. Preferably, the fluid volume added to the interior cavity of the sac at each step is in the region of 250-750 mi and more preferably in the region of 350-650 ml and more 30 preferably still is about 500 ml. The volume selected is dependent on the size of the distensible sac and may vary accordingly, the volume introduced into the sac is sufficient to maintain distension throughout the decellularisation process. Preferably, the fluid volume around the exterior of the sac is sufficient to cover the whole 35 of the distended fluid filled sac. It is convenient whilst refreshing the fluid inside the sac to simultaneously replace the fluid surrounding the exterior of the sac.
    5 Once the process of decellularisation is complete the distensible sac may be dissected to form a flattened sheet or it may remain intact as a sac depending on its intended subsequent use. 5 The bladder is an extremely compliant organ and is able to expand to more than 15 times its contracted volume, it this property that has been exploited in the method of the present invention. By distending the intact bladder to stretch and thin the bladder wall, solutions were able to permeate throughout the thinned wall, resulting in complete decellularisation whilst surprisingly retaining properties of native bladder tissue. 10 Preferably, the distensible membranous sac is a whole bladder. Preferably, the distensible membranous sac is derived from a pig. 15 However, it will be appreciated that the method of the present invention is equally applicable a membranous sac derived from a human or any other animal that has physical parameters, for example size, that are compatible with a human bladder. Thus the method of the invention may be used to decellularise donor human bladders. It may also be used to, for example, replace the bladder from another animal such as a cat with 20 a membranous sac derived from a cat or another species which has approximately the same size as a cat's bladder. The interspecies cross is not intended to limit the scope of the invention. Porcine whole bladder is the particularly preferred tissue for use in the method of the 25 present invention since not only is there a ready supply but its size, physical and mechanical characteristics are similar to humans which makes it compatible as a human bladder tissue replacement. Preferably, the method of decellularisation of a tissue comprising a distensible 30 membranous sac, comprises: (i) distending the distensible membranous sac by introducing a sufficient volume of a buffer solution at a mild alkaline pH which includes active amounts of a proteolytic inhibitor into the sac whilst simultaneously immersing the sac in the same buffer solution; 35 (ii) removing said buffer from both the interior cavity of the sac and the surrounding exterior area and replacing it with an anionic detergent at a 6 mild alkaline pH at a concentration which is sufficient to effect decellularisation but which maintains the histoarchitecture of the biological material ; (iii) removing said detergent from the interior of the sac and its exterior and 5 replacing it with a washing buffer solution at a mild alkaline pH both with and without active amounts of proteolytic inhibitors so as to wash both the interior and exterior surfaces of the distensible membranous sac; (iv) removing said washing buffer from both the interior of the sac and its exterior and replacing it with a solution comprising one or more enzymes 10 selected from the group comprising DNase Type 1, DNase Type 11, and/or Rnase and optionally; (v) removing the solution comprising one or more enzymes from the interior of the sac and its exterior and optionally placing the biological material in a cryoprotectant medium or storage medium or other suitable protective 15 medium for later use. Preferably, the method further includes the step of modifying the decellularised matrix with a suitable agent to enhance immunoacceptability of the matrix on implantation. For example and without limitation, the tissue matrix may be treated enzymatically with a 20 galactosidase or a glycosidase digestion to remove a-gal epitopes (Gal al-3 Gal p1 4Glc NAC-R). Alternatively, it may be treated chemically with cross-linking agents such as glutaraldehyde or other aliphatic and aromatic diamine compounds that provide additional cross-linking through side chain groups of aspartic and glutamic acid residues of the collagen peptide. It will be appreciated that any suitable agent that is capable of 25 enhancing immunoacceptability and thus reducing the likelihood of post implantation rejection and/or inflammation will be applicable for use in the method of the present invention. Preferably, the method further includes the step of recellularisation. Recellularisation 30 can be either in vitro or in vivo and may be enhanced by a suitable agent appropriately administered either in vitro or in vivo. The agent may also be coated directly onto the tissue matrix prior to implantation to encourage recellularisation. As regards the solutions and concentrations of components in the method of the 35 invention, preferably the buffer solution is hypotonic or isotonic. It will be appreciated that each may be used either as the sole buffer or in combination at different stages of 7 the method and that use of hypotonic or isotonic buffer is not intended to limit the scope of the present application. Preferably, the proteolytic inhibitors are ethylene diamine tetraacetic acid (EDTA) and 5 Aprotinin. We have found Aprotinin particularly effective as a proteolytic inhibitor and of particular utility because of its low toxicity, stability in solution at different pHs and stability at a variety of different temperatures. Typically, EDTA is used at a concentration range of 1 to 100 mM or 0.01-1.0 % (w/v) and 10 typically at 10mM or 0.1% and Aprotinin at a concentration range of 1-100 KlU and typically at 1OKIU. Preferably, the mild alkaline conditions of step (i) are in the range of pH above 7.0 and up to pH 10.0, and more preferably are at pH 8.0. 