FORM 2
THE PATENTS ACT, 1970
[39 OF 1970]
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]
“PROCESSING AND PRESERVATION OF ACELLULAR TISSUE GRAFT, AND APPLICATIONS THEREOF”
NAME OF THE APPLICANT:
NOVO TISSUE BANK AND RESEARCH CENTRE PRIVATE LIMITED
ADDRESS:
Ground floor, Anita Apartments, Shivaji Nagar Road, Vakola, Santacruz East, Mumbai – 400055
NATIONALITY: IN
The following specification particularly describes the invention and the manner in which it is to be performed.

TECHNICAL FIELD
The present disclosure relates to the field of tissue processing. The present disclosure particularly relates to a process of preparation and preservation of an acellular tissue graft, the acellular tissue graft obtained by said process, and a kit for preparing and preserving the acellular tissue graft.
BACKGROUND OF THE DISCLOSURE
The ever increasing and the ageing yet active population represents increased incidences of rotator cuff tears, large tendon ruptures and burn injuries. The incidence of rotator cuff tears in India is increasing drastically over the years. It is estimated that during the last one year alone, the incidence of rotator cuff tears in India is about 39,000, and about 12,000 surgeries are carried out every year for fixing rotator cuff tears out of which about 1/3rd of the surgeries require grafts. Similarly, other tissue injuries requiring grafts are increasing.
The currently available options for soft tissue defect reconstruction include split thickness skin grafts, local skin flap coverage techniques, free tissue transfer, autografts and xenografts. Each have its own set of disadvantages like donor site morbidity, risk of flap/graft complication, graft rejection etc. Some defects cannot rely on autografts at all. Apart from donor site morbidity, the use of autografts for massive rotator cuff tears is accompanied by a high rate of post-operative retears due to increased tension and stiffness at the reconstruction site.
An allograft is intended for use as an “onlay” on top of a repair or as a bridge between tissues for various reconstructive surgeries including but not limited to burn injuries, orthopaedic surgeries, plastic surgery, chronic wounds, oral surgery, abdominal surgery, otolaryngeology. The use of these grafts eliminates or decreases the need for an autograft whose use is often criticised for additional surgical morbidity and time. The need for enhanced outcomes of biologic augmentations in reconstructive surgeries necessitates development of safe, effective and affordable allograft choice.

Acellular tissue matrix or scaffolds are generally derived from human skin, tendons or from animal origin and more recently through plant origin. These grafts offer the versatility to repair numerous reconstructive defects. An acellular tissue graft is typically devoid of cellular components that cause an exuberant inflammatory response. An ideal acellular matrix or scaffold for tissue repair should be biologic, sterile, non-inflammatory, and have sufficient suture retention strength.
Post retrieval of the tissue from its source, the currently available techniques for obtaining and preserving acellular tissue grafts/scaffolds possess limitations in terms of risk of infection, risk of immunogenic reaction that leads to graft rejection, limited customization, difficulty in storage, requiring pre-operative preparation, poor/reduced structural and biomechanical properties as compared to the native tissues, and increased cost due to additional steps. For instance, employing endonucleases such as DNAse and/or RNAse in decellularization step makes the procedure cost-intensive and also increases overall time required for decellularization. Further, inefficient washing of the tissues with washing solutions after endonuclease treatment may lead to an incompatibility of graft tissue in the host and elicit an immunogenic response in vivo. Further, commonly employed preservation techniques such freeze-drying or cryopreservation at -80°C, are not only expensive but also affect the structural integrity and mechanical strength of the tissue. Alternatively, preservation of decellularized tissue using solvents/chemicals like acetone, alcohol, and/or sodium benzoate causes irreversible denaturation of the tissue scaffolds requiring rehydration for end applications.
Thus, there is a pressing need for efficient processing and preservation methods of acellular tissue grafts or scaffolds wherein said acellular tissue grafts can be later used for effective reconstruction of tissue defects. The present disclosure overcomes the said need for efficient processing and preservation of acellular tissue grafts/scaffolds.

SUMMARY OF THE DISCLOSURE
Accordingly, the present disclosure relates to a process for preparation and preservation of an acellular tissue graft comprising:
- subjecting a tissue to decellularization with:
a) first decellularization agent comprising a hypertonic solution or a
hypotonic solution, and
b) a second decellularization agent comprising a detergent selected from
anionic detergent, or non-ionic detergent, or a combination thereof, to prepare
the acellular tissue graft;
and
- subjecting said acellular tissue graft to preservation with a preservant
comprising a hygroscopic solvent.
The present disclosure further relates to an acellular tissue graft prepared and preserved according to the process described above.
The present disclosure also relates to a kit for preparing and preserving an acellular tissue graft comprising:
- a first decellularization agent comprising a hypertonic solution or a hypotonic solution;
- a second decellularization agent comprising a detergent selected from one or more of anionic detergent, non-ionic detergent and a combination thereof;
- a preservant comprising a hygroscopic solvent; and
- a package insert or label comprising instructions for preparing and preserving the acellular tissue graft according to the process described above.
The present disclosure additionally relates to use of the acellular tissue graft prepared and preserved according to the process described above for reconstructive orthopaedics, craniofacial or maxillofacial surgery, dentistry, plastic surgery and wound care.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the present 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, where:
Figure 1 illustrates an embodiment/schematic of the present process comprising the processing (preparation and preservation) of human full thickness skin graft (FTSG) to obtain human acellular dermal allograft (processed human dermis).
Figure 2 illustrates the preparation and preservation of human full thickness skin graft (FTSG), wherein: (A) is an image of human full thickness skin graft [biological tissue]; (B) is an image of human acellular dermal graft obtained after treatment with combination of hypertonic solution (NaCl) and mild anionic detergent (SDS) showing no epidermis; and (C) is an image of ready to be packaged human acellular dermal graft obtained after preservation (treatment with hygroscopic solvent - glycerol).
Figure 3 provides light microscope images of H & E-stained dermal samples. Figure 3(A) and (B) provide H & E-stained images of control sample (unprocessed FTSG). Figure 3(C) and (D) provide H & E-stained images of the present glycerol preserved human acellular dermal graft (gly-HAD) sample. Figure 3(E) provides H & E-stained image of freeze-dried human acellular dermal graft (FD-HAD) sample.
Figure 4 provides images of biomechanical evaluation of processed glycerol preserved human acellular dermal graft (gly-HAD) samples on Universal testing machine, wherein (A) is an image of the present irradiated gly-HAD clamped onto the UTM grippers; (B) is an image of the present irradiated gly-HAD with single end vertical suture (i.e., single end sutured gly-HAD) subjected to suture retention test; and (C) is an image of the present irradiated gly-HAD with vertical sutures on

