DESC:TECHNICAL FIELD
The present disclosure broadly relates to the fields of medical dressings and wound management. Particularly, the disclosure provides a placental tissue based composite dressing and method(s) for preparation thereof, which enable production of dressings specifically characterized by the features of porous structure, cross-linkage and thickness ranging from about 0.1cm to about 2cm. The 3D, sponge-like, porous structure of the dressings of the present disclosure makes them fit for tissue regeneration and healing applications. Particularly, the dressings of the present disclosure may be applied to facilitate tissue healing in conditions such as but not limited to deep cavity wounds and ulcers.
BACKGROUND OF THE DISCLOSURE
Chronic non-healing ulcers is known to affect about 0.6% - 3% of individuals over 60 years of age increasing to 5% of individuals aged over 80 years. Estimates suggest that 15% of the older adults in the US suffer from chronic wounds and every year, 2 to 3 million more people are diagnosed with various types of chronic wounds every year. About 0.2-1 individuals per 1000 have been reported to suffer from chronic ulcers in the rest of the Western world. Gupta et al reported the prevalence of chronic ulcers in India to be about 4.5 per 1000 people. The incidence of these ulcers are rising because of an increase in the aging population which is expected to grow substantially during the next several decades and increases in risk factors such as smoking, diabetes, obesity, immobilization, high blood pressure. While the overall prevalence of ulcers is not affected by social class, ulcers tend to take longer to heal in lower socioeconomic classes. Chronic ulcers are very debilitating and usually lead to a poor quality of life in several domains compared to age matched controls. Patients experience symptoms which negatively affect life such as pain, itchiness, leg swelling, odour, restricted mobility, sleep disturbances. Reduced mobility due to leg ulceration can restrict working capacity in younger patients and the unpleasant odour has been reported to cause social embarrassment leading to higher anxiety, depression and altered body image. A negative emotional impact on life with symptoms such as anger, depression, and social isolation was reported by 68% of patients.
Deep chronic wounds are characterized by the loss of extracellular matrix and in combination with the exposure of structures such as bone and tendon. The healing of such wounds poses a major challenge as the development of granulation tissue and subsequent reepithelialisation over these structures is extremely slow and often may not happen at all. If such deep ulcers do not heal they might lead to amputation, which is often the case of diabetic ulcers. Patients undergoing such amputations have a higher 5 year mortality rate.
In order to facilitate healing of deep ulcers or wounds, it is desirable to apply thick, porous dressings that allow for infiltration of cells at the wound sites. Membrane based dressings in the art do not provide sufficient thickness to facilitate sufficient degree of cell attachment, cell growth and granulation.
Such thick-porous sponge like dressings, that are capable of retaining structural integrity at the site of application are therefore the need of the hour.
SUMMARY OF THE DISCLOSURE
Addressing the requirement for placenta based thick-porous sponge like dressings, the present disclosure provides a placental tissue derived cross-linked porous dressing having thickness ranging from about 0.1cm to about 2cm.
In some embodiments, the dressing is prepared from placental tissue as a whole or from fetal component(s) and/or maternal component(s) thereof; wherein the fetal component(s) is selected from amnion membrane and chorion membrane or a combination thereof; and the maternal component is selected from maternal decidua and umbilical cord or a combination thereof.
In some embodiments, the dressing is composed completely of placental tissue or a combination of placental and other biomaterials selected from a group comprising collagen, cellulose, gelatin, alginate, chitosan, silk, fibrin, guar gum and xanthum gum or any combination thereof.
In some embodiments, the dressing has porosity ranging from about 90% to about 99.5%
The present disclosure further provides a method for preparation of the placental tissue derived cross-linked porous dressing described above, said method comprising
processing placental tissue into a form selected from a group comprising dried cryo-milled membrane, coarse dried powder and non-dried tissue;
preparing porous matrix from the processed placental tissue; and
subjecting the porous matrix to cross-linking to obtain the placental tissue derived cross-linked porous dressing.
In some embodiments, the dried cryo-milled membrane form of placental tissue is prepared by a method comprising
preparing dried amnion membrane from a placental tissue sample or preparing dried chorion membrane from a placental tissue sample or both; and
subjecting the dried membrane(s) to cryo-milling.
The preparation of the dried chorion membrane, in some embodiments, further comprises -
treating the saline washed chorion membrane with alkali and
washing the alkali treated chorion with buffer prior to contacting the washed chorion membrane with the antibiotic(s).
In some embodiments, preparing the coarse dried powder or a non-dried tissue form of the placental tissue comprises
chopping the placental tissue;
washing the chopped placental tissue with solvent selected from a group comprising water, sodium dodecyl sulphate and ethanol or any combination thereof;
freezing the washed placental tissue;
optionally lyophilizing the frozen tissue; and
optionally powdering the lyophilized tissue
to obtain the placental tissue in the form of a coarse dried powder or a non-dried tissue.
In some embodiments, preparing a porous matrix from the processed placental tissue comprises
preparing a solution of another biomaterial;
preparing a suspension of the processed placental tissue and the biomaterial solution;
subjecting the suspension to freezing followed by lyophilization to obtain the porous matrix.
In some embodiments, preparing a porous matrix from the processed placental tissue comprises
mixing the processed placental tissue with a solvent to form a mixture;
homogenizing the mixture;
casting the homogenized mixture onto a mould; and
freezing followed by lyophilization of the contents of the mould
to obtain the porous matrix.

In some embodiments, preparing a porous matrix from the processed placental tissue comprises
preparing a mixture of the processed placental tissue in an acid;
homogenizing the mixture to obtain a homogenized mixture;
adding a digesting enzyme to the homogenized mixture;
adding cold buffer to the digested mixture;
neutralizing the mixture by addition of alkali;
casting the homogenized mixture onto a mould;
freezing of the contents of the mould; and
lyophilizing the frozen contents of the mould
to obtain the porous matrix.
The dressing of the present disclosure is cross-linked in order to confer to the dressing the property of integrity when put to application at the wound site. In some embodiments, subjecting the porous matrix as prepared by the above method(s) to cross-linking comprises -
submerging the porous matrix in cross-linking solution;
washing the porous matrix treated with the cross-linking solution; and
subjecting the washed porous matrix to freezing followed by lyophilization to obtain the placental tissue derived cross-linked porous dressing.
The present disclosure further provides an in-vitro method of promoting cell attachment and cell growth in an injured tissue comprising application of the placental tissue derived cross-linked porous dressing to the tissue.
Also provided is an in-vitro method of promoting granulation in an injured tissue comprising application of the of the placental tissue derived cross-linked porous dressing to the tissue.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:
FIG 1. depicts the glass mould set employed for setting the porous matrix of the dressing of the present disclosure.
FIG 2. depicts physical appearance of dressing containing various amounts of placental powder in combination with gelatin - A) Dressings with thickness of 0.1 cm, B) Dressings with thickness of 0.5 cm and C) Dressings with thickness of 2 cm.
FIG 3. depicts SEM image of the dressing containing 10% processed placental tissue in combination with gelatin.
FIG 4. depicts (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentrations of coarse dried powder by homogenizing and freezing at -20°C before lyophilization.
FIG 5. depicts (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentration of coarse dried powder by homogenization and freezing at -80°C before lyophilization.
FIG 6. depicts SEM images of dressings made from different starting concentration of coarse dried powder by homogenization and freezing at -20°C before lyophilization.
FIG 7. depicts (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentration of non-dried tissue by homogenization and freezing at -20°C before lyophilization.
FIG 8. depicts (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentration of non-dried tissue by homogenization and freezing at -80°C before lyophilization.
FIG. 9 depicts SEM of dressings made from different starting concentration of non-dried tissue by homogenization and freezing at -20°C before lyophilization.
FIG 10. depicts (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentration of coarse dried powder by solubilizing and freezing at -20°C before lyophilization.
FIG 11. depicts (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentration of coarse dried powder by solubilizing and freezing at -80°C before lyophilization.
FIG 12. depicts SEM of dressings made from different starting concentration of concentration of coarse dried powder by solubilizing and freezing at -20°C before lyophilization.
FIG 13. depicts (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentration of non-dried tissue by solubilizing and freezing at -20°C before lyophilization.
FIG 14. depicts. (A) 2cm thick dressings; and (B) 0.1cm dressings made from different starting concentration of non-dried tissue by solubilizing and freezing at -80°C before lyophilization.
FIG 15. depicts SEM images of dressings made from different starting concentration of non-dried tissue by solubilizing and freezing at -20°C before lyophilization.
FIG 16. depicts dressing comprising non-dried tissue at a concentration of 15% and frozen at -20°C before lyophilization with and without cross-linking. The crosslinked dressing is stable (left panel) while non-crosslinked dressing is not stable (right panel).
FIG 17. depicts cell morphology observed in L929 cells after 48 hrs of incubation A) with 0.5% placental powder in gelatin combined dressing conditioned media B) control.
FIG 18. depicts results of alamar blue reduction assay in L929 cells incubated with 0.5% placental powder in gelatin sponge conditioned media
FIG 19. Epithelial cell and Fibroblast cell attachment and growth at day 1 and day 5 in 15% wet tissue homogenized sponge frozen at -20°C.
FIG 20. A) Non-healing ulcers devoid of granulation tissue after debridement with exposed tendon. B) Application of placental sponge over the ulcer. C) Excellent granulation tissue suitable for grafting after two rounds of NPWT. D) Excellent update of split-thickness skin graft.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the phrases/terms ‘placental tissue derived cross-linked porous dressing’, ‘placental tissue-based dressing’, ‘dressing’, ‘porous dressing’, ‘porous matrix’, ‘sponge’, ‘porous sponge’ used interchangeably, refer to the 3D porous dressing of the present disclosure formed from placental tissue.
As used herein, the term ‘biomaterial’ refers to a natural or synthetic material that is suitable for introduction into or contacting with the living tissue.