15 Preferably, the incubation period of step (i) of the method is for between 8 to 24 hours and more preferably is for 14 hours. Preferably, the anionic detergent is sodium dodecyl sulphate (SDS) or sodium 20 deoxycholate. Preferably, SDS is used at a concentration equal to or below 0.1% (w/v), and equal to or above 0.03% (w/v). 25 Reference herein to the term % (w/v) refers to the percentage in weight (grams) per unit volume (100 mi), thus 0.1% w/v is equivalent to 0.1 gm dissolved in 100 ml. Preferably, sodium deoxycholate is used at a concentration equal to or below 2.0% (w/v) and equal to or above 0.5% (w/v). 30 Preferably, the incubation period of step (ii) of the method is for between 20 to 28 hours and more preferably is for 24 hours. Preferably, the mild alkaline conditions of step (ii) are in the range of pH above 7.0 and 35 up to pH 10.0, and more preferably are at pH 8.0.
    8 Preferably, the washing step (iii) of the method involves multiple washes, typically X3 washes with physiological buffered saline (preferably phosphate buffered saline PBS, 0.01M phosphate buffer, 0.137M NaCl) containing protease inhibitors (0.1% EDTA and 10KIU/ml Aprotinin), and further, multiple washes, typically X3 washes with physiological 5 buffered saline without the protease inhibitors. Preferably, the mild alkaline conditions of step (iii) are in the range of pH above 7.0 and up to pH 10.0, and more preferably are at pH 8.0. 10 Preferably, the incubation step (iv) of the method is for 4-6 hours at temperature range of between 200C to 45 OC and preferably at 370C. The DNase Type 1, DNase Type 11 or Rnase are employed in an amount effective so as to eliminate nucleic acids and provide a tissue matrix of limited calcification potential. 15 Accordingly any other agents which are capable of the same function are included within the scope of the present invention. Preferably, DNAse I is used at a concentration range of 5.0-100 igIml and typically at 20l-tg/ml and RNAse A at a concentration range of 0.1-10 tg/ml and typically at 1 1 g/ml. 20 The decellularisation process includes osmotic lysis of the cells, solubilisation of cell fragments using SDS, protease inhibitors to inhibit autolysis and nucleases to digest nuclear materials. All stages of the process are carried out using a whole, full thickness distensible sac that is substantially distended during all steps so that its walls are 25 stretched and thinned so allow solutions to permeate throughout the thinned wail, resulting in complete decellularisation thereof. The method of the present invention provides a process whereby intact whole porcine bladders are distended in a series of decellularisation solutions such that 30 decellularisation of the full thickness bladder wall is achieved. Moreover, the resultant biomaterial retained the physical and structural features of the native bladder tissue and contained no residual cell or nuclear bodies. Results have shown that the underlying bladder histoarchitecture was retained and that the biomaterial produced by the method of the invention when compared to fresh porcine bladder tissue showed no differences in 35 the overall compliance; ultimate tensile strength or ability of the decellularised matrix to retain sutures under force. Furthermore, the bladder biomaterial was biocompatible with 9 cells, as homologous smooth muscle cells were able to repopulate the matrix after 21 days in culture. Our results have shown there was no significant difference in the ultimate tensile 5 strength of the distensible sac following decellularisation and in terms of its potential applications in vivo, there was no significant difference in the ability of the decellularised matrix to retain sutures under force compared to fresh bladder tissue. According to a further aspect of the invention there is provided a natural biomaterial 10 product comprising bladder tissue having a DNA content of less than 0.2 gg/mg dry weight, a suture retention strength (Fmax) of between 3-6 N, ultimate tensile strength (apex to base) of between 1-4 MPa, failure strength (apex to base) of between 70-150%. Preferably, the natural biomaterial product is a whole bladder or is a patch or portion of 15 bladder tissue. It will be appreciated that the natural biomaterial produced by the method of the present inventions retains the above characteristics and parameters and as such may be used either as the whole bladder for transplantation or experimental purposes alternatively patches or portions of the bladder may be used for any of the various surgical uses and scenarios as hereinbefore mentioned. 