both ends (i.e., double end sutured gly-HAD) subjected to suture retention strength test.
Figure 5 is graph showing ultimate load-to-failure comparison between the present glycerol preserved human acellular dermal graft (gly-HAD) sample and freeze-dried human acellular dermal graft (FD-HAD) sample.
Figure 6 is a graph showing suture retention strength evaluation for single end sutured gly-HAD and double end sutured gly-HAD.
Figure 7 shows a schematic representation of mechanism of replacement of water molecules by glycerol molecules and formation of the intrachain hydrogen bonds with the collagen fiber of the acellular dermal graft.
DESCRIPTION OF THE DISCLOSURE
As used herein, the term ‘decellularization’ refers to a process of eliminating cells and their components (e.g., nuclear and cellular debris especially DNA, RNA, etc.) from the extracellular matrix (ECM) of a biological tissue to yield an acellular ECM scaffold.
As used herein, the phrases ‘acellular tissue graft’, ‘acellular tissue scaffold’, ‘acellular graft’, ‘acellular scaffold’, ‘decellularized tissue’, ‘decellularized graft’ or the likes are used interchangeably and refer to the result/product of the decellularization process.
The present disclosure relates to the processing and preservation of acellular tissue graft. The process is efficient, simple, cost effective and provides an acellular tissue graft having desired biomechanical strength with retained acellular matrix ultrastructure. The acellular tissue graft provides for effective reconstruction of tissue defects including but not limited to rotator cuff tears, burns and wounds, and oral recession defects.

The present disclosure particularly provides a process of preparation and preservation of an acellular tissue graft comprising:
- subjecting a tissue to decellularization with:
a) first decellularization agent comprising a hypertonic solution or a
hypotonic solution, and
b) a second decellularization agent comprising a detergent selected from
one or more of anionic detergent, non-ionic detergent and a combination
thereof, to prepare the acellular tissue graft;
and
- subjecting said acellular tissue graft to preservation with a preservant
comprising a hygroscopic solvent.
In the present process, the decellularization of the biological tissue by the specific combination of hypertonic or hypotonic treatment along with detergent treatment is highly effective in removal of nuclear and cellular debris, and the preservation through a hygroscopic solvent such as glycerol enables long-term storage of the graft at room temperature in hydrated condition thereby preserving the strength while reducing the pre-op graft preparation time in surgical applications.
In some embodiments of the process, the first decellularization agent comprises the hypertonic solution or the hypotonic solution at a concentration ranging from about 0M to about 2M, including all the values in the range, for instance, about 0M, about 0.01M, about 0.02M, about 0.03M, about 0.04M, about 0.05M, about 0.06M, about 0.07M, about 0.08M, about 0.09M, about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.9M, or about 2M and including subranges of the range between 0.05M to about 2M.
In some embodiments, the hypertonic solution is at a concentration ranging from about 1M to about 2M, including all the values in the range. In some embodiments,

the hypertonic solution is at a concentration of above 0.15M to about 2M, including all the values in the range.
In some embodiments, the hypotonic solution is at a concentration ranging from about 0.05M to about 0.07M, including all the values in the range. In some embodiments, the hypotonic solution is at a concentration of 0M to below 0.15M, including all the values in the range.
In some embodiments, the hypertonic solution is selected from a group comprising sodium chloride (NaCl) solution, potassium chloride (KCl) solution, magnesium chloride (MgCl2) solution, calcium chloride (CaCl2) solution, potassium phosphate (K3PO4) solution, sodium phosphate (Na3PO4) solution, magnesium phosphate [Mg3(PO4)2] solution and combinations thereof. In some embodiments, the hypotonic solution is selected from a group comprising sodium chloride (NaCl) solution, purified water, tris-HCl, potassium chloride (KCl) solution, magnesium chloride (MgCl2) solution, calcium chloride (CaCl2) solution, potassium phosphate (K3PO4) solution, sodium phosphate (Na3PO4) solution, magnesium phosphate [Mg3(PO4)2] solution.
In some embodiments, the first decellularization agent is a 0.05M to 2M NaCl solution.
In some embodiments, the first decellularization agent is a 1M to 2M hypertonic solution.
In some embodiments, the first decellularization agent is a hypertonic solution which is 1M to 2M NaCl. In some embodiments, the first decellularization agent is a hypertonic solution which is 1.2M to 1.8M NaCl. In some embodiments, the first decellularization agent is a hypertonic solution which is 1.4M to 1.6M NaCl. In some embodiments, the first decellularization agent is a hypertonic solution which is 1.5M NaCl.

In some embodiments, the first decellularization agent is a hypertonic solution which is 1M to 2M NaCl in water or a buffer. In some embodiments, the first decellularization agent is a hypertonic solution which is 1.5M NaCl in water or a buffer.
In some embodiments, the first decellularization agent is a hypotonic solution which is 0.01M to 0.1M Tris-HCl solution.
In some embodiments, the first decellularization agent is a hypotonic solution which is 0M purified water.
In some embodiments of the present process, the second decellularization agent comprises the detergent in an amount ranging from about 0.1% to about 1%, including all the values in the range, for instance, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0% and including subranges of the range between about 0.1% to about 1%.
In some embodiments, the anionic detergent is selected from a group comprising sodium dodecyl sulfate (SDS), sodium deoxycholate, ammnonium lauryl sulfate (ALS), sodium myreth sulfate and combinations thereof. In some embodiments, the non-ionic detergent is selected from a group comprising 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol (Triton X-100), polyoxyethylene (20) sorbitan monolaurate [Tween-20], cetyltrimethyl ammonium bromide (CTAB) and combinations thereof.
In some embodiments, the second decellularization agent is 0.1% to 1% anionic detergent. In some embodiments, the second decellularization agent is 0.1% to 1% SDS.