Reference to ‘placental tissue’ throughout the present disclosure envisages placental tissue of human origin, discarded during birth and collected after consent from the donor and approval from the relevant ethics committee at the medical facility that the placental tissue is procured from. ‘Placental tissue’ as employed in the present disclosure comprises one or more of amnion, chorion, maternal decidua and umbilical cord.
The term ‘processed placental tissue’ refers to placental tissue (fresh or preserved) that is processed into any one of the following forms - dried cryo-milled membrane, coarse dried powder and non-dried tissue – said forms being further described below.
The term ‘sponge solution’ refers to the solution of processed placental tissue in a suitable solvent prior to being subjected to freezing and/or lyophilization.
Unless otherwise defined, reference to ‘water’ as a solvent for dissolution of any substance or as a washing liquid envisages the possibility of the water being any of tap water, purified water, RO water, distilled water, double distilled water or deionized water.
Unless otherwise defined, process steps such as but not limited to ‘washing’, ‘shaking’, ‘heating’, ‘freezing’, ‘lyophilization’, ‘drying’ envisage said steps being performed as per standard practices in the art.
‘Lyophilization’ as referred to in the present disclosure, refers to the process of freezing the material being subjected to the lyophilization, then reducing the pressure and adding heat to allow the frozen water in the material to sublimate. Steps of lyophilization as referred to above may be performed in any standard lyophilizer, through routine cycles, without any restriction on temperature as long as a freeze-dried material is yielded.
The steps of ‘washing’ as referred to in the present disclosure, has been defined in terms of the solvent or washing liquid employed for washing. The volume of solvent or washing liquid in each of said steps is enough to completely submerge the tissues in sufficient excess of the solvent or washing liquid to achieve efficient washing.
With respect to the use of various well-known components in the present disclosure, whose concentrations are not particularly defined herein, said components are deemed to be used in concentrations that are well known to a person skilled in the art in the context of medical/pharmaceutical applications such as those described in the present disclosure.
As used throughout the present disclosure, ranges are a shorthand for describing each and every value within the range. Any value within the range can be selected as the terminus of the range.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. The use of the expression ‘about’ refers to values ±20% of the values defined immediately following said term. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Particularly provided by the present disclosure is a placental tissue derived cross-linked porous dressing having thickness ranging from about 0.1cm to about 2cm.
In some embodiments, thickness of the dressing is about 0.1cm, about 0.25cm, about 0.5cm, about 0.75cm, about 1cm, about 1.25cm, about 1.5cm, about 1.75cm or about 2.0cm.
The thickness of the placental tissue derived cross-linked porous dressing depends on the depth of the wound/ulcer to which it is to be applied.
The placental tissue derived cross-linked porous dressing is specifically characterized by uniform thickness across its length and breadth.
The placental tissue derived cross-linked porous dressing of the present disclosure is prepared from placental tissue as a whole or specifically from fetal and/or maternal components thereof. Fetal components of the placental tissue include the amnion and chorion membranes. Maternal components of the placental tissue include the decidua and umbilical cord.
In some embodiments, the placental tissue derived cross-linked porous dressing of the present disclosure comprises placental tissue selected from a group comprising amnion, chorion, maternal decidua and umbilical cord or any combination thereof.
In some embodiments, the placental tissue derived cross-linked porous dressing is composed completely of placental tissue or comprises placental tissue in combination with other biomaterials.
In an embodiment, the other biomaterial is selected from a group comprising collagen, cellulose, gelatin, alginate, chitosan, silk, fibrin, guar gum and xanthum gum or any combination thereof. In some embodiments, the other biomaterial may further include other biomaterials which are capable of forming a porous matrix when pre-treated and subjected to lyophilization.
In some embodiments, the dressing comprises the placental tissue at a concentration ranging from about 0.5% to about 100%, and the other biomaterial at a concentration ranging from about 0% to about 99.5%.
In an exemplary embodiment, placental tissue derived cross-linked porous dressing comprises placental tissue in combination with gelatin, wherein the placental tissue specifically comprises the amnion and chorion membranes of the placenta.
In some embodiments, the placental tissue derived cross-linked porous dressing has porosity ranging from about 90% to about 99.5%. In a non-limiting embodiment, the dressing is opaque off-white and semi-occlusive.
The placental tissue derived cross-linked porous dressing of the present disclosure is fit for tissue healing or tissue regeneration applications at sites such as but not limited to deep/chronic wounds and ulcers. Accordingly, the placental tissue derived cross-linked porous dressing of the present disclosure is non-cytotoxic in nature. The placental tissue derived cross-linked porous dressing is characterized by its 3D, sponge-like structure, making it amenable for said applications. The sponge-like structure serves as a scaffold for cell attachment and cell growth.
In some embodiments of the present disclosure, the placental tissue derived cross-linked porous dressing facilitates granulation at the site of application. Said dressing of the present disclosure is capable of acting as a scaffold which has cell attachment and cell growth properties.
While the thickness of the dressing makes it suitable for application at deep chronic wounds or ulcers, by virtue of the dressing being formed from placental tissue, the dressing allows for interaction with both healing as well as non-healing tissue. Moreover, in view of the cross-linkage present in the dressing, the dressing retains its structure for a considerable period of time without collapsing once applied to the wound site.
In an embodiment, the dressing of the present disclosure may be applied independently or in combination with Negative-Pressure Wound Therapy (NPWT). Combinatorial therapy with NPWT been shown to accelerate granulation over exposed tendons and bone, thereby reducing the number of changes of the dressing and further, preventing the adherence of NPWT foam materials to the wound site.
The present disclosure further provides a method for preparation of the placental tissue derived cross-linked porous dressing, said method comprising
processing placental tissue into a form selected from a group comprising dried cryo-milled membrane, coarse dried powder and non-dried tissue;
preparing porous matrix from the processed placental tissue; and
subjecting the porous matrix to cross-linking to obtain the placental tissue derived cross-linked porous dressing.
In some embodiments, the placental tissue employed as starting material is selected from freshly sourced placental tissue and preserved placental tissue. In a non-limiting embodiment, placental tissue preserved up to a period of upto about 1 year to about 2 years may be employed for the preparation of the dressing of the present disclosure.
In exemplary embodiments, the placental tissue is selected from donors free of infectious diseases. In preferred embodiments placenta having yellow colour and malodour are avoided. Further, only intact placental tissues are selected to prepare the dressing of the present disclosure.
In some embodiments, the dried cryo-milled membrane, coarse dried powder and non-dried tissue form of the placental tissue may be composed of constituents of placental tissue as a whole or one or more of amnion, chorion, umbilical cord and maternal decidua.
Once the porous matrix is obtained, in order to retain structural, physical and spatial integrity, the porous matrix is subjected to cross-linking.
While any method known in the art may be employed in order to facilitate cross-linking, in some embodiments, the cross-linking is achieved by treating the porous matrix with cross-linking agents selected from a group comprising 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Glutaraldehyde, Genepin, Transglutaminase, Dialdehyde Starch or any combination thereof. In some embodiments, the present disclosure also provides for employment of any other agent capable of cross-linking the porous matrix formed from placenta and/or other biomaterial such as but not limited to gelatin or collagenous materials.
In some embodiments, subjecting the porous matrix to cross-linking comprises
submerging the porous matrix in cross-linking solution;
washing the porous matrix treated with the cross-linking solution; and
subjecting the washed porous matrix to freezing followed by lyophilization to obtain the placental tissue derived cross-linked porous dressing.
In some embodiments, the cross-linking solution comprises about 0.5 % to 10 % cross-linking agent in a solvent such as but not limited to alcohol. In some embodiments, the cross-linking agent is selected from a group comprising 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), Glutaraldehyde, Genepin, Transglutaminase and Dialdehyde Starch or any combination thereof. In some embodiments, the solvent is selected from a group comprising ethanol, isopropanol and water or any combination thereof.
In preferred embodiments, the cross-linking solution comprises about 1% EDC in about 90% ethanol.
In some embodiments, the porous matrix is submerged in cross-linking solution for about 4 hours to about 36 hours, preferably about 24 hours.
In some embodiments, the porous matrix treated with the cross-linking solution is washed with water about 1 to about 2 times, preferably 2 times in wash cycles of about 30 minutes to 60 minutes, each at about 50 rpm to about 150 rpm, preferably about 100 rpm.
In some embodiments, the washed porous matrix is subjected to freezing at a temperature ranging from about -20°C to about 80°C for about 4 hours to about 48 hours.
In further embodiments, the frozen porous matrix is subjected to lyophilization for about 16 hours to about 48 hours, preferably at least about 24 hours, to obtain the placental tissue derived cross-linked porous dressing. The lyophilization may be performed in any standard lyophilizer, through routine cycles, without any restriction on temperature as long as the freeze-dried placental tissue derived cross-linked porous dressing of the present disclosure is yielded.
Aforesaid steps of cross-linking followed by freezing and lyophilization yielding the final dressing of the present disclosure remaining common, the steps of processing of placental tissue and preparing porous matrix from the processed placental tissue may have the different variations, all yielding the dressing of the present disclosure.
In some embodiments, as defined above, the placental tissue may be processed into the form of dried cryo-milled membrane. In some embodiments, the cryo-milled membrane may be prepared from placental tissue selected from a group comprising amnion, chorion, maternal decidua and umbilical cord or any combination thereof.
In some embodiments, the dried cryo-milled membrane form of placental tissue is prepared by a method comprising
preparing dried amnion membrane from a placental tissue sample; and/or
preparing dried chorion membrane from a placental tissue sample; and
subjecting the dried amnion membrane and/or the dried chorion membrane to cryo-milling.
In some embodiments, the dried cryo-milled membrane form of placental tissue is prepared by a method comprising
preparing dried chorion membrane from a placental tissue sample; and
subjecting the dried chorion membrane to cryo-milling.