20 The biocompatible implant material produced by the method of the present invention is characterised by a DNA content of less than 0.2 jig/mg dry weight, that is to say it is substantially deceilularised. It is also characterised by a suture retention strength (Fmax) of between 3-6 N, ultimate tensile strength (apex to base) of between 1-4 MPa, 25 failure strength (apex to base) of between 70-150% all of these parameters indicate that the mechanical strength of the biomaterial product is comparable to natural bladder that has not undergone a decelluiarisation process according to the method of the invention. The biomaterial of the present invention is stable, non-cytotoxic and retains the strength 30 and compliance properties of the native bladder, indicating potential use in a range of surgical procedures and as a scaffold for bladder tissue-engineering. According to a yet further aspect of the invention there is provided a tissue matrix obtainable by the method of the present invention for use as a transplant tissue. 35 According to a yet further aspect of the invention there is provided use of a tissue matrix obtainable by the method of the present invention as a transplant tissue.
    10 Preferably, the tissue matrix is a bladder tissue matrix. Preferably, the bladder tissue matrix is used for reconstructive surgery and in particularly 5 in reconstruction surgery of congenital or acquired bladder defects or bladder augmentation. Initial studies in vitro using alternative biomaterials have proved encouraging in so far as normal urothelial cells (NUC) have been shown to readily attached and grow as a 10 monolayer on the surface of PermacolTM, however smooth muscle (SM) cells could only survive when co-cultured with NUC (Kimuli et al BJU Int. 94, 859, 2004) and even when co-cultured with NUC cells the SM cells could not infiltrate the Permacol
    TM
    . These observations confirm similar finding with SIS matrices and NUC and SM cells (Zhang et al J Urol, 928, 164, 2000). A functionally normal urinary bladder distends passively as it 15 fills with urine, and contracts voluntarily to void. To carry out this normal function the bladder requires a normal SM cell component. We have found that the tissue matrices produced by the present invention could sustain SM cell proliferation, thus making it a suitable biomaterial for urological tissue engineering. Thus the improved method of decellularisation provided in the present invention shows that distensible sacs treated 20 with the method can, when implanted sustain two of the essential cell types vital for normal function. According to a yet further aspect of the invention there is provided a tissue matrix obtainable by the method of the present invention for use in tissue repair. 25 According to a yet further aspect of the invention there is provided use of a of a tissue matrix obtainable by the method of the present invention in tissue repair. It is envisaged that the processed bladder biomaterial will have uses in tissue repair of 30 for example and without limitation as pubovaginal slings and in vaginal repair. It is also envisaged that the bladder biomaterial obtainable by the method of the present invention and having the unique characteristics as hereinbefore described has potential applications in gastroenterology including the repair of abdominal wall defects, such as those caused by trauma, tumour resection, and also in chest wall repair as pericardial 35 patches following tumour excision and could potentially be used for cardiac repair.
    11 According to a yet further aspect of the invention there is provided a tissue matrix obtainable by the method of the present invention for use as in wound healing or wound repair. 5 According to a yet further aspect of the invention there is provided use of a of a tissue matrix obtainable by the method of the present invention as wound healing or repair material. Preferably, the processed bladder biomaterial is in the form of a sheet or patch. 10 In wound care, natural biomaterials can be used in the repair of defects occurring as a result of burns, venous ulcers, diabetic ulcers and large full-thickness defects such as those occurring following acute injury, 15 According to a yet further aspect of the invention there is provided a tissue matrix obtainable by the method of the present invention for use as in cosmetic surgery as augmentation material According to a yet further aspect of the invention there is provided use of a of a tissue 20 matrix obtainable by the method of the present invention in cosmetic surgery as augmentation material. Cosmetic applications for the processed bladder biomaterial include eyebrow augmentation, repair of nasal septum, cleft palate repair, breast surgery, lip, cheek and 25 chin augmentation. The compliance the biomaterial we have developed also makes it a suitable material for soft and connective tissue coverage, where intimate contact between the matrix and wound surface is required. A custom made patch could be fashioned to fit to the 30 margins of ulcers or wounds with the confidence that it would lie against deep structures in order to protect and encourage healing. This may be of particular benefit in traumatic tissue loss where myocutaneous flaps are currently used to cover exposed bony or tendonous structures. The ability of our biomaterial to conform to underlying structures would make it ideal for augmentation cystoplasty, urothelial coverage in detrusor 35 myectomy and other urological reconstructive procedures.