In some embodiments, the second decellularization agent is 0.2% to 0.8% anionic detergent. In some embodiments, the second decellularization agent is 0.2% to 0.8% SDS.
In some embodiments, the second decellularization agent is 0.4% to 0.6% anionic detergent. In some embodiments, the second decellularization agent is 0.4% to 0.6% SDS.
In some embodiments, the second decellularization agent is 0.5% anionic detergent. In some embodiments, the second decellularization agent is 0.5% SDS.
One of the crucial aspects of processing biological tissue for graft preparation is decellularization which involves elimination of cellular components with minimal adverse impact on the matrix ultrastructure. In order to effectively remove nuclear and cellular debris, the combination of hypertonic treatment with detergent treatment was used in the present process. Particularly, in the present process, the combination of first decellularization agent (hypertonic or hypotonic solution treatment) and second decellularization agent (detergent treatment) provides for complete de-epidermization of the biological tissue. For instance, employing hypertonic solutions (e.g., about 1M to 2M NaCl solution) create an osmotic gradient that drives the epidermal cells to shrink and disassociate from the dermis. Hypertonic solution further facilitates cytolysis and therefore resulting in decellularization. Coupling hypertonic or hypotonic solution treatment with detergent treatment (e.g., anionic detergent) lead to cell lysis and reduction in bioburden by solubilizing the peptidoglycan cell wall, phospholipid cell membrane and disrupting the nuclear material. In an embodiment, Figure 2 demonstrates processing of human full thickness skin graft, wherein Figure 2B depicts the human acellular dermal graft obtained after treatment with combination of hypertonic solution and mild anionic detergent.

In an embodiment, the Figures 3(A) to 3(D) provide images of histological evaluation of hematoxylin and eosin-stained dermal samples, wherein it is observed that the glycerol preserved acellular dermal grafts (Figures 3C and 3D) prepared according to the process of the present disclosure show absence of epidermis with intact collagen network similar to that of the control dermal graft (Figures 3A and 3B) indicating retained flexibility and strength and devoid of any nuclear remains presenting the acellular nature.
In some embodiments of the present process, the preservant is a hygroscopic solvent at a concentration ranging from about 25% to about 60% in a buffer or purified water, including all the values in the range, for instance, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, and so on and so forth, up until about 60% and including subranges such as about 25% to 45%, about 25% to 40%, and so on. In some embodiments, the hygroscopic solvent is selected from a group comprising glycerol, sorbitol, sucrose, glucose, mannitol, ethylene glycol, D-galactose and combinations thereof.
Acellular dermal matrix from human origin is composed of Collagen type I and III. These collagen structures are stabilized by hydrogen bonding. The collagen fiber has a triple-helix structure stabilized by two types of hydrogen bonds. The first type relates directly to the hydrogen bonds between two amino acids of collagen, in different chains, and the second one is related to hydrogen bonds formed between the water molecules with carbonyl and/or hydroxyl groups in inter and intra chain links. Therefore, water content plays a crucial role in maintaining the collagen structure. Dermal matrix has a total water content of about 73.3% ± 3.32 of its weight: which includes loose bound water and tightly bound water.

The purpose of preservation step is to ensure the water activity reduces to inhibit the growth of microorganisms and to maintain the hydrogen bonding that confers to the mechanical strength of the tissue for desired end application.
In some embodiments of the present process, the acellular tissue graft is subjected to preservation with about 25% to about 60% glycerol in a buffer or purified water.
In some embodiments of the present process, the acellular tissue graft is subjected to preservation with about 25% to 50% hygroscopic solvent. In some embodiments of the present process, the preservation is carried out with glycerol at a concentration of about 25% to 50%.
In some embodiments of the present process, the acellular tissue graft is subjected to preservation with about 25% to 40% hygroscopic solvent. In some embodiments of the present process, the preservation is carried out with glycerol at a concentration of about 25% to 40%.
In some embodiments of the present process, the acellular tissue graft is subjected to preservation with about 25% to 35% hygroscopic solvent. In some embodiments of the present process, the preservation is carried out with glycerol at a concentration of about 25% to 35%.
In some embodiments of the present process, the acellular tissue graft is subjected to preservation with about 30% hygroscopic solvent. In some embodiments of the present process, the preservation is carried out with 30% glycerol.
The inventors of the present disclosure have surprisingly identified that preservation of decellularized tissue with about 25% to about 60% glycerol, preferably about 25% to about 45% glycerol, replaces the tightly bound water and forms intrachain hydrogen bonds with the collagen fiber and thus maintains the integrity of the acellular matrix.

The water activity (aw) of 25% to 60% glycerol is lower than the aw of the tissue and therefore can replace water until it equilibrates with the relative water activity. So, the glycerol solution can absorb several grams of water before it reaches its saturation. Given the already diluted glycerol solution (25% to 60%, preferably 25% to 45%), at a room temperature of about 25°C with 40-50% of relative humidity, it is far from its saturation limit and can possibly replace the tightly bound water in the collagen matrix. Direct mixing of said glycerol solution with the decellularized tissue allows fastest replacement of the inherent water molecules with the glycerol molecules which compete with water to form hydrogen bonds with carbonyl and/or hydroxyl groups in collagen. The glycerol molecules displace water molecules by forming more stable or stronger hydrogen bonds due to its multiple hydroxyl groups and its ability to establish more extensive bonding networks.
In an embodiment, Figure 7 shows a schematic representation of mechanism of replacement of water molecules by glycerol molecules and formation of the intrachain hydrogen bonds with the collagen fiber of the acellular dermal graft.
The inventors of the present disclosure have further identified that preservation of decellularized tissue with about 25% to about 60% glycerol, preferably about 25% to about 45% glycerol provides bacteriostatic properties. Common skin flora such as Staphylococcus epidermidis, Staphylococcus aureus, Acinetobacter, Pseudomonas species, etc require a certain minimum water activity (ranging from about 0.86 to 0.91) for its growth. The preservation solution, containing about 25% to about 60% glycerol, have reduced water activity compared to the minimum water activity requirement of the skin microflora. Thus, employing said concentration of glycerol inhibits the growth of microorganisms.
In some embodiments, the average batch bioburden of the prepared and preserved acellular dermal graft samples of the present disclosure was found to be less than 1000 CFU/ graft. The bioburden result as verified by sterility testing was adequate