In some embodiments, the dried cryo-milled membrane form of placental tissue is prepared by a method comprising
preparing dried amnion membrane from a placental tissue sample; and
subjecting the dried amnion membrane to cryo-milling.
In some embodiments, the dried cryo-milled membrane form of placental tissue is prepared by a method comprising
preparing dried amnion membrane from a placental tissue sample;
preparing dried chorion membrane from a placental tissue sample; and
subjecting the dried amnion membrane and the dried chorion membrane to cryo-milling.
The dried amnion and chorion membranes may be prepared from the same placental tissue sample or different tissue samples. In some embodiments, the dried amnion membrane and the dried chorionic membrane are optionally decellularized.
While the amnion and chorion membranes may be isolated, processed for removal of unwanted material and dried by any method known in the art, in a preferred embodiment, preparing the dried amnion and/or chorion membrane(s) comprises
separating the amnion and/or chorion membrane(s) from rest of the placental tissue;
washing the separated membrane(s) with saline;
contacting the washed membrane with antibiotics(s);
scraping the membrane(s) on one or both sides;
washing the scraped membrane(s); and
drying the washed membrane(s) to obtain the dried amnion and/or chorion membrane(s).
Steps of the aforesaid method may be performed in any order. In an embodiment, each of the steps of washing as described above may be repeated about 2 times to about 5 times.
In an embodiment, the separation of the amnion and/or chorion membrane(s) from rest of the placental tissue is carried out by conventional techniques well known and understood by a person skilled in the art. The mode and manner in which this step is carried out does not adversely affect the preparation of the dressing of the present disclosure.
In an embodiment, the dried amnion and chorion membranes may be prepared simultaneously by parallel processing of both the membranes, or, sequentially i.e. one followed by the other. In some embodiments, the membranes are optionally decellularized by treatment with agents such as but not limited to Sodium Dodecyl Sulphate (SDS).
In some embodiments, the membrane(s) are washed with the saline at a temperature of about 25°C to about 45°C preferably about 37°C and maintained under shaking condition of about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 30 mins to about 2 hours, preferably about 1 hour.
In some embodiments, the saline comprises about 4 % NaCl to about 18% NaCl, preferably about 5.8 % NaCl.
In some embodiments, the antibiotic(s) is selected from a group comprising ofloxacin, pencillin, streptomycin and amphotericin B or any combination thereof.
In some embodiments, the membrane(s) are contacted with the antibiotic(s) at a temperature of about 25°C to about 37°C preferably about 37°C and maintained under shaking condition of, about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 30 mins to about 2 hours, preferably about 1 hour.
In some embodiments, in the preparation of the dried chorion membrane, prior to contacting the washed chorion membrane with antibiotics, the saline washed chorion membrane is treated with alkali and the alkali treated membrane is thereafter washed with buffer.
In a non-limiting embodiment, the alkali is selected from a group comprising Sodium Hydroxide (NaOH) and Potassium Hydroxide (KOH) or a combination thereof.
In some embodiments, the buffer for washing the alkali treated chorion membrane is selected from a group comprising Phosphate Buffer Saline (PBS), Hanks Balanced Salt Solution (HBSS) and Ringers Lactate, or any combination thereof.
In some embodiments, the membrane is contacted with the alkali at a temperature of about 25°C to about 45°C for about 1 minute to 30 minutes.
In some embodiments, the alkali treated membrane is washed with the buffer(s) at a temperature of about 25°C to about 45°C, preferably about 37°C and maintained under shaking conditions of about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 5 minutes to about 2 hours.
In some embodiments, the alkali is added to the membrane(s) post which the membrane is placed on a shaker at room temperature for about 1 minute. After about 1 minute, the alkali is decanted and addition of buffer is carried out, post which the membrane is again placed on the shaker at room temperature for about 5 minutes.
In some embodiments, in the preparation of the dried amnion membrane, the scraping is performed on the stromal side of the membrane to remove the spongy layer.
In some embodiments, in the preparation of the dried chorion membrane, the scraping is performed on both surfaces of the membrane to remove the trophoblast layer and the spongy layer.
In some embodiments, the washing after scraping of the membranes is performed with purified water, RO water, distilled water, double distilled water, deionized water or any combination thereof. In a preferred embodiment, the washing is performed with deionized water.
In some embodiments, said washing of the membrane(s) is carried out at 37°C and maintained under shaking condition about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 10 minutes to about 2 hours.
In some embodiments, after washing the membranes are dried in a hot air oven at a temperature of about 37°C to about 45°C, preferably about 37°C for about 2 hours to about 4 hours, preferably about 2 hours.
The present disclosure therefore provides a process for preparing dried amnion membrane comprising
separating the amnion and/or chorion membrane(s) from rest of the placental tissue;
washing the separated membrane with saline;
contacting the washed membrane with antibiotics(s);
scraping the stromal side of the membrane to remove the spongy layer;
washing the membrane lacking the spongy layer; and
drying the washed membrane to obtain the dried amnion membrane.
In another embodiment, the present disclosure provides a process for preparing dried chorion membrane comprising
separating the amnion and/or chorion membrane(s) from rest of the placental tissue;
washing the separated membrane(s) with saline;
treating the saline washed membrane with alkali;
washing the alkali treated membrane with buffer;
contacting the washed membrane with antibiotics(s);
scraping both sides of the membrane to remove the trophoblast layer and the spongy layer;
washing the membrane lacking the trophoblast layer and the spongy layer; and
drying the washed membrane to obtain the dried chorion membrane.
In an embodiment, the steps of the above methods may be performed in any order and each step of washing may be repeated about 2 to about 5 times.
Once the dried amnion and chorion membrane(s) are obtained, they are subjected to cryo-milling to obtain the amnion and chorion membrane(s) in powdered form. The cryo-milling may be performed by any method commonly practiced in the art. Without any specific restriction on the instruments or conditions employed, the cryo-milling is intended to yield a completely dry powder of the membrane(s) which can be stored and reconstituted in a suitable solvent for preparing a porous matrix therefrom. In some embodiments, the cryo-milling process may be performed in a single instrument or as two separate steps – i.e. freezing followed by powdering or crushing. In some embodiments, the amnion and chorion membrane(s) are subjected to cryo-milling together or separately. In case the cryo-milling of the amnion and chorion membrane(s) is performed separately, the powders of the amnion and chorion membrane(s) are combined post cryo-milling. In some embodiments, the cryo-milled membrane comprises the amnion and chorion membranes at a ratio ranging from about 99:1 to about 1:99, preferably about 2.5:1.
In an embodiment, the cryo-milling is performed for about 4 minutes to about 10 minutes.
In some embodiments, the cryo-milling is repeated about 2 times to about 4 times.
In some embodiments, post cryo-milling, the obtained powders are further subjected to drying at a temperature ranging from about 37°C to about 45°C, preferably about 37°C.
In some embodiments, the dried powder of the dried amnion and chorion membrane(s) is passed through a sieve having pore size 60 micron to about 300 micron, preferably about 200 micron.
In some embodiments, once obtained, the dried powder of the dried amnion and chorion membrane(s) is transferred to a sealed container such as but not limited to a centrifuge tube and the container is stored it in a cool dry place.
While the above embodiments describe the process to prepare cryo-milled membrane(s) comprising dried amnion and/or chorion membranes, similar process may be performed to prepare cryo-milled membrane(s) comprising one or more of amnion, chorion, maternal decidua and umbilical cord or any combination thereof.
In some embodiments, as defined above, the placental tissue may be processed into the form of a coarse dried powder or a non-dried tissue. Said forms of processed placental tissue may be prepared from a single placental tissue sample or a combination of multiple placental tissue samples.
In some embodiments, the coarse dried powder or non-dried tissue form of placental tissue is prepared by a method comprising
chopping the placental tissue;
washing the chopped placental tissue with solvent selected from a group comprising water, sodium dodecyl sulphate and ethanol or any combination thereof;
freezing the washed placental tissue; optionally lyophilizing the frozen tissue; and
optionally powdering the lyophilized tissue
to obtain the placental tissue in the form of a coarse dried powder or a non-dried tissue.
Steps of the aforesaid method may be performed in any order. In an embodiment, the steps of washing may be performed separately for each solvent and each such step of washing may be repeated about 2 times to about 5 times.
In some embodiments, the coarse dried powder form or non-dried tissue form of the placental tissue is prepared by
washing the placental tissue with water;
chopping the placental tissue;
washing the chopped placental tissue with solvent selected from a group comprising water, sodium dodecyl sulphate and ethanol or any combination thereof;
freezing the washed placental tissue;
optionally lyophilizing the frozen tissue; and
optionally powdering the lyophilized tissue
to obtain the placental tissue in the form of a coarse dried powder or a non-dried tissue.
In some embodiments, the coarse dried powder form or non-dried tissue form of the placental tissue is prepared by
washing the placental tissue with water;
chopping the placental tissue into fine pieces;
washing the chopped placental tissue with water;
washing the chopped placental tissue with sodium dodecyl sulphate, followed by water;
washing the chopped placental tissue with ethanol followed by water;
freezing the washed placental tissue;
optionally lyophilizing the frozen tissue; and
optionally powdering the lyophilized tissue
to obtain the placental tissue in the form of a coarse dried powder or a non-dried tissue.
As mentioned above, each step of washing in the afore-defined method may be repeated about 2 times to about 5 times.
In some embodiments, the sodium dodecyl sulphate is employed in the form of a solution in water; wherein the concentration of sodium dodecyl sulphate in the solution ranges from about 0.5% to about 1.5% preferably 1%. The step of washing with sodium dodecyl sulphate decellularizes the tissues. Therefore, the step of washing with sodium dodecyl sulphate may be referred to as decellularization of the tissue(s).
In some embodiments, the concentration of ethanol employed for washing ranges from about 70% to about 90%, preferably about 75%
In some embodiments, post the chopping, the pieces of tissue are washed with water under shaking condition about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 30 mins to about 2 hours, preferably about 1 hour.