    12 According to a yet further aspect of the invention there is provided a tissue matrix obtainable by the method of the present invention for use as a scaffold for tissue engineering. 5 According to a yet further aspect of the invention there is provided use of a tissue matrix obtainable by the method of the present invention as a scaffold for tissue-engineering. According to a yet further aspect of the invention there is provided a tissue matrix obtainable by the method of the present invention for use as an in vitro model to 10 investigate bladder function. According to a yet further aspect of the invention there is provided use of a tissue matrix produced by the method of the present invention as an in vitro model to investigate bladder function. 15 According to a yet further aspect of the invention there is provided a method of implantation of a tissue matrix in bladder reconstruction or replacement surgery comprising: (i) immersing the distensible membranous sac in a buffer solution at a mild 20 alkaline pH which includes active amounts of a proteolytic inhibitor; (ii) distending the distensible membranous sac by introducing a sufficient volume of the same buffer solution into the interior cavity of the sac and; (iii) continuing decellularisation of the sac by replacing and introducing fresh solutions both around the exterior surface of the sac and into the sac 25 interior itself so as to maintain distension of the sac during recellularisation and;. (iv) implanting either the whole decellularised distensible sac or a portion thereof into a donor 30 As previously mentioned steps (i) and (ii) may be reversed. According to a yet further aspect of the invention there is provided a method of wound healing and/or tissue repair comprising: (i) immersing the distensible membranous sac in a buffer solution at a mild 35 alkaline pH which includes active amounts of a proteolytic inhibitor; 13 (ii) distending the distensible membranous sac by introducing a sufficient volume of the same buffer solution into the interior cavity of the sac; (iii) continuing decellularisation of the sac by replacing and introducing fresh solutions both around the exterior surface of the sac and into the sac 5 interior itself so as to maintain distension of the sac during recellularisation and;. (iv) placing a portion or patch of the decellularised sac onto or around a wound or attaching said portion or patch on or about the tissue to be repaired. 10 Preferably, any of the further aspects of the invention further include any one or more of the features recited in the first aspect of the invention. The method of the present invention, products produced thereby and the use of the 15 products provide a decellularised, biocompatible bladder matrix which retains properties of native bladder tissue. It is envisaged that the acellular matrix retaining the major structural components and strength of the urinary bladder can be used to produce a functional, tissue-engineered 20 construct for use in bladder repair and as an in vitro model to study host cell-matrix interactions and the role of mechanical forces on bladder tissue functionality. Surgeons who have felt the bladder biomaterial obtainable by the method of the present invention have reported that the strength, feel, compliance and elasticity of the material 25 makes it particularly well suited for any surgical procedures where PermacolTM and other natural biomaterials are being used currently. indeed it has been indicated that the compliance of the material of the present invention makes it a more attractive material to use than available prior art materials. 30 Furthermore experimental evidence suggests that decellularised bladder material has a good shelf life even after as long as 30 months storage.