to support gamma sterilization with the sterility assurance level was 10-6 and ensure preservation of graft for at least up to 2 years at room temperature and preferably up to 3 to 5 years.
Employing glycerol at specific concentration range of about 25% to about 60%, preferably between 25% to 45% in the present preservation process particularly overcomes the shortcomings associated with high concentrations (e.g., more than 60%) of glycerol, which if not washed properly before implantation, can interfere with the incorporation and healing in the recipient. Further, using higher concentrations increases the pre-operative preparation time by at least >15 minutes which is unfavourable and reduces ease of use of the acellular tissue graft. On the other hand, lower concentration of glycerol (e.g., about 25% to 45%) according to the present process has specific advantage in that it can be removed easily within quick 2-10 minutes of rinse in saline. Even if traces of the glycerol remain in the acellular tissue graft, they are not in the higher concentration range that could hinder host cell incorporation and proliferation.
In some embodiments, the present process of preparing and preserving acellular tissue graft does not contain the step of enzyme/endonuclease treatment (e.g., DNAase/RNAse).
In some embodiments of the present process, subjecting the tissue with the first decellularization agent is carried out for a duration of about 24 hours to about 28 hours, including all the values in the range, for instance, about 24 hours, about 25 hours, about 26 hours, about 27 hours or about 28 hours, and including subranges of the range between 24 hours to 28 hours, and at a temperature ranging from about 36.2°C to about 37.2°C, including all the values in the range, for instance, about 36.2°C, about 36.3°C, about 36.4°C , about 36.5°C, about 36.6°C, about 36.7°C, about 36.8°C, about 36.9°C, about 37.0°C, about 37.1°C or about 37.2°C, and including subranges of the range between 36.2°C to 37.2°C. In some embodiments, the treatment of the tissue with the first decellularization solution is carried out in

an incubator. In some embodiments, the tissue is treated with 1M to 2M hypertonic solution for about 26 hours at about 37°C in an incubator.
In some embodiments of the present process, subjecting the tissue with the second decellularization agent is carried out for a duration of about 30 minutes to about 120 minutes, including all the values in the range, for instance, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, and so on and so forth, up until about 120 minutes and including subranges of the range between about 30 minutes to about 120 minutes, and at a temperature ranging from about 25°C to about 37°C, including all the values in the range, for instance, about 25°C, about 26°C, about 27°C , about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C or about 37°C and including subranges of the range between about 25°C to about 37°C. In some embodiments, the treatment of the tissue with the second decellularization solution is carried out in an ultrasonicator. In some embodiments, the tissue is treated with 0.1% to 1% anionic detergent for about 60 minutes at about 37°C in an ultrasonicator.
In some embodiments of the present process, subjecting the acellular tissue graft to preservation with the preservant is carried out for a time duration ranging from about 0.5 hours to about 72 hours, including all the values in the range, for instance, about 0.5 hours, about 1 hours, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, and so on and so forth, up until about 72 hours and including subranges of the range between about 0.5 hours to about 72 hours, and at a temperature ranging from about 25°C to about 37.2°C, including all the values in the range, for instance, about 25°C, about 26°C, about 27°C, about 28°C, and so on and so forth, up until about 37.2°C and including subranges of the range between about 25°C to about 37.2°C. In some embodiments, the acellular tissue graft is preserved by soaking said graft in the preservant in an

ultrasonicator, waterbath shaker, rotary shaker, beaker with magnetic stirrer/ agitator. In some embodiments, the acellular tissue graft is preserved by soaking said graft in the preservant at least two times. In some embodiments, the acellular tissue graft is preserved by soaking said graft in 25% to 60% glycerol at about 25°C for about 30 minutes twice in an ultrasonicator. Soaking said graft in 25% to 60% glycerol at about 25°C for about 30 minutes twice under ultrasonication conditions allows sufficient time for glycerol to penetrate the acellular tissue matrix.
In some embodiments of the present process, the process comprises rinsing the tissue with a rinsing solution after subjecting the tissue to decellularization with the first decellularization agent, or the second decellularization agent, or each of the first decellularization agent and the second decellularization agent. In some embodiments, said rinsing solution is selected from a group comprising NaCl solution, phosphate buffered saline (PBS), and Hanks balanced salt solution. In some embodiments, the rinsing is carried out with agitation and/or ultrasonication at a temperature of about 25°C to 37.2°C, including all the values in the range, for instance, about 25°C, about 26°C, about 27°C, about 28°C, and so on and so forth, up until about 37.2°C and including subranges of the range between about 25°C to about 37.2°C, and for a time duration of about 30 minutes to about 60 minutes, including all the values in the range, for instance about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes and so on and so forth, up until about 60 minutes and including subranges of the range between about 30 minutes to about 60 minutes, to remove the residual decellularizing agents.
In some embodiments of the present process, the prepared acellular tissue graft is disinfected before subjecting the same to preservation. In some embodiments, the disinfection is carried out using a disinfectant selected from a group comprising isopropyl alcohol, ethanol, hydrogen peroxide (H2O2), peracetic acid, and combinations thereof for a temperature of about 25°C to about 37.2°C, including

all the values in the range, for instance, about 25°C, about 26°C, about 27°C, about 28°C, and so on and so forth, up until about 37.2°C and including subranges of the range between about 25°C to about 37.2°C, for a time duration of about 30 minutes to 120 minutes, including all the values in the range, for instance about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes and so on and so forth, up until about 120 minutes and including subranges of the range between about 30 minutes to about 120 minutes. In some embodiments, the disinfection is carried out with 70% isopropyl alcohol at a temperature of about 25°C to about 37.2°C for a duration of about 30 minutes to 60 minutes. In some embodiments, the disinfection is carried out with ethanol at a temperature of about 25°C to about 37.2°C for a duration of about 30 minutes to 60 minutes. Using isopropyl alcohol or ethanol for disinfection removes loose bound water thereby facilitating greater penetration of the preservant such as glycerol into the dermal matrix. This aids the preservant to replace the tightly bound water and forming intrachain hydrogen bonds with the collagen fiber.
In some embodiments of the present process, the tissue is cleaned prior to the decellularization. In an embodiment, the cleaning comprises removing of subcutaneous fat by gentle mechanical scraping.
In some embodiments of the present process, the tissue is subjected to preprocessing prior to the decellularization. In an embodiment, said preprocessing comprises:
i) cutting the tissue into patches with sizes ranging from about 3x5 cm to about 10x10 cm, including all the values in the range, for instance, about 4x5 cm, about 4x6 cm, about 4x7 cm, about 4x8 cm, about 4x9 cm, about 4x10 cm, about 5x6 cm, about 5x7 cm, about 5x8 cm, about 5x9 cm, about 5x10 cm and so on and so forth, up until about 10x10 cm and including subranges of the range between 3x5 cm to about 10x10 cm; and
ii) storing the cut biological tissue at a temperature ranging from about 0°C to about 10°C, including all the values in the range, for instance, about 1°C, about