Post water wash, the chopped pieces are subjected to decellularization treatment with Sodium Dodecyl Sulphate (SDS) under shaking condition about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 16 hours to about 48 hours.
Post Sodium Dodecyl Sulphate (SDS) treatment the tissue pieces are washed with water as described above and then in ethanol under shaking condition about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 12 hours to about 24 hours.
Further, after the step of washing with ethanol, the tissue pieces are washed with water about 2 times under shaking condition about 50 rpm to about 150 rpm, preferably about 100 rpm for a time period of about 30 mins to about 2 hours, preferably about 1 hour. The tissues are then transferred to a sterile container and stored at -200C.
In some embodiments, the freezing is carried out at a temperature ranging from about -20°C to about -80°C, for about 4 hours to about 48 hours.
In further embodiments, the lyophilization is carried out for about 16 hours to about 48 hours, preferably at least about 24 hours.
The step of powdering the lyophilized tissue pieces, in some embodiments, may be performed by any method routinely practiced in the art to crush or powder substances such as but not limited to manual crushing or subjecting to grinding or milling.
Stopping the above method at freezing yields the placental tissue in the form of a non-dried tissue for further processing into a porous matrix.
In some embodiments, the non-dried tissue form of the placental tissue is prepared by
washing the placental tissue with water;
chopping the placental tissue into fine pieces;
washing the chopped placental tissue with water;
washing the chopped placental tissue sodium dodecyl sulphate, followed by water;
washing the chopped placental tissue with ethanol followed by water; and
freezing the washed placental tissue
to obtain the placental tissue in the form of a non-dried tissue.
Lyophilization of the frozen tissue and powdering of the lyophilized tissue yields the placental tissue in the form of a coarse dried powder for further processing into a porous matrix.
In some embodiments, the coarse dried powder form of the placental tissue is prepared by
washing the placental tissue with water;
chopping the placental tissue into fine pieces;
washing the chopped placental tissue with water;
washing the chopped placental tissue sodium dodecyl sulphate, followed by water;
washing the chopped placental tissue with ethanol followed by water;
freezing the washed placental tissue;
lyophilizing the frozen tissue; and
powdering the lyophilized tissue
to obtain the placental tissue in the form of a coarse dried powder.
In some embodiments, the lyophilized tissue may be stored and powdered just before further use.
In some embodiments, once obtained, the frozen non-dried tissue or the coarse dried powder, is transferred to a sealed container such as but not limited to a centrifuge tube and the container is stored in a cool dry place, preferably at a temperature of about -20°C until further use.
Once the placental tissue has been processed by any of the above methods into a form selected from dried cryo-milled membrane(s), coarse dried powder or non-dried tissue, the processed placental tissue is further subjected to preparation of a porous matrix by lyophilization. The lyophilized porous matrix hence obtained is then be subjected to cross-linking as defined above.
The porous matrix may be prepared from any form of the placental tissue by any of the following process variants, all yielding a 3D porous matrix, having thickness ranging from about 0.1cm to about 2cm.
In some embodiments, the method of the present disclosure is directed towards preparation of porous matrix composed entirely of placental tissue or placental tissue in combination with other biomaterials.
In some embodiments, the processed placental tissue is combined with other biomaterials to prepare the porous matrix.
In some embodiments, preparing a porous matrix from the processed placental tissue comprises-
preparing a solution of another biomaterial;
preparing a suspension of the processed placental tissue and the biomaterial solution; and
subjecting the suspension to freezing followed by lyophilization obtain the porous matrix.
In some embodiments, the processed placental tissue employed in the above method is in the form selected from cryo-milled membrane(s), coarse dried powder and non-dried tissue.
In an exemplary embodiment, the processed placental tissue employed in the above method is in the form of dried cryo-milled membrane(s).
In an embodiment, the other biomaterial is selected from a group comprising cellulose, collagen, cellulose, gelatin, alginate, chitosan, silk, fibrin, guar gum and xanthum gum or any combination thereof. In some embodiments, the other biomaterial may further include other biomaterials which are capable of forming a porous matrix when pre-treated and subjected to lyophilization.
In an exemplary embodiment, the other biomaterial is gelatin.
In a non-limiting embodiment, the biomaterial solution is prepared by mixing the biomaterial with water, subjecting the mixture to agitation, optionally followed by heating at a temperature of about 50°C to about 60 °C for about 60 minutes to about 4 hours, preferably at about 60°C for about 2 hours.
In some embodiments, the biomaterial solution comprises the biomaterial at a concentration ranging from about 2% to about 5 %.
In some embodiments, the suspension of the processed placental tissue and the biomaterial solution comprises the processed placental tissue at a concentration ranging from about 1% to about 5% when the processed placental tissue is in the form of coarse dried powder and, at a concentration ranging from about 5% to about 25% when the processed placental tissue is in the form of a non-dried tissue.
In preferred embodiments, the processed placental tissue is in the form of a powder of the cryo-milled amnion and chorion membrane(s) and the suspension of the processed placental tissue and the biomaterial solution comprises the processed placental tissue at a concentration ranging from about 0.5% to about 10 % when the processed placental tissue is in the form of a powder of the cryo-milled membrane(s).
In some embodiments, the suspension is subjected to freezing at a temperature ranging from about -20°C to about -80°C for about 4 hours to about 48 hours.
In further embodiments, the frozen suspension is subjected to lyophilization for about 16 hours to about 48 hours, preferably 24 hours to obtain the porous matrix.
In some embodiments, preparing a porous matrix composed of 100% placental tissue comprises methods selected from lyophilization alone, and enzyme solubilization followed by lyophilization.
In an embodiment, preparing a porous matrix composed of 100% placental tissue from the processed placental tissue by lyophilization alone comprises -
mixing the processed placental tissue with a solvent to form a mixture;
homogenizing the mixture;
casting the homogenized mixture onto a mould; and
freezing followed by lyophilization of the contents of the mould
to obtain the porous matrix.
In some embodiments, the processed placental tissue employed in the above method is in the form selected from cryo-milled membrane(s), coarse dried powder and non-dried tissue.
In an exemplary embodiment, the processed placental tissue employed in the above method is in the form of coarse dried powder or non-dried tissue.
In another embodiment, when the processed placental tissue is in the form of non-dried tissue, it is thawed and then minced or chopped into fine pieces before mixing with the solvent to form a mixture.
In some embodiments, the solvent is selected from a group comprising purified water, RO water, distilled water, double distilled water, deionized water or any combination thereof.
In some embodiments, the suspension of the processed placental tissue comprises the processed placental tissue at a concentration ranging from about 1% to about 5% when the processed placental tissue is in the form of cryo-milled membrane(s) or coarse dried powder and, at a concentration ranging from about 5% to about 25% when the processed placental tissue is in the form of a non-dried tissue.
In some embodiments, the homogenization is performed on ice. In a non-limiting embodiment, the homogenization is performed for about 2 minutes to about 10 minutes, preferably about 5 minutes.
In some embodiments, the freezing is conducted at a temperature ranging from about -20°C to about -80°C, for about 4 hours to about 48 hours.
In some embodiments, the lyophilization is conducted for about 16 hours to about 48 hours, preferably about 24 hours.
In an embodiment, preparing a porous matrix composed of 100% placental tissue from the processed placental tissue by enzyme solubilization followed by lyophilization comprises
preparing a mixture of the processed placental tissue in an acid;
homogenizing the mixture to obtain a homogenized mixture;
subjecting the homogenized mixture to enzyme digestion;
adding cold buffer to the digested mixture;
neutralizing the mixture by addition of alkali;
casting the neutralized mixture onto a mould;
freezing of the contents of the mould; and
lyophilizing the frozen contents of the mould
to obtain the porous matrix.
In another embodiment, when the processed placental tissue is in the form of non-dried tissue, it is thawed and then minced or chopped into paste or fine pieces before mixing with the acid to form a mixture.
In some embodiments, the suspension of the processed placental tissue and the biomaterial solution comprises the processed placental tissue at a concentration ranging from about 1% to about 5% when the processed placental tissue is in the form of cryo-milled membrane(s) or coarse dried powder and, at a concentration ranging from about 5% to about 15% when the processed placental tissue is in the form of a non-dried tissue.
In some embodiments, the acid in the above method is selected from a group comprising hydrochloric acid (HCl) and acetic acid or a combination thereof.
In some embodiments, the homogenization is performed on ice. In a non-limiting embodiment, the homogenization is performed for about 2 minutes to about 10 minutes, preferably about 5 minutes.
In some embodiments, the digesting enzyme in the above method is selected from a group comprising pepsin and papain or a combination thereof. Depending on the enzyme used, pH of the mixture may be adjusted to favour its optimum activity before or after addition of the enzyme. In some embodiments, the enzyme digestion may be performed for about 48 hours to about 72 hours.
In some embodiments, the buffer in the above method is selected from a group comprising Phosphate Buffer Saline (PBS) and Hanks Balanced Salt Solution (HBSS) or a combination thereof.
In some embodiments, the alkali in the above method is selected from a group comprising sodium hydroxide (NaOH) and Potassium Hydroxide (KOH) or a combination thereof.
In some embodiments, the freezing is conducted at a temperature ranging from about -20°C to about -80°C, preferably about -20°C, for about 4 hours to about 48 hours.
In some embodiments, the lyophilization is conducted for about 16 hours to about 48 hours, preferably about 24 hours.
The porous matrix may be arrived at by applying any combination of the form of processed placental tissue and method for preparation of the porous matrix as described above. All said combinations yield a porous matrix, having a 3D, sponge like structure of a uniform thickness of about 0.1cm to about 2cm. In order for said structure to maintain integrity when put to application in facilitating tissue healing, the porous matrix is mandatorily subjected to cross-linking as defined in the above embodiments. The cross-linking yields the dressing of the present disclosure and lends to the dressing structural integrity even when put to application in an aqueous environment such as but not limited to deep wounds/body cavity.