    14 BRIEF DESCRIPTION OF THE DRAWiNGS Figure 1 shows a photomicrograph of dissected bladder showing the five anatomical regions of the bladder that underwent biomechanical characterisation. Tissue strips 5 from all regions were collected in both transverse (T) and base-to-apex (B-A) directions. Figure 2 shows histological characterisation of fresh (A, B) and decellularised (C, D) porcine bladder. Sections were stained with haematoxylin and eosin (A and C) and Hoeschst 33258 (B and D). Scale bar - 100pm. 10 Figure 3 shows histological characterisation of fresh (A and C) and decellularised (B and D) porcine bladder. Paraffin-wax-embedded sections were stained with Van Gieson (A and B) and Miller's Elastin stain (C and D) [SM: Smooth Muscle, C: Collagen matrix]. Scale bar - 100pm. 15 Figure 4 shows immunoperoxidase labelling of fresh (A, C, E and G) and decelluiarised (B, D, F and H) porcine bladder. Sections labelled with antibodies against collagen type I (A and B), smooth muscle actin (C and D), vimentin (E and F) and desmin (G and H) were counterstained with haematoxylin and show a qualitative reduction in smooth 20 muscle actin and desmin. Figure 5 shows immunoperoxidase labelling of fresh (A and C) and decellularised (B and D) porcine bladder. Sections labelled with antibodies against collagen type IV (A and B) and laminin (C and D) were counterstained with haematoxylin. 25 Figure 6 shows immunofluorescence labelling of SM cells labelled with an antibody to smooth muscle actin (A). Hoescht 33258 dye was used to identify all nuclei in an equivalent field of view (B). Scale bar 50pm. 30 Figure 7 (A) shows SM cell proliferation assessed by MTT assay over 7 days and Figure 7 (B) on day 7. The graphs show that in the presence of 10% (v/v) serum, proliferation of SM cells was not inhibited by medium preconditioned by incubation with the decellularised matrix. Absorbance was measured in replicates of six and the calculated SEM plotted as x and y error bars. 35 15 Figure 8 shows a phase-contrast micrograph showing porcine SM cells growing up to the decellularised matrix (DM) indicating that the matrix was not cytotoxic. Scale bar 50pm. Figure 9 shows an H&E stained section showing porcine SM cells seeded onto the 5 decellularised porcine bladder matrix. (A) After 14 days in culture, there is a single layer of cells lining the surface of the matrix and some cells are seen to have infiltrated. (B) Further matrix penetration has occurred after 21 days. The matrix showed no signs of degradation. Scale bar 100pm. 10 Figure 10 shows the phases of biological tissue stress-strain behaviour. Figure 11 shows biomechanical testing of fresh and decellularised porcine bladder tissue (n=6). Each test was performed in two directions: apex-to-base and transverse; and on samples from 5 different anatomical regions of the bladder. Results are presented as 15 mean values (+/- 95% Cl). Figure 12 shows determination of the (A) suture retention strength and (B) burst pressure of fresh and decellularised porcine bladder tissue. Suture retention tests were performed on bladder strips and burst pressure tests on intact bladder specimens. In 20 each case, n=6 and the results are presented as mean values (+/- 95% Cl). Figure 13 shows a burst pressure rig for testing of fresh and decellularised bladder burst testing. 25 Figure 14 is a bar chart comparison representation of suture retention strength between fresh bladder, decellularised bladder, fresh pericardium, decellularised pericardium and SISTM and PERMACOL TM implants respectively. Figure 15 is a bar chart comparison representation of mean elastin phase slope between 30 fresh bladder, decellularised bladder, fresh pericardium, decellularised pericardium and SISTM and PERMACOL TM implants respectively. Figure 16 is a bar chart comparison representation of mean transition stress between fresh bladder, decellularised bladder, fresh pericardium, decellularised pericardium and 35 SISTM and PERMACOL T M implants respectively.
    16 Figure 17 is a bar chart comparison representation of mean transition strain between fresh bladder, decellularised bladder, fresh pericardium, decellularised pericardium and SIS TM and PERMACOL TM implants respectively. 5 Figure 18 is a bar chart comparison representation of mean thickness between fresh bladder, decellularised bladder, fresh pericardium, decellularised pericardium and SISTM and PERMACOL TM implants respectively. Figure 19 is a bar chart comparison representation of mean collagen slope between 10 fresh bladder, decellularised bladder, fresh pericardium, decellularised pericardium and SISTM and PERMACOL T M implants respectively. Figure 20 is a bar chart comparison representation of mean ultimate tensile strength between fresh bladder, decellularised bladder, fresh pericardium, decellularised 15 pericardium and SIS T M and PERMACOL TM implants respectively. Figure 21 is a bar chart comparison representation of mean failure strength between fresh bladder, decellularised bladder, fresh pericardium, decellularised pericardium and
    SIS
    T M and PERMACOL TM implants respectively. 20 Figure 22 shows explanted fresh bladder tissue stained with (a) haematoxylin and eosin (b) Anti-CD3 (pan T-cell) and (c) Anti f4180 (macrophage). Magnification is at x 40 and arrows indicate implanted tissue. 25 Figure 23 shows explanted acellular bladder biomaterial stained with (a) haematoxylin and eosin (b) Anti-CD3 (pan T-cell) and (c) Anti f4/80 (macrophage). Magnification is at x 40 and arrows indicate implanted biomaterials. Figure 24 shows a bar chart comparison representation of mean elastin phase slope 30 between fresh decellularised bladder and decellularised bladder that has been stored for 6, 9 and 30 months respectively. Figure 25 shows a bar chart comparison representation of mean collagen phase slope between fresh decellularised bladder and decellularised bladder that has been stored for 35 6, 9 and 30 months respectively.