2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C or about 10°C and including subranges of the range about 0°C to about 10°C, in physiological solution with antibiotics.
In some embodiments of the present process, the preserved acellular tissue graft is packaged and stored at a temperature ranging from about 4°C to about 35°C followed by performing terminal sterilization. In some embodiments, the preserved acellular tissue graft is subjected to:
- removal of excess solution(s) from the preserved acellular tissue graft;
- packaging of the preserved acellular tissue graft under aseptic conditions in inert packages;
- storing the preserved and packed acellular tissue graft at a temperature ranging from about 4°C to about 35°C, including all the values in the range, for instance, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C or about 34°C or about 35°C and including subranges of the range between 4°C to 35°C; and
- performing terminal sterilization.
In an embodiment, removing the excess solutions is carried out by spinning, blotting or a combination thereof; and the terminal sterilization is carried out with 25 kiloGrays (kGy) dose of gamma radiation from cobalt-60 source. In another embodiment, terminal sterilization by validated gamma radiation dose eliminates the risk of disease transmission and infection.
In some embodiments, the prepared and preserved acellular tissue graft is an acellular tissue allograft, acellular tissue xenograft or acellular collagen scaffold.

In some embodiments of the present process, the tissue is a human tissue, animal tissue or plant tissue. In some embodiments, the tissue is selected from a group comprising skin or dermis, blood vessels, pericardium, heart valves, tendons, ligaments and bones. In some embodiments, the tissue employed in the present process is human skin or dermis, bovine or porcine intestinal sub mucosal membrane or urinary bladder. In an embodiment, the tissue is human skin or dermis.
In an embodiment of the present process, the tissue is human full thickness skin graft (human FTSG).
In some embodiments, the prepared and preserved acellular tissue graft is human acellular dermal graft. In some embodiments of the present process, the tissue is human skin and the prepared and preserved acellular tissue graft is human acellular dermal graft.
In an embodiment, Figure 1 of the present disclosure illustrates the processing (preparation and preservation) of human skin tissue (human FTSG) to obtain acellular human dermal allograft (processed human dermis).
In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- subjecting the tissue to decellularization with:
a) about 0.05M to about 2M NaCl solution, and
b) 0.1% to about 1% SDS, to prepare an acellular tissue graft; and
- subjecting said acellular tissue graft to preservation with 25% to 60%
glycerol.
In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- subjecting a biological tissue to decellularization with:
a) about 1M to about 2M NaCl solution, and

b) 0.1% to about 1% SDS, to prepare an acellular tissue graft; and
- subjecting said acellular tissue graft to preservation with 25% to 60%
glycerol.
In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- subjecting a biological tissue to decellularization with:
a) about 0.05M to about 0.07M NaCl solution, and
b) 0.1% to about 1% SDS, to prepare an acellular tissue graft; and
- subjecting said acellular tissue graft to preservation with 25% to 60%
glycerol.
In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- subjecting a human full thickness skin graft (human FTSG) to
decellularization with:
a) about 0.05M to about 2M NaCl solution, and
b) 0.1% to about 1% SDS, to prepare an acellular human FTSG; and
- subjecting said acellular human FTSG to preservation with 25% to 60%
glycerol.
In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- subjecting a dermal tissue to decellularization with:
a) hypertonic solution of about 1M to about 2M NaCl solution or
hypotonic solution of about 0M to below 0.15M NaCl solution, and
b) about 0.1% to about 1% SDS, to prepare the acellular dermal graft;
and
- subjecting the acellular dermal graft to preservation with 25% to 45%
glycerol.

In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- subjecting a dermal tissue to decellularization with:
a) hypertonic solution of about 1M to about 2M NaCl solution or
hypotonic solution of about 0M to below 0.15M NaCl solution, and
b) about 0.1% to about 1% SDS, to prepare the acellular dermal graft;
and
- subjecting the acellular dermal graft to preservation with 25% to 35%
glycerol.
In some embodiments of the present disclosure, the process of preparation and preservation of the human FTSG comprises:
- subjecting a dermal tissue to decellularization with:
a) hypertonic solution of about 1M to about 2M NaCl solution or
hypotonic solution of about 0M to below 0.15M NaCl solution, and
b) about 0.1% to about 1% SDS, to prepare the human FTSG;
and
- subjecting the human FTSG to preservation with 25% to 45% glycerol.
In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- subjecting a tissue to decellularization with:
a) about 0.05M to about 2M NaCl solution for a duration of about 24 hours to about 28 hours at a temperature ranging from about 36.2°C to about 37.2°C, and
b) 0.1% to about 1% SDS for a duration of about 30 minutes to about 120 minutes at a temperature ranging from about 25°C to about 37.2°C, to prepare an acellular tissue graft; and
- subjecting said acellular tissue graft to preservation with 25% to 60%
glycerol at least two times for a duration of about 0.5 hours to about 72
hours at a temperature ranging from about 25°C to about 37.2°C.

In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft comprises:
- cutting the tissue into patches of size ranging from about 3x5 cm to about 10x10 cm, and storing the biological tissue at a temperature ranging from about 0°C to about 10°C in physiological solution with antibiotics;
- subjecting the biological tissue to:

a) decellularization with about 1M to about 2M NaCl solution or about 0.01M to about 0.1M Tris-HCl solution for a duration of about 24 hours to about 28 hours at a temperature ranging from about 36.2°C to about 37.2°C, followed by rinsing the biological tissue with 0.9 % NaCl solution; and
b) decellularization with 0.1% to about 1% SDS for a duration of about 0.5 hours to about 2 hours at a temperature ranging from about 25°C to about 37.2°C, followed by rinsing the biological tissue with 0.9 % NaCl solution, to prepare the acellular tissue graft;

- subjecting said acellular tissue graft to preservation by soaking the same in about 25% to 60% of glycerol at least two times for a duration ranging from about 30 minutes to 60 minutes at a temperature ranging from about 25°C to 37.2°C;
- removing excess solutions from the preserved acellular tissue graft followed by packaging under aseptic conditions in inert packages; and
- storing the acellular tissue graft at a temperature ranging from about 4°C to about 35°C followed by terminal sterilization.
In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft does not involve any surgical or therapeutic step/method. In some embodiments of the present disclosure, the process of preparation and preservation of the acellular tissue graft employs biological tissue sourced from a tissue bank or any other facility which procures/stores the biological tissue.