In some embodiments, the dressing of the present disclosure finds application in wound healing or tissue regeneration. The dressing is capable of acting as a scaffold for cell attachment and cell growth and further facilitates granulation.
In an embodiment, the present disclosure relates to the use of the placental tissue derived cross-linked porous dressing of the present disclosure in the facilitating the treatment of deep chronic wounds such as but not limited to ulcers. Said use envisages both independent use of the dressing and its use in combination with Negative-Pressure Wound Therapy (NPWT).
In some embodiments, the present disclosure relates to an in-vitro method of promoting cell attachment and cell growth in a tissue comprising application of the of the placental tissue derived cross-linked porous dressing of the present disclosure to the tissue. In some embodiments, the tissue may be in a state corresponding to an injured tissue in vivo.
In some embodiments, the present disclosure further relates to an in-vitro method of promoting granulation in a tissue comprising application of the placental tissue derived cross-linked porous dressing of the present disclosure to the tissue. In some embodiments, the tissue may be in a state corresponding to an injured tissue in vivo.
In non-limiting embodiments, in the aforesaid methods, the tissues that the dressing is applied to are in vitro models of tissue injury or trauma.
In some embodiments, the placental tissue derived cross-linked porous dressing of the present disclosure promotes cell attachment, cell growth and granulation in tissues that have been damaged, injured or stressed due to any reason, and as a result comprise no cells or reduced number of cells when compared to original tissue in its healthy state. As the dressing of the present disclosure is a placental tissue derived dressing, it comprises growth factors and other cell signalling biomolecules that allows for enhanced cell to cell interaction and acts as a scaffold that promotes cell attachment, growth and granulation. The dressing of the present disclosure is therefore useful in treating or managing all conditions where it is needed to trigger tissue regeneration through cell infiltration.
While the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof has been shown by way of examples and drawings and are described in detail below. The Examples are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.
EXAMPLES
The placenta employed in the below Examples is discarded placenta procured from hospitals, with the consent of the donor. The procurement of placenta was approved by the Rangadore Ethics committee. The approval number obtained for the experiments described below id RMHEC/03/2015 dated 30/09/2015.
EXAMPLE 1: Preparation of placental tissue in the form of cryo-milled membrane
Amnion processing
The placenta was taken from the transport container and placed with the amnion side up in a sterile autoclaved stainless steel tray (SS tray).
The SS tray was filled with deionized water (DI H2O) and the placenta was gently massaged to remove the blood clots. Identifying an area from where the amniotic membrane could be separated from the chorion, the amniotic membrane layer was gently separated from the chorion using slow but continuous motion. Care was taken not to tear the membrane. In case of any tear, the separation process was started afresh.
The separated amnion membrane layer was placed in the autoclaved SS container with epithelial side facing up and about 200 ml of saline was added to the SS container. The SS container was thereafter position on a shaker set at a temperature of about 37°C and maintained under shaking condition of about 100 rpm for about 1 hour, after which the saline was decanted.
Another 200 ml of saline was then added to the SS container and the steps of placing on a shaker and decantation of saline were repeated.

After decantation of the saline, about 100 ml of antibiotic ofloxacin (200mg/100ml) was added to the SS container and the container was placed on a shaker set at a temperature of about 37°C and maintained under shaking condition of about 100 rpm for about 1 hour, after which the antibiotic was decanted.
After treatment with the antibiotic, the amnion was removed from the SS container and placed in a flat autoclaved SS tray with the epithelial side facing down.
The stromal side was scraped gently to remove the spongy layer, post which the amnion was placed in an SS container. About 200 ml of DI H2O at was added to the container.
This container was placed on a shaker set at 37°C and maintained, under shaking condition, of about 100 rpm, for about 10 minutes.
After about 10 minutes, the DI H2O was decanted and about 200 ml of DI H2O was added to the container. The step of placing on a shaker and decantation of the water was repeated twice.
Post decantation of the DI H2O, the amnion was spread on sterile teflon sheets with the epithelial side down. The sterile teflon sheets were placed in a hot air oven set at a temperature of about 37°C for about 2 hours. After drying, the dried membrane was removed gently from the teflon sheets. The dried membrane was transferred to a separate sterilized container and stored at a temperature of about 25°C.
Chorion Processing
The chorion was separated by cutting the chorion along the chorionic plate from the placenta from which amnion was removed already.
The chorion was placed in the autoclaved SS container with spongy side facing up and about 200 ml of 1M sodium chloride (NaCl) (saline) was added to the container. The container was placed on a shaker set at temperature of about 37°C and maintained, under shaking condition, of about 100 rpm for about 1 hour, post which the saline was decanted.
Another 200 ml of saline was then added to the SS container and the steps of placing on a shaker and decantation of saline were repeated.
After decantation of the saline, about 200ml of about 0.5M NaOH was added to the SS container, which was then placed on the shaker at room temperature (RT) for about 1 minute. After about 1 minute, the NaOH was decanted and about 200 ml of 1X PBS was added to the container. The container with the chorion membrane and PBS was placed on the shaker at room temperature for about 5 minutes, post which the PBS was decanted.
After decantation of the PBS, the chorion was removed from the SS container and placed in a flat autoclaved SS tray with the trophoblast layer facing up.
The chorion was scraped gently to remove the trophoblast layer, post which the chorion was flipped and subjected to scraping to remove the spongy layer.
After removal of spongy layer and trophoblast layer, the membrane was then placed in an SS container about 100 ml of antibiotic ofloxacin (about 200mg/100ml) was added to the SS container and the container was placed on a shaker set at a temperature of about 37°C and maintained under shaking condition of about 100 rpm for about 1 hour. The Ofloxacin was then decanted.
About 200 ml of DI H2O was added to the container set at 37°C and maintained, under shaking condition, of about 100 rpm for about 10 minutes.
After about 10 minutes, the DI H2O was decanted and about 200 ml of DI H2O was added to the container. The step of placing on a shaker and decantation of the water was repeated twice.
Post decantation of the DI H2O, the chorion was spread on sterile teflon sheets with the trophoblast layer down. The sterile teflon sheets were placed in a hot air oven set at a temperature of about 37°C for about 2 hours. After drying, the dried membrane was removed gently from the teflon sheets. The dried membrane was transferred to a separate sterilized container and stored at a temperature of about 25°C.
Cryo-milling of placental membranes
The dried amnion and chorion membranes prepared as described above were taken and cut into small pieces.
About 0.7gms of the pieces of the dried amnion and chorion membranes were loaded into a vial and cryo-milled using the following parameters: Cycles-1, Precool-5, Run Time-6 mins, Cool Time-2 mins, Rate-12 CPS.
After the run, the vial was removed. If big pieces were seen, some amount of the membrane was removed and the cryo-mill was run again.
The grinding step was repeated till all samples were ground. The ground powder was transferred to a centrifuge tube.
The powder was dried in a hot air oven set at a temperature of about 37°C for not more than about 1 hour with the cap of the tube open.
After drying, the lid of the tube was tightly closed and the tube was stored in a cool dry place.
The cryo-milled powder was passed through a 200 micron sieve and the the sieved powder was transferred to a new tube. The lid was closed tightly. The tube was labelled and stored in a cool dry place.
EXAMPLE 2: Preparation of placental tissue in the form of coarse dried powder
The placenta was taken from the transport container and placed with the amnion side up in a sterile autoclaved stainless steel tray (SS tray).
The SS tray was filled with 1000ml of deionized water (DI H2O) and the placenta was gently massaged to remove the blood clots, post which the DI H2O was discarded.
The steps of addition of DI H2O and massaging the placenta was repeated about two times.
After the DI H2O was discarded, the placental tissue was taken and chopped into fine pieces. These pieces were washed about 2 times in about 500ml of DI H2O in a shaker at about 100 rpm for about 1 hour each.
After washing with DI H2O, the fine pieces of membrane were washed in 500ml of 1% Sodium Dodecyl Sulphate (SDS) made in DI H2O in a shaker at about 100 rpm, for about 16 hours.
After said step of washing with SDS, these pieces were again washed about 3 times in about 500ml of DI H2O in a shaker at about 100 rpm for about 1 hour each.
The tissue pieces were then washed with 500ml of about 75% ethanol overnight in shaker at 100rpm. After said step of washing with ethanol, these pieces were again washed about 2 times in 500ml of DI H2O in a shaker at about 100 rpm for about 1 hour each. The tissues were then transferred to a sterile container and stored at -200C.
The frozen tissues were thereafter transferred into a lyophilizer and subjected to lyophilization for at least about 24 hours. The lyophilized tissues were crushed into a coarse powder using scapel blade or scissors. The resultant dried coarse powder was removed and stored at about -200C until further use.
EXAMPLE 3: Preparation of placental tissue in the form of non-dried tissue
The placenta weighing about was taken from the transport container and placed with the amnion side up in a sterile autoclaved stainless steel tray (SS tray). Using sterile surgical blade, the umbilical cord was removed.
The SS tray was filled with 1000ml of deionized water (DI H2O) and the placenta was gently massaged to remove the blood clots, post which the DI H2O was discarded.
The steps of addition of DI H2O and massaging the placenta was repeated about two times.
After the DI H2O was discarded, the placental tissue was taken and chopped into fine pieces. These pieces were washed about 2 times in 500ml DI H2O in a shaker at about 100 rpm for about 1 hour each.
After washing with DI H2O, the fine pieces of membrane were washed in about 500ml of about 1% Sodium Dodecyl Sulphate (SDS) made in DI H2O in shaker at about 100 rpm for 16 hours.
After said step of washing with SDS, these pieces were again washed about 3 times in DI H2O in a shaker at about 100 rpm for about 1 hour each.