    17 Figure 26 shows a bar chart comparison representation of mean transition stress between fresh decellularised bladder and decellularised bladder that has been stored for 6, 9 and 30 months respectively. 5 Figure 27 shows a bar chart comparison representation of mean ultimate tensile strength between fresh decellularised bladder and decellularised bladder that has been stored for 6, 9 and 30 months respectively. Figure 28 shows a bar chart comparison representation of mean transition strength 10 between fresh decellularised bladder and decellularised bladder that has been stored for 6, 9 and 30 months respectively. Figure 29 shows a bar chart comparison representation of mean failure strength between fresh decellularised bladder and decellularised bladder that has been stored for 15 6, 9 and 30 months respectively. DETAILED DESCRIPTION Specimen procurement 20 Whole porcine bladders were obtained from a local abattoir within 4 h of slaughter and transported to the laboratory on ice in sterile transport medium (Hanks' balanced salt solution (HBSS) containing 10mM HEPES pH 7.6 and 10 KlU/ml Aprotinin (Trasylol, Bayer, Berkshire, UK). 25 Porcine Bladder decellularisation Intact bladders were washed in phosphate buffered saline (PBS) containing 0.1% w/v ethylene diamine tetra-acetic acid (EDTA) and Aprotinin (10 KIU/ml) to inhibit protease activity. Subsequent treatments, unless otherwise stated, were all carried out with protease inhibition. At each stage the intact bladder was distended with up to 500 ml 30 buffer through a funnel inserted into the bladder neck, closed with Nalgene TM forceps (VWR International Ltd, Poole, UK) and immersed in the same solution. The bladders were decellularised by incubating the bladder for 24 h at 4"C in hypotonic Tris buffer (10 mM Tris, pH 8.0) followed by distension and incubation with agitation on an orbital shaker for 24 h in 0.1% (w/v) sodium dodecyl sulphate (SDS) in hypotonic Tris buffer at 35 room temperature. Bladders were washed in PBS without protease inhibition, before being incubated for 24 h in 50 U.mr 1 deoxyribonuclease I (Sigma, Poole, UK) and 1 Uml 1 18 ribonuclease A (Sigma, Poole, UK) in 10mM Tris-HCI pH 7.5 with gentle agitation at 37*C. Bladders were sterilized by incubation in 0.1% (v/v) peracetic acid in PBS for 3 h and finally, were washed in sterile PBS once for 24 h followed by 3 periods of 1 h under aseptic conditions. The resulting material was stored in PBS at 4*C for at least 6 months 5 with no change in appearance or handling. Histology and microscopy Fresh and decellularised tissue samples were fixed in 10% (vlv) neutral buffered formalin, dehydrated and embedded in paraffin wax. Haematoxylin and eosin staining 10 was used to evaluate the cellular content and general histoarchitecture of the porcine bladders. Miller's elastin staining was used to evaluate the elastin content and the Van Gieson technique was used for the identification of collagen I fibres (Bancroft and Stevens, Theory and Practise of Histological Techniques. London, Churchill Livingstone, 1990). 15 Immuno-labelling of specific proteins was performed using an indirect immunoperoxidase method as previously described (Booth et al, Lab. Investigation 76, 843, 1997). Tris-buffered saline [(TBS), 0.05M Tris-HOl, 0.15M NaCI, pH 7.6] was used as the diluent and wash buffer throughout. Non-specific background staining was 20 eliminated by blocking with 10% (vlv) rabbit serum. Monoclonal antibodies were obtained against collagen type 1 (COL 1), smooth muscle actin (1A4), laminin (LAM89) (Sigma, Poole, UK), collagen type IV (clV22), vimentin (V9) and desmin (D33) (Dako, High Wycombe, UK).Sections were incubated sequentially in primary antibody for 1 h, biotinylated rabbit ant-mouse Ig (F (ab')2 fragments (Dako, High Wycombe, UK) for 30 25 min and strepavidin/HRP ABC complex (Dako, High Wycombe, UK) for 30 min, with washing between each step. Bound antibody was visualized using a 3,3' diaminobenzidene substrate (DAB) reaction catalysed by H 2 0 2 . Sections were counterstained with haematoxylin, before being dehydrated, cleared and mounted in DPX (Sigma, Poole, UK). Omission of the primary antibody from the labelling protocol 30 and the use of irrelevant primary antibodies served as negative controls. Biochemical analysis Three porcine bladders were decellularised for biochemical analysis and comparison with six fresh, untreated porcine bladders. Unless otherwise stated, test solutions for 35 analysis were prepared from samples of fresh and decellularised matrix that had been 19 freeze-dried to constant weight, hydrolysed by incubation with 6M HCI for 4 hours at 120*C and neutralised to pH 7 with NaOH. Glycosaminoglycan assay 5 The amount of sulphated sugars (GAGs) was determined by dimethylmethylene blue binding (Enobakhare et al, Anal.Biochem. 