The present disclosure further relates to an acellular tissue graft processed and preserved according to the process described above.
In some embodiments of the present disclosure, the acellular tissue graft is human acellular dermal graft.
The acellular tissue graft of the present disclosure is devoid of epidermis, cellular and nuclear remains representing the acellular nature of the graft and thus does not produce immunogenic reactions as observed in conventional tissue grafts or scaffolds which lead to graft rejection. Said acellular tissue graft has intact collagen network similar to native tissues and thus have enhanced structural and biomechanical properties when compared to conventional tissue grafts or scaffolds.
In an embodiment, the Figures 3(A) to 3(D) provide images of histological evaluation of hematoxylin and eosin-stained dermal samples, wherein it is observed that the glycerol preserved acellular dermal grafts (Figure 3C and 3D) prepared according to the process of the present disclosure shows absence of epidermis with intact collagen network similar to that of the control dermal graft (Figure 3A and 3B) indicating retained flexibility and strength and devoid of any nuclear remains presenting the acellular nature. On the other hand, the conventional freeze-dried dermal grafts (Figure 3E), though acellular in nature, were more brittle and less flexible than native dermal graft.
The present disclosure also relates to a kit for preparing and preserving an acellular tissue graft.
In some embodiments of the present disclosure, said kit for preparing and preserving the acellular tissue graft comprises:
- a first decellularization agent comprising a hypertonic solution or a hypotonic solution;

- a second decellularization agent comprising a detergent selected from one or more of anionic detergent, non-ionic detergent and a combination thereof;
- a preservant comprising a hygroscopic solvent; and
- a package insert/label/instruction manual comprising instructions for preparing and preserving the acellular tissue graft.
In some embodiments of the present disclosure, the kit for preparing and preserving the acellular dermal graft comprises:
- about 0.05M to about 2M of the first decellularization agent/solution;
- about 0.1% to about 1% of the second decellularization agent/solution;
- about 25% to 60% of the preservant solution; and
- a package insert/label/instruction manual comprising instructions for preparing and preserving the acellular dermal graft according to the process described above.
In some embodiments of the present disclosure, the kit for preparing and preserving the acellular dermal graft comprises:
- about 0.05M to about 2M NaCl solution;
- about 0.1% to about 1% SDS;
- about 25% to 60% of glycerol; and
- a package insert/label/instruction manual comprising instructions for preparing and preserving the acellular dermal graft according to the process described above.
The present disclosure further relates to use of the acellular tissue graft prepared and preserved according to the process described above, for reconstructive orthopaedics, craniofacial or maxillofacial surgery, dentistry, plastic surgery and wound care. In some embodiments, the present disclosure provides use of the acellular tissue graft in tissue defect reconstruction including but not limited to soft tissue defect reconstruction such as rotator cuff tear reconstruction.

In some embodiments, the acellular tissue graft prepared and preserved according to the present process can be used in clinical applications as provided in Table 1.

The present disclosure further relates to the following numbered embodiments describing the non-limiting features of the disclosure –
1. A process of preparation and preservation of an acellular tissue graft comprising: - subjecting a tissue to decellularization with:
a) first decellularization agent comprising a hypertonic solution or a hypotonic solution, and
b) a second decellularization agent comprising a detergent selected from one or more of anionic detergent, non-ionic detergent and a combination thereof, to prepare the acellular tissue graft;
and

- subjecting said acellular tissue graft to preservation with a preservant comprising a hygroscopic solvent.
2. The process of embodiment 1, wherein the first decellularization agent is at
a concentration ranging from about 0M to about 2M; or
wherein the hypertonic solution is at a concentration of above 0.15M to about 2M, or about 1M to about 2M; or
wherein the hypotonic solution is at a concentration of 0M to below 0.15M, or about 0.05M to about 0.07M.
3. The process of embodiment 1, wherein the hypertonic solution is selected from a group comprising sodium chloride (NaCl) solution, potassium chloride (KCl) solution, magnesium chloride (MgCl2) solution, calcium chloride (CaCl2) solution, potassium phosphate (K3PO4) solution, sodium phosphate (Na3PO4) solution, magnesium phosphate [Mg3(PO4)2] solution; and wherein the concentration of the hypertonic solution is ranging from about 1M to about 2M.
4. The process of embodiment 1, wherein the hypotonic solution is selected from a group comprising (NaCl) solution, purified water, tris-HCl , potassium chloride (KCl) solution, magnesium chloride (MgCl2) solution, calcium chloride (CaCl2) solution, potassium phosphate (K3PO4) solution, sodium phosphate (Na3PO4) solution, magnesium phosphate [Mg3(PO4)2] solution; and wherein the concentration of the hypotonic solution is about 0.05M to about 0.07M.
5. The process of embodiment 3, wherein the hypertonic solution is sodium chloride solution; or wherein the hypertonic solution is sodium chloride in water or a buffer.
6. The process of embodiment 1, wherein the detergent is at a concentration of about 0.1% to about 1%.
7. The process of embodiment 1, wherein the anionic detergent is selected from a group comprising sodium dodecyl sulfate (SDS), sodium deoxycholate, ammonium lauryl sulfate (ALS), sodium myreth sulfate and combinations thereof.