The tissue pieces were then washed with about 75% ethanol overnight in shaker at 100 rpm. After said step of washing with ethanol, these pieces were again washed about 2 times in 500ml of DI H2O in a shaker at about 100 rpm for about 1 hour each. The tissues were then transferred to a sterile container and stored at -200C.
EXAMPLE 4: Preparation of cross-linked porous matrix from placental tissue in the form of cryo-milled membrane
A sterile 500 ml glass bottle that had never been in contact with detergent or soap on was placed on the working bench.
About 20 g of gelatin powder was weighed and added to a labelled bottle in the laboratory hood. About 500 ml endotoxin-free water was added to the bottle and the bottle was swirled to mix the contents.
The solution was heated about 60°C for about 2 hours within about 2 hours after mixing. The resultant about 4% gelatin solution was cooled to room temperature on the bench. The about 4% gelatin solution was then stored in a bottle at a temperature of about 4°C to about 8°C in a refrigerator until use. The solution was used within two months of preparation.
Two clean glass plates and one spacer which corresponds to the required thickness of the porous matrix were taken to be used as a mould for setting the porous matrix. Non-stick film such as parafilm was affixed to the sides of glass plate. The parafilm was placed and secured on the contact surfaces of the glass plates. The spacer was placed between two glass plates such that both parafilm lined surfaces faced each other to form the innerside of the mould as shown in Figure 1. 3 kinds of separate moulds were prepared having thickness of 0.1 cm, 0.5 cm and 2 cm. The above-described mould was used for a thickness of 0.1 cm, 12 well plates were used for a thickness of 0.5cm and 5ml screw vials were used for a thickness of 2cm.
The gelatin stock solution was thawed at about 37°C for a period of about 1 hour. The cryo-milled membrane powder was weighed based on the concentration required and the amount of sponge solution to be prepared.
In order to prepare a solution comprising 0.5 % cryo-milled membrane powder, about 0.5 gm of the powder was weighed and added to about 100ml of the gelatin stock solution. Similarly, solutions comprising 2% and 4 % powder were prepared.
The cryo-milled membrane powder was mixed in gelatin solution and mixing was performed by shaking for about 5 minutes till a homogeneous suspension was obtained.
The sponge solution was poured in the cavity of the mould (separate moulds of each thickness for solutions having concentration of placental powder – 0.5%, 2%, 4%), and immediately after filling, the mould was placed in a -80°C freezer for minimum of about 5 hours to about 6 hours.

The frozen sponge solution was removed from the mould and placed in a petri dish. The frozen sponge solution was lyophilized for at least about 16 hours.
Post lyophilization, the porous matrix was subjected to cross-linking. About 200ml of a cross-linking solution of about 1% EDC in about 90% ethanol was prepared. The lyophilized porous matrix was submerged in cross-linking solution and the submerged mixture was placed on a shaker for about 24 hours at room temperature. After about 24 hours, the porous matrix was taken out and washed with DI water two times in wash cycles of about 30 minutes each at 100 rpm.
After completion of washing, the porous matrix was taken out from the water and placed in a petri dish for subsequent freezing at about -20°C for about 5 hours to about 6 hours. The frozen cross-linked porous matrix was finally lyophilized for at least about 16 hours. The cross-linked porous matrix is seen in Figure 2. Figure 3 shows the SEM image of the dressing containing 0.5% placental tissue.
EXAMPLE 5: Preparation of cross-linked porous matrix from placental tissue in the form of coarse dried powder of amnion, chorion, maternal decidua and umbilical cord by lyophilization alone
The coarse dried powder of placental tissue as prepared in Example 2 was weighed out based on the concentration required and the amount of sponge solution and added to an appropriate amount of water.
In order to prepare a solution comprising 1% of the coarse dried powder of placental tissue, about 1 gm of the powder was weighed and added to about 100ml of water. In a similar manner, solutions having 2%, 3%, 4% and 5% concentration of the coarse dried powder of placental tissue were prepared – each to be set separately.
The solution was homogenized on ice for about 5 minutes. The homogenized solution was cast into petri dishes.
The petri dishes were divided into two batches – one batch was subjected to freezing at -20°C or -80°C for at least 4 hours.
The frozen sponge solution was lyophilized for at least about 16 hours, preferably about 24 hours.
Post lyophilization, the porous matrix was subjected to cross-linking. About 500 ml of a cross-linking solution of about 1% EDC in about 90% ethanol was prepared. The lyophilized porous matrix was submerged in the cross-linking solution and the submerged mixture was placed on a shaker for about 24 hours at room temperature. After about 24 hours, the porous matrix was taken out and washed with DI water two times in two wash cycles of about 30 minutes each.
After completion of washing, the porous matrix was taken out from the water and placed in a petri dish for subsequent freezing at about -20°C for about 5 hours to about 6 hours. The frozen cross-linked porous matrix was finally lyophilized for at least about 16 hours.
Figures 4 and 5 depict the physical appearance of the dressing made from coarse dried powder at all tested concentrations of the processed placental tissue by freezing at -20°C and -80°C before lyophilization and cross-linking. Figure 6 depicts SEM images the dressings made from coarse dried powder by freezing at -20°C before lyophilization.
EXAMPLE 6: Preparation of cross-linked porous matrix from placental tissue in the form of non-dried tissues of amnion, chorion, maternal decidua and umbilical cord by lyophilization alone
Frozen tissue as prepared in Example 3 was thawed and minced into fine pieces using scalpel blade or scissors.
The minced tissue was weighed out based on the concentration required and the amount of sponge solution and added to an appropriate amount of water.
In order to prepare a solution comprising 5% placental tissue, about 5 gm of the minced tissue was weighed and added to about 100ml of water. In a similar manner, solutions having 10% 15%, 20% and 25% concentration of placental tissue were prepared – each to be set separately.
The solution was homogenized on ice for about 5 minutes. The homogenized solution was cast into petri dishes.
The petri dishes were divided into two batches – one batch was subjected to freezing at -20°C or -80°C for at least 4 hours.
The frozen sponge solution was removed from the mould and placed in a petri dish. The frozen sponge solution was lyophilized for atleast 16 hours
Post lyophilization, the porous matrix was subjected to cross-linking. About 500 ml of a cross-linking solution of about 1% EDC in about 90% ethanol was prepared. The lyophilized porous matrix was submerged in cross-linking solution and the submerged mixture was placed on a shaker for about 24 hours at room temperature. After about 24 hours, the porous matrix was taken out and washed with DI water two times in wash cycles of about 30 minutes each.
After completion of washing, the porous matrix was taken out from the water and placed in a petri dish for subsequent freezing at about -20°C for about 5 hours to about 6 hours. The frozen cross-linked porous matrix was finally lyophilized for at least about 16 hours.
Figures 7 and 8 depict the physical appearance of the dressing made from non-dried tissue at all tested concentrations of the processed placental tissue by freezing at -20°C and -80°C before lyophilization and cross-linking. Figure 9 depicts SEM images the dressings made from non-dried tissue by freezing at -20°C before lyophilization.
EXAMPLE 7: Preparation of cross-linked porous matrix by enzyme solubilization of coarse dried powder of amnion, chorion, maternal decidua and umbilical cord followed by lyophilization
The placental powder as prepared in Example 2 was weighed out based on the concentration required and the amount of sponge solution and added to an appropriate amount of water.
In order to prepare a solution comprising 3% of the coarse dried powder of placental tissues, about 3 gm of the powder was weighed and added to about 100ml of 0.1N HCl and the mixture was allowed to sit for about 1 hour. In a similar manner, solutions having 2%, 3%, 4% and 5% concentration of the coarse dried powder of placental tissue were prepared – each to be set separately.
The mixture was homogenized on ice for about 5 minutes. pH of the mixture was adjusted to 2 and pepsin ( 10% to the weight of the placental tissue) was added to the mixture. The tissue was allowed to be digested for about 48 hours. About 10ml of cold 10X PBS was added to the digested mixture. pH of the mixture was neutralized to about 7.4 using about 9 ml 1N NaOH.
The homogenized solution was cast into petri dishes. The petri dishes were divided into two batches – one batch was subjected to freezing at -20°C or -80°C for at least 4 hours.
The frozen sponge solution was lyophilized overnight for at least 16 hours.
Post lyophilization, the porous matrix was subjected to cross-linking. About 500 ml of a cross-linking solution of about 1% EDC in about 90% ethanol was prepared. The lyophilized porous matrix was submerged in cross-linking solution the submerged mixture was placed on a shaker for about 24 hours at room temperature. After about 24 hours, the porous matrix was taken out and washed with DI water two times in wash cycles of about 30 minutes each.
After completion of washing, the porous matrix was taken out from the water and placed in a petri dish for subsequent freezing at about -20°C for about 5 hours to about 6 hours. The frozen cross-linked porous matrix was finally lyophilized for at least about 16 hours.
Figures 10 and 11 depict the physical appearance of the dressing made from the coarse dried powder at all tested concentrations of the processed placental tissue by freezing at -20°C and -80°C before lyophilization and cross-linking. Figure 12 depicts SEM images the dressings made from coarse dried powder by freezing at -20°C before lyophilization.
EXAMPLE 8: Preparation of cross-linked porous matrix by enzyme solubilization of non-dried amnion, chorion, maternal decidua and followed by lyophilization
Frozen tissue as prepared in Example 3 was thawed and minced into fine pieces using scalpel blade or scissors.
The minced tissue was weighed out based on the concentration required and the amount of sponge solution and added to an appropriate amount of water.
In order to prepare a solution comprising 5% of the placental tissue, about 5 gm of the minced tissue was weighed and added to about 100ml of 0.1N HCl and the mixture was allowed to sit for about 1 hour. In a similar manner, solutions having 10% 15%, 20% and 25% concentration of placental tissue were prepared – each to be set separately.