243, 189, 1996; Farndale et al, Biochim. Biophys. Acta., 883, 173, 1986). Briefly, test solutions were incubated with the dimethylmethylene blue solution and the absorbance read at 525nm. The amount of GAGs was calculated by interpolation from a standard curve prepared using chondroitin 10 sulphate and phosphate assay buffer (0.1M sodium di-hydrogen orthophosphate, O.1M di-sodium hydrogen orthophosphate, pH6.8) over a range of concentrations. Hydroxyproline assay The amount of hydroxyproline was determined using a method based on that described 15 elsewhere (Brown et al, Biotechniques 30, 38, 2001; Edwards et al, Clin Chem Acta 104, 161, 1980; Stegemann and Stadler Clin Chem Acta, 18, 267, 1967). A range of hydroxyproline standards were prepared using trans-4-hydroxy-L-proline in hydroxyproline assay buffer [0.17M citric acid, 0.8% (v/v) acetic acid, 0.6M sodium acetate, 0.57M sodium hydroxide and 20% (v/v) propan-1-ol pH6]. 50jl of each 20 standard and test solution was aliquoted into a clear flat bottomed 96 well plate. Oxidation was achieved by adding 100pl of chloramine T solution to each well. The plate was gently shaken for five minutes and 1 00pl of Ehrlich's reagent added. The plate was covered and incubated at 600C for 45 minutes, before reading the absorbance at 570nm. A standard curve of hydroxyproline concentrations was plotted using the 25 standard solutions and the amount of hydroxyproline present in the test samples determined. To measure the amount of denatured hydroxyproline, fresh and decellularised tissue samples that had been freeze-dried to constant weight were digested with a-chymotrypsin prior to analysis (Bank et al, Matrix Biol. 16, 233, 1997). 30 DNA assay Fresh and decellularised tissue samples that had been freeze-dried to constant weight were digested in papain buffer at 60"C for 24 hours as previously described (Kim et al Anal Biochem, 174, 168, 1988; Labarca et al Anal Biochem, 102, 344 1980). Test solutions were incubated with Hoechst 33258 dye solution and using a fluorometer, the 35 plate was read using excitation at 365nm and emission at 458nm. The amount of DNA 20 was calculated by interpolation from a standard curve prepared using calf thymus DNA solubilised in Tris buffered saline pH 7.6 over a range of concentrations. EXAMPLE I 5 The bladder wall is composed primarily of collagen, elastin, and smooth muscle and is organised in two major layers: the lamina propria and the detrusor. The lamina propria consists of the urothelium, which lines the luminal surface, and an underlying connective tissue matrix that contains a dense layer of randomly oriented collagen fibres in which the capillary network of the bladder is embedded. The majority of the lamina propria is 10 constituted by a thick layer of collagen that functions to maintain the shape of the bladder wall and to limit its overall compliance (ratio of maximum volume divided by pressure). The detrusor muscle layer provides the contraction during voiding, and is composed of muscle fibres of 50 to 150 pm in diameter, and 20-50 pm apart and interconnected with collagen bundles. Histological analysis of the decellularised matrix 15 showed that whilst the urothelium and smooth muscle cells had been removed, the underlying histoarchitecture was retained. (See Figure 2 and 3 and Example 6) EXAMPLE 2 Glycosaminoglycans (GAGs) are the main component of the ground substance in which 20 cells, collagen (comprised of hydroxyproline, proline and glycine) and elastin fibres are embedded. Compared to fresh bladder tissue, the proportion of hydroxyproline and GAGs in decellularised tissue samples relative to total dry weight, was significantly higher due to the loss of other soluble proteins and cell components (Table 1). Table 1 below shows the biochemical characterisation of fresh and decellularised porcine 25 bladder tissue.