8. The process of embodiment 1, wherein the non-ionic detergent is selected from a group comprising 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polyoxyethylene (20) sorbitan monolaurate, cetyltrimethyl ammonium bromide (CTAB) and combinations thereof.
9. The process of embodiment 1, wherein the hygroscopic solvent is at a concentration of about 25% to about 60%.
10. The process of embodiment 1, wherein the hygroscopic solvent is selected from a group comprising glycerol, sorbitol, sucrose, glucose, mannitol, ethylene glycol, D-galactose and combinations thereof.
11. The process of embodiment 1, wherein subjecting the tissue with the first decellularization agent is carried out for a duration of about 24 hours to about 28 hours at a temperature ranging from about 36.2°C to about 37.2°C;
and wherein subjecting the tissue with the second decellularization agent is carried out for a duration of about 0.5 hours to about 2 hours at a temperature ranging from about 25°C to about 37.2°C.
12. The process of embodiment 1, wherein subjecting the acellular tissue graft to preservation with the preservant is carried out for a duration ranging from about 0.5 hours to 72 hours at a temperature ranging from about 25°C to about 37.2°C.
13. The process of embodiment 1, wherein the process comprises rinsing the tissue with a rinsing solution after subjecting the tissue to decellularization with the first decellularization agent, or the second decellularization agent, or both;
wherein the rinsing solution is selected from a group comprising NaCl solution, phosphate buffered saline (PBS), Hanks balanced salt solution and combinations thereof.
14. The process of embodiment 1, wherein the acellular tissue graft is
disinfected before subjecting said acellular tissue graft to preservation;
and wherein the disinfection is carried out for a duration of about 30 minutes to 120 minutes using a disinfectant selected from a group

comprising isopropyl alcohol, hydrogen peroxide, peracetic acid and combinations thereof.
15. The process of embodiment 1, wherein the process comprises cleaning the biological tissue prior to the decellularization, wherein said cleaning comprises removing subcutaneous fat by gentle mechanical scraping.
16. The process of embodiment 1, wherein the preserved acellular tissue graft is packaged and stored at a temperature ranging from about 4°C to about 35°C followed by terminal sterilization.
17. The process of embodiment 1, wherein the tissue is a human tissue, animal tissue or plant tissue.
18. The process of embodiment 17, wherein the tissue is selected from a group comprising skin or dermis, blood vessels, pericardium, heart valves, tendons, ligaments and bones.
19. The process of any of the embodiments 1 to 18, wherein the tissue is skin tissue or dermis or human full thickness skin graft (FTSG); and wherein the prepared and preserved acellular tissue graft is a human acellular dermal graft.
20. The process of any of the embodiments 1 to 19, wherein the process comprises:
- subjecting a dermal tissue to decellularization with:
a) about 0.05M to about 2M NaCl solution, and
b) 0.1% to about 1% SDS, to prepare an acellular dermal graft; and
- subjecting said acellular dermal graft to preservation with 25% to 60%
glycerol.
21. A kit for preparing and preserving an acellular tissue graft comprising:
- a first decellularization agent comprising a hypertonic solution or a hypotonic solution;

- a second decellularization agent comprising a detergent selected from one or more of anionic detergent, non-ionic detergent and a combination thereof;
- a preservant comprising a hygroscopic solvent; and
- a package insert or label comprising instructions for preparing and preserving the acellular tissue graft by the process according to any of the above embodiments 1 to 20.

22. The kit of embodiment 21, wherein the first decellularization agent is about 0.05M to about 2M NaCl solution, the second decellularization agent is about 0.1% to about 1% SDS, and the preservant is about 25% to about 60% glycerol.
23. An acellular tissue graft prepared according to the process of any of the above embodiments 1 to 20.
24. The acellular tissue graft of embodiment 23, wherein the acellular tissue graft is a human acellular dermal graft.
25. Use of the acellular tissue graft prepared and preserved according to any of
the above embodiments 1 to 20 or use of the acellular tissue graft of any of
embodiments 23 or 24 for reconstructive orthopaedics, craniofacial or
maxillofacial surgery, dentistry, plastic surgery and wound care.
While the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof have been shown by way of examples (and drawings) described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims. The present disclosure is therefore further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

EXAMPLES
The present disclosure is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.
Example 1
Preparation and preservation of acellular human dermal graft
Human full thickness skin grafts (FTSGs) were obtained from tissue bank (Novo Tissue Bank And Research Centre Pvt. Ltd., Mumbai, Maharashtra). The FTSGs were removed from the transportation container in the biosafety cabinet and placed with its reticular side down on a clean sterile stainless-steel tray and cut into rectangular patches with sizes ranging from 3x5 cm to 10x10 cm. A small cut was made on the bottom right corner of the graft to distinguish the reticular and papillary surfaces after processing. The grafts were then stored at 0-10°C in physiological solution with antibiotics prior to processing. Antibiotics were employed in the storage due to the presence of collagen degrading proteases from existing bioburden. Tissues were kept at these low temperatures for maximum 7 days until further processing and preservation.
The human Full thickness Skin Grafts (FTSGs) were cleaned of any subcutaneous fat by gentle mechanical scraping. The tissues were treated with 1.5M hypertonic solution of NaCl in purified water for 26 hours at 37°C in an incubator. The tissues were rinsed with 0.9 % NaCl in sterile Phosphate Buffered Saline (PBS) having pH 7 accompanied with ultrasonication to remove residual decellularizing reagent. The tissues were then treated with 0.5% SDS (anionic detergent) in purified water for 1 hour at 37°C in an ultrasonicator to obtain human acellular dermal grafts devoid of cellular components. The combination of treatment with hypertonic solution and mild anionic detergent enables removal of epidermis. Decellularization was followed by thorough rinsing with 0.9 % NaCl in sterile Phosphate Buffered Saline (PBS) having pH 7 accompanied with agitation and /or ultrasonication to remove the residual decellularizing reagents.