The solution was homogenized on ice for about 5 minutes. The homogenized solution was cast into petri dishes.
The mixture was homogenized on ice for about 5 minutes. pH of the mixture was adjusted to 2 and about pepsin (2% to the weight of the placental tissue) was added to the mixture. The tissue was allowed to be digested for about 48 hours. About 10 ml of cold 10X PBS was added to the digested mixture. pH of the mixture was neutralized to about 7.4 using about 9 ml 1N NaOH.
The homogenized solution was cast into petri dishes. The petri dishes were divided into two batches – one batch was subjected to freezing at -20°C or -80°C for at least 4 hours.
The frozen sponge solution was removed from the mould and placed in a petri dish. The frozen sponge solution was lyophilized for at least 16 hours.
Post lyophilization, the porous matrix was subjected to cross-linking. About 500ml of a cross-linking solution of about 1% EDC in about 90% ethanol was prepared. The lyophilized porous matrix was submerged in cross-linking solution and the submerged mixture was placed on a shaker for about 24 hours at room temperature. After about 24 hours, the porous matrix was taken out and washed with DI water two times in wash cycles of about 30 minutes each.
After completion of washing, the porous matrix was taken out from the water and placed in a petri dish for subsequent freezing at about -20°C for about 5 hours to about 6 hours. The frozen cross-linked porous matrix was finally lyophilized at a at least about 16 hours.
Figures 13 and 14 depict the physical appearance of the dressing made from non-dried tissue at all tested concentrations of the processed placental tissue by freezing at -20°C and -80°C before lyophilization and cross-linking. Figure 15 depicts physical appearance of the dressings made from non-dried tissue by solubilizing and freezing at -20°C before lyophilization.
EXAMPLE 9: Physical Properties of the dressing
The thickness and porosity of the dressings of the present disclosure as obtained in Examples 4-8 were measured and are provided in Tables 1 and 2 below.
Table 1: Thickness of the dressings of the present disclosure
Form of processed placental tissue used as starting material Processing method (prior to cross-linking) Concentration of processed placental tissue in sponge solution Thickness (cm)
Cryo-milled membrane Mixing with other biomaterial before lyophilization 0.5%
2%
4 % 0.561±0.003
0.57±0.004
0.576±0.001
Coarse dried powder Lyophilization alone after freezing at -20°C. 1%
2%
3%
4%
5% 0.325±0.019
0.391±0.023
0.577±0.076
0.550±0.045
0.541±0.046
Coarse dried powder Lyophilization alone after freezing at -80°C. 1%
2%
3%
4%
5% 0.298±0.0273
0.38251±0.063
0.404±0.0711
0.490±0.084
0.503±0.056
Non-dried tissue Lyophilization alone after freezing at -20°C. 5%
10%
15%
20%
25% 0.318±0.007
0.537±0.04
0.588±0.06
0.520±0.091
0.530±0.075
Non-dried tissue Lyophilization alone after freezing at -80°C. 5%
10%
15%
20%
25% 0.270±0.02
0.507±0.07
0.585±0.05
0.510±0.06
0.5190±0.09
Coarse dried powder Enzyme solubilization and freezing at -20°C 1%
2%
3%
4%
5% 0.439±0.03
0.417±0.08
0.478±0.07
0.490±0.10
0.486±0.11
Coarse dried powder Enzyme solubilization and freezing at -80°C 1%
2%
3%
4%
5% 0.357±0.06
0.449±0.09
0.462±0.06
0.484±0.16
0.473±0.23
Non-dried tissue Enzyme solubilization and freezing at -20°C 5%
10%
15%
20%
25% 0.484±0.02
0.478±0.05
0.522±0.08
0.490±0.09
0.515±0.04
Non-dried tissue Enzyme solubilization and freezing at -80°C 5%
10%
15%
20%
25% 0.357±0.06
0.449±0.09
0.512±0.12
0.490±0.03
0.487±0.06

As can be seen from Table 1, all process variants described above yield a dressing of thickness ranging from about 0.29cm to about 0.58cm.
Table 2: Porosity of the dressings of the present disclosure
Form of processed placental tissue used as starting material Processing method (prior to cross-linking) Concentration of processed placental tissue in sponge solution Porosity (%)
Cryo-milled membrane Mixing with other biomaterial before lyophilization 10%;
33%;
50% 96.2±0.28;
94.6±1.1;
93.1±0.77
Coarse dried powder Simple lyophilization after freezing at -20°C. 1%
2%
3%
4%
5% 99.1±0.11
98.5±0.35
97.8±0.77
97.1±0.81
96.36±0.46
Coarse dried powder Simple lyophilization after freezing at -80°C. 1%
2%
3%
4%
5% 98.9±0.16
97.2±0.17
96.8±0.88
96.1±0.19
95.8±0.09
Non-dried tissue Simple lyophilization after freezing at -20°C. 5%
10%
15%
20%
25% 99.2±0.34
97.7±0.22
96.5±0.42
95.9±0.58
95.5±0.41
Non-dried tissue Simple lyophilization after freezing at -80°C. 5%
10%
15%
20%
25% 98.9±0.45
97.5±0.30
96.0±0.13
95.5±0.62
95.2±0.21
Coarse dried powder Enzyme solubilization and freezing at -20°C 1%
2%
3%
4%
5% 99.3±0.04
98.5±0.15
97.6±0.51
96.8±0.23
95.9±0.31
Coarse dried powder Enzyme solubilization and freezing at -80°C 1%
2%
3%
4%
5% 99.2±0.21
98.1±0.36
97.2±0.62
96.5±0.78
95.5±0.21
Non-dried tissue Enzyme solubilization and freezing at -20°C 5%
10%
15%
20%
25% 99.1±0.16
98.6±0.23
97.8±0.17
96.5±0.32
95.8±0.47
Non-dried tissue Enzyme solubilization and freezing at -80°C 5%
10%
15%
20%
25% 99.2±0.26
98.3±0.15
97.4±0.17
96.2±0.33
95.8±0.25

As can be seen from Table 2, all process variants described above yield a dressing of porosity ranging from about 93.1% to about 99.3%.
EXAMPLE 10: Cytotoxicity assay of dressing of the present disclosure
An established cell line of L-929 mouse fibroblasts was used for evaluation of cytotoxicity of the dressing. Sponge conditioned media were prepared by incubating 0.1 gm of gamma sterilized dressing as prepared in Example 5 in about 1ml of DMEM media with about 10% FBS for about 24 hours at about 37°C in sterile conditions. The L-929 cells were seeded in 12 well plates at a density of about 100K cells per well and cultured for about 24 hours. The media was removed from the wells and cells were incubated with either sponge conditioned media + fresh media at a ratio of 1:3 or control/normal media. After about 48 hours, the cell morphology was observed under optical microscope. The cells morphology of L929 in both sponge conditioned media and controls were similar (Figure 17). The cell viability was also compared between conditioned media using alamar dye assay. No difference in % alamar blue was observed in L929 cells incubated with sponge conditioned media and controls (Figure 18). Observations from cell morphology and alamar blue assay demonstrated that the dressing of the present disclosure is not cytotoxic.
EXAMPLE 11: Cell attachment and cell growth properties of dressing of the present disclosure
Sterilized pieces of the dressing of the present disclosure as prepared in Example 7 were seeded either with about 1×105 cells/ml of human amnion epithelial cells or about 1×106 cells/ml of human dermal fibroblasts and cultured for up to about 5 days. The cells in the scaffold were stained with the fluorescent dye Fluorescein Diacetate and imaged in a fluorescence microscope. At day 1, it was evident that both epithelial cells and fibroblast cells were able to attach to the dressing (Figure 19). After 5 days, the number of epithelial and fibroblast cells were higher in the scaffolds, indicating the proliferation of cells.
Thus, the dressing of the present disclosure is capable of acting as scaffold which has cell attachment and cell growth properties.
EXAMPLE 12: Efficacy of the dressing of the present disclosure in promoting healing of ulcer
A 66 year old male had a non-healing deep ulcer in the left foot with exposed tendon. The ulcers were treated with regular dressing for over 3 months with no improvement. After 3 months, the treatment module was changed to incorporate use of the dressing of the present disclosure in place of the regular dressing.
The ulcer was initially debrided to remove the slough and dead tissue and irrigated with saline. Figure 20A shows the regions of white area in debrided area which were devoid of granulation tissue and the presence of exposed tendon.
The dressing of the present disclosure as prepared in Example 6 was cut into the size and shape of the ulcer. The cut dressings were moistened in saline and then placed onto the ulcer (Figure 20B). Similarly, the black foam of the NPWT kit was cut into the size and shape of the ulcer and placed on top of the placental dressing. Transparent dressings were applied on top of the foam dressings and pressed down firmly, to ensure no air leaks. Then the drain tube was attached to the foam area. Suction tubing was then attached to canister and pressure of the NPWT unit was set to continuous suction of 120mmHg.
After ensuring that there were no leaks, the wound area was covered with gauzes. After about 7 days the patient underwent another round of NPWT. After about 6 days, the dressing was removed. The ulcer areas showed remarkable granulation (Figure 20C). The entire area had granulated well and the previously exposed tendon area was also covered by granulation tissue. The ulcer areas were grafted with split thickness graft. Uptake of the graft was excellent (Figure 20D). No local adverse event was observed with the application of the dressing.

EXAMPLE 13: Synergy between thickness and cross-linking in the dressing
In order to check and understand the importance of cross-linking and thickness of the dressing of the present disclosure, 2 dressings were prepared employing non-dried tissue as starting material. A solution comprising 10% of non-dried tissue was prepared and the dressing was prepared as per Example 8. For preparation of one set of dressings, the process was stopped before cross-linking.