    权利要求:
    Claims (5)
    [1] 1. A product comprising decellularised a whole bladder, the product being characterised by a DNA content of less than 0.2 p.glmg dry weight, a suture retention strength (Fmax) of between 3-6 N, ultimate tensile strength (apex to base) of between 1 5 4 MPa, failure strength (apex to base) of between 70-150%.
    [2] 2. A product according to claim 1, further including any one or more of the following characteristics selected from the group comprising: (i) a DNA content of less than 0.1 pg/mg dry weight; 10 (ii) a hydroxyproline content of between 75-90 gg/mg dry weight; (iii) glycosaminoglycans content of between 50-60 gg/mg dry weight; and (iv) a burst pressure similar to that of fresh bladder tissue.
    [3] 3. A product according to either claim 1 or 2, wherein the burst pressure is about 15 2000 mmHg.
    [4] 4. A product according to any of one of claims I to 3 that is devoid of urothelium and has no cells present within underlying tissue. 20
    [5] 5. A product according to any one of claims 1 to 4 for use in any one or more of the functions selected from the group comprising (i) transplant tissue; (ii) implant material; (iii) reconstruction of congenital or acquired bladder defects; 25 (iv) tissue repair; (v) wound healing or wound repair; (vi) cosmetic surgery as augmentation material.; (vii) a scaffold for tissue-engineering; and (viii) as an in vitro model to investigate bladder function. 30
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    同族专利:
    公开号 | 公开日
    AU2007231131A1|2007-10-04|
    US20150050359A1|2015-02-19|
    US20150018966A1|2015-01-15|
    CA2653551A1|2007-10-04|
    EP2004250B1|2014-08-13|
    WO2007110634A3|2008-05-08|
    GB2433745A|2007-07-04|
    EP2004250A2|2008-12-24|
    GB2433745B|2007-12-05|
    AU2007231131B2|2013-03-14|
    GB0606231D0|2006-05-10|
    GB0706014D0|2007-05-09|
    US8828448B2|2014-09-09|
    PL2004250T3|2015-01-30|
    US9180144B2|2015-11-10|
    ES2520019T3|2014-11-11|
    WO2007110634A2|2007-10-04|
    AU2013200822B2|2013-06-13|
    US9180143B2|2015-11-10|
    CA2653551C|2016-04-19|
    US20090130221A1|2009-05-21|
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    法律状态:
    2013-10-10| FGA| Letters patent sealed or granted (standard patent)|
    2020-10-22| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|
    优先权:
    申请号 | 申请日 | 专利标题
    GBGB0606231.9A|GB0606231D0|2006-03-29|2006-03-29|Improvements relating to decellurisation of tissue matrices for bladder implantation|
    GB0606231.9||2006-03-29||
    AU2007231131A|AU2007231131B2|2006-03-29|2007-03-28|Improvements relating to decellularisation of tissue matrices for bladder implantation|
    AU2013200822A|AU2013200822B2|2006-03-29|2013-02-14|Improvements Relating to Decellularisation of Tissue Matrices for Bladder Implantation|AU2013200822A| AU2013200822B2|2006-03-29|2013-02-14|Improvements Relating to Decellularisation of Tissue Matrices for Bladder Implantation|
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