Both of said dressings were exposed to aqueous media for about 5 minutes. At the end of said time period, when the said dressings were squeezed about 5 times, it was observed that the dressing devoid of cross-linking underwent dissolution in the aqueous media (Figure 20). This shows that in the absence of cross-linking, the efficacy of the dressings when applied to wounds/body cavities will reduce drastically since the structural integrity of the dressing cannot be maintained for sufficient period of time. ,CLAIMS:1. A placental tissue derived cross-linked porous dressing having thickness ranging from about 0.1cm to about 2cm.
2. The placental tissue derived cross-linked porous dressing as claimed in claim 1, wherein the dressing has uniform thickness across its length and breadth.
3. The placental tissue derived cross-linked porous dressing as claimed in claim 1, wherein the dressing is prepared from placental tissue as a whole or from fetal component(s) and/or maternal component(s) thereof.
4. The placental tissue derived cross-linked porous dressing as claimed in claim 3, wherein the fetal component(s) is selected from amnion membrane and chorion membrane or a combination thereof; and the maternal component is selected from maternal decidua and umbilical cord or a combination thereof.
5. The placental tissue derived cross-linked porous dressing as claimed in claim 1, wherein the dressing is composed completely of placental tissue or a combination of placental and other biomaterials selected from a group comprising collagen, cellulose, gelatin, alginate, chitosan, silk, fibrin, guar gum and xanthum gum or any combination thereof.
6. The placental tissue derived cross-linked porous dressing as claimed in claim 5, wherein the dressing comprises the placental tissue at a concentration ranging from about 0.5% to about 100% and the other biomaterial at a concentration ranging from about 0% to about 99.5%.
7. The placental tissue derived cross-linked porous dressing as claimed in claim 1, wherein the dressing has porosity ranging from about 90% to about 99.5%
8. A method for preparation of the placental tissue derived cross-linked porous dressing as claimed in claim 1, said method comprising
processing placental tissue into a form selected from a group comprising dried cryo-milled membrane, coarse dried powder and non-dried tissue;
preparing porous matrix from the processed placental tissue; and
subjecting the porous matrix to cross-linking to obtain the placental tissue derived cross-linked porous dressing.
9. The method as claimed in claim 8, wherein the dried cryo-milled membrane form of placental tissue is prepared by a method comprising
preparing dried amnion membrane from a placental tissue sample or preparing dried chorion membrane from a placental tissue sample or both; and
subjecting the dried membrane(s) to cryo-milling.
10. The method as claimed in claim 9, wherein preparing the dried amnion and/or chorion membrane(s) form of the placental tissue comprises
separating the amnion and/or chorion membrane from rest of the placental tissue;
washing the separated membrane with saline;
contacting the washed membrane with antibiotic(s);
scraping the membrane on one or both sides
washing the scraped membrane(s); and
drying the washed membrane(s) to obtain the dried amnion and/or chorion membrane(s).
11. The method as claimed in claim 10, wherein preparation of the dried chorion membrane further comprises -
treating the saline washed chorion membrane with alkali and
washing the alkali treated chorion with buffer prior to contacting the washed chorion membrane with the antibiotic(s).
12. The method as claimed in claim 10, wherein the washing of the scraped membrane(s) is performed with purified water, RO water, distilled water, double distilled water and deionized water or any combination thereof; wherein the antibiotic(s) is selected from a group comprising ofloxacin, pencillin, streptomycin and amphotericin B or any combination thereof.
13. The method as claimed in claim 11, wherein the alkali is selected from a group comprising Sodium Hydroxide (NaOH) and Potassium Hydroxide (KOH) or a combination thereof; and wherein the buffer for washing the alkali treated chorion membrane is selected from a group comprising Phosphate Buffer Saline (PBS) and Hanks Balanced Salt Solution or a combination thereof.
14. The method as claimed in claim 9, wherein the cryo-milling is performed for about 4 minutes to about 10 minutes.
15. The method as claimed in claim 8, wherein preparing the coarse dried powder or a non-dried tissue form of the placental tissue comprises
chopping the placental tissue;
washing the chopped placental tissue with solvent selected from a group comprising water, sodium dodecyl sulphate and ethanol or any combination thereof;
freezing the washed placental tissue;
optionally lyophilizing the frozen tissue; and
optionally powdering the lyophilized tissue
to obtain the placental tissue in the form of a coarse dried powder or a non-dried tissue.
16. The method as claimed in claim 15, wherein preparing the non-dried tissue form of the placental tissue comprises
washing the placental tissue with water;
chopping the placental tissue into fine pieces;
washing the chopped placental tissue with water;
washing the chopped placental tissue with sodium dodecyl sulphate, followed by water;
washing the chopped placental tissue with ethanol followed by water; and
freezing the washed placental tissue
to obtain the placental tissue in the form of a non-dried tissue.
17. The method as claimed in claim 15, wherein preparing the coarse dried powder form of the placental tissue comprises
washing the placental tissue with water;
chopping the placental tissue into fine pieces;
washing the chopped placental tissue with water;
washing the chopped placental tissue with sodium dodecyl sulphate, followed by water;
washing the chopped placental tissue with ethanol followed by water;
freezing the washed placental tissue;
lyophilizing the frozen tissue; and
powdering the lyophilized tissue
to obtain the placental tissue in the form of a coarse dried powder.
18. The method as claimed in claim 15, wherein the placental tissue is selected from a group comprising amnion, chorion, maternal decidua and umbilical cord or any combination thereof.
19. The method as claimed in claim 8, wherein preparing a porous matrix from the processed placental tissue comprises -
preparing a solution of another biomaterial;
preparing a suspension of the processed placental tissue and the biomaterial solution;
subjecting the suspension to freezing followed by lyophilization to obtain the porous matrix.
20. The method as claimed in claim 19, wherein the other biomaterial is selected from a group comprising collagen, cellulose, gelatin, alginate, chitosan, silk, fibrin, guar gum and xanthum gum or any combination thereof; and wherein the biomaterial solution comprises the biomaterial at a concentration ranging from about 2% to about 5%.
21. The method as claimed in claim 19, wherein the processed placental tissue is in the form of dried cryo-milled membrane(s); and wherein the suspension of the processed placental tissue and the biomaterial solution comprises the processed placental tissue in the form of cryo-milled membrane(s) at a concentration ranging from about 0.5% to about 10%.
22. The method as claimed in claim 8, wherein preparing a porous matrix from the processed placental tissue comprises
mixing the processed placental tissue with a solvent to form a mixture;
homogenizing the mixture;
casting the homogenized mixture onto a mould; and
freezing followed by lyophilization of the contents of the mould
to obtain the porous matrix.
23. The method as claimed in claim 22, wherein the solvent is selected from a group comprising purified water, RO water, distilled water, double distilled water and deionized water or any combination thereof.
24. The method as claimed in claim 8, wherein preparing a porous matrix from the processed placental tissue comprises
preparing a mixture of the processed placental tissue in an acid;
homogenizing the mixture to obtain a homogenized mixture;
adding a digesting enzyme to the homogenized mixture;
adding cold buffer to the digested mixture;
neutralizing the mixture by addition of alkali;
casting the homogenized mixture onto a mould;
freezing of the contents of the mould; and
lyophilizing the frozen contents of the mould
to obtain the porous matrix.
25. The method as claimed in any one of claims 22 or 24, wherein the processed placental tissue is in the form of coarse dried powder or non-dried tissue.
26. The method as claimed in claim 22 or 24, wherein the mixture of processed placental tissue and solvent or the mixture of processed placental tissue and acid comprises the processed placental tissue at a concentration ranging from about 1% to about 5% when the processed placental tissue is in the form of cryo-milled membrane(s) or coarse dried powder; wherein the mixture of processed placental tissue and acid comprises the processed placental tissue at a concentration ranging from about 5% to about 25% when the processed placental tissue is in the form of a non-dried tissue.
27. The method as claimed in claim 24, wherein the acid is selected from a group comprising hydrochloric acid (HCl) and acetic acid or a combination thereof; wherein the digesting enzyme is selected from a group comprising pepsin and papain or a combination thereof; wherein the buffer is selected from a group comprising Phosphate Buffer Saline (PBS) and Hanks Balanced Salt Solution or a combination thereof; and wherein the alkali is selected from a group comprising sodium hydroxide (NaOH) and Potassium Hydroxide (KOH) or a combination thereof.
28. The method as claimed in any one of claims 22 or 24, wherein the homogenization is performed on ice; and the homogenization is performed for about 2 minutes to about 10 minutes, preferably about 5 minutes.
29. The method as claimed in claim 8, wherein subjecting the porous matrix to cross-linking comprises -
submerging the porous matrix in cross-linking solution;
washing the porous matrix treated with the cross-linking solution; and
subjecting the washed porous matrix to freezing followed by lyophilization to obtain the placental tissue derived cross-linked porous dressing.
30. The method as claimed in claim 29, wherein the cross-linking solution comprises about 0.5% to 10% cross-linking agent selected from a group comprising EDC, Glutaraldehyde, Genepin, Transglutaminase and Dialdehyde Starch or any combination thereof; wherein the cross-linking solution is prepared in a solvent selected from a group comprising ethanol, isopropanol and water or any combination thereof; wherein
the porous matrix is submerged in cross-linking solution for about 4 hours to 36 hours; and wherein the porous matrix treated with the cross-linking solution is washed with water.
31. The method as claimed in any one of claims 15, 19, 22, 24 or 29 wherein the freezing is at a temperature ranging from about -20°C to about -80°C and time period ranging from about 4 hours to about 48 hours.
32. The method as claimed in any one of claims 15, 19, 22, 24 or 29 wherein the lyophilization is for time period ranging from about 16 hours to about 48 hours.
33. An in-vitro method of promoting cell attachment and cell growth in an injured tissue comprising application of the placental tissue derived cross-linked porous dressing as claimed in claim 1 to the tissue.
34. An in-vitro method of promoting granulation in an injured tissue comprising application of the of the placental tissue derived cross-linked porous dressing as claimed in claim 1 to the tissue.