The human acellular dermal grafts were disinfected with 70% isopropyl alcohol for 30 min at room temperature. The human acellular dermal grafts were soaked in 30% of glycerol in purified water in an ultrasonicator twice for about 30 minutes each at about 25°C to obtain glycerol preserved human acellular dermal graft (gly-HAD). Excess reagents were removed by dry spinning and/or blotting followed by packaging of the glycerol preserved human acellular dermal graft (gly-HAD) under aseptic conditions in inert packages and stored at room temperature not exceeding 35°C followed by terminal sterilization.
Figure 1 illustrates the schematic flow of processing (preparation and preservation) of human full thickness skin graft (FTSG) to obtain acellular human dermal allograft (processed human dermis). Figure 2 demonstrates the experimental preparation and preservation of human full thickness skin graft (FTSG). Particularly, Figure 2A is shows human FTSG. Figure 2B is the human acellular dermal graft obtained after treatment with combination of hypertonic solution (1.5M NaCl) and mild anionic detergent (0.5% SDS) showing no epidermis, and Figure 2C the ready to be packaged human acellular dermal graft after preservation step (treatment with hygroscopic solvent – 30% glycerol).
Example 2
Comparison of present preservation process with conventional process
There are various preservation techniques available to maintain the shelf life of grafts. The effect of two preservation methods i.e., conventional freeze drying/lyophilization and preservation by hygroscopic solvent in accordance with the present process along with their quality (e.g. histological evaluation) and effects on the final biomechanical properties of the grafts were evaluated.
Preservation by freeze-drying/ lyophilization (conventional preservation process) Preservation by freeze-drying involves removal of water from the specimen under vacuum leading to final specimen with reduced water activity facilitating increased shelf life at room temperature. The decellularized/acellular dermal grafts prepared

according to Example 1 were disinfected with 70% isopropyl alcohol and stored at -80°C for 18-24 hours followed by the drying cycle in Martin Christ Alpha 1-4 LSC Plus. The program for the lyophilizer was set such that the final residual moisture content of the grafts after freeze drying was 2% to 6%. The freeze-dried human acellular dermis grafts (FD-HAD) were then heat sealed in impervious pouches and stored at room temperature not exceeding 35°C followed by terminal sterilization.
Preservation by hygroscopic solvent (present process)
The hygroscopic solvent - glycerol was used as the preserving solution due to its unique hygroscopicity in addition to non-toxicity, compatibility with tissues, low molecular weight and safety. The decellularized/acellular dermal grafts which were subjected to preservation using glycerol [named as glycerol preserved HAD (gly-HAD)] according to Example 1 were studied for the effects on the final biomechanical properties. The results of the comparative evaluation are discussed in Example 3.
Example 3
Evaluation of quality of the acellular dermal grafts
HISTOLOGICAL EVALUATION:
In the present study, it was hypothesized that the dermal graft processing steps should render the tissue acellular along with intact collagen network. To verify the same, histological evaluation was conducted. Hematoxylin and Eosin staining (H & E) is a common method used to provide a clear microanatomy of the tissues. Dermal tissue samples were fixated with 10% formalin solution. After processing and sectioning, histological cross-sections were stained with the H & E method and visualized under 10X & 40X magnification. Tissue histology of control (unprocessed FTSG), glycerol preserved human acellular dermal graft (gly-HAD) obtained by the present process, and freeze-dried human acellular dermal graft (FD-HAD) were compared for remnants of cellular components and collagen banding.

Figure 3 provides images of light microscope analysis of H & E-stained dermal samples. Figures 3(A) and (B) provide H & E-stained images of control unprocessed FTSG sample. Figures 3(C) and (D) provide H & E-stained images of the present glycerol preserved human acellular dermal graft (gly-HAD) sample. Figure 3(E) provides H & E-stained images of freeze-dried human acellular dermal graft (FD-HAD) sample.
As can be seen from Figure 3, the light microscope analysis of H & E-stained dermal samples mapped out the clear histological differences in processing methods. Scanning across the control unprocessed FTSG sample [Figures 3(A) and (B)] delineated the epidermis with numerous purple-stained nuclear materials embedded in pink collagen network. The epidermis was followed by thick horizontal collagen network of papillary dermis progressing into thicker network of reticular dermis. On the contrary, the glycerol preserved human acellular dermal graft (gly-HAD) sample [Figures 3(C) and (D)] showed absence of epidermis with pink stained collagen network devoid of any nuclear remains presenting the acellular nature. The collagen fibrils were observed as intact intricate pattern arranged in horizontal fashion similar to that of the control indicating retained flexibility and strength. However, the freeze-dried human acellular dermal graft (FD-HAD) sample [Figure 3(E)] showed pink stained collagen fiber network reoriented from their normal horizontal pattern with absence of cellular components. Though acellular in nature, freeze drying yielded dermal grafts which were more brittle and less flexible than native dermal graft.
BIOBURDEN TESTING:
Bioburden of the specimen is the number of viable microorganisms present in the specimen after completion of processing steps (preparation and preservation) and prior to sterilization. It is also known as ‘contamination level’ of the specimen. Bioburden testing is a crucial step to assess the efficiency of the graft processing method. It is also important in formulating the specific parameters of sterilization method in order to achieve implantable tissue graft. Vacuum filtration technique is

an accepted method of bioburden evaluation by ISO 11737-1 standards, to ensure validated sterilization dose are derived to render the dermal graft sterile. In the present study, microbiological analysis of processed glycerol preserved non-radiated dermal graft sample (gly-HAD) was performed using vacuum filtration technique followed by incubation of the membrane in Tryptic soy agar for 14 days at 30°C to 35°C. Table 2 shows the bioburden results of the acellular dermal graft samples of the present disclosure.
Table 2: Bioburden testing results

Sample No. Dimensions (L x W x T) mm Bioburden (CFU/ unit)
1 5 x 5 x 1.2 455
2 5 x 5 x 1.2 491
3 3 x 7 x 1.2 510
4 2.5 x 2.5 x 1.5 250
5 7.5 x 7.5 x 1.3 700
Average Bioburden 481 CFU/unit
Dermal grafts from mammalian origin are inherently characterized by numerous bacteria, spores and fungi. In the present study, average batch bioburden as evaluated was found to be 480 CFU/ graft. Since the average is less than 1000 CFU/ graft and the sterility assurance level is -6, the reference table by ANSI/AAMI/ISO 11137 standards was followed to determine the appropriate applied dose as 25kGy.
STERILITY TESTING:
The processed and packaged acellular glycerol preserved dermal grafts were maintained at -78.5°C using dry ice and subjected to 25kGy dose of gamma radiation from 60cobalt source. Low temperature treatment was employed to prevent the collagen fibril damage associated with gamma radiation. Processed glycerol

preserved dermal samples which were gamma radiated with 25kGy dose were tested for presence of viable microorganisms using recommendations in ISO 11737-2. The radiated samples were incubated in sterile Tryptic soy broth at 30°C to 35°C for 14 to 21 days in order to validate the sterilization dose as per ISO 11137-2.
Implantable medical biomaterials are terminally sterilized to achieve a Sterility Assurance Level (SAL) of 10-6, which implies a probability of finding not more than one viable microorganism in one million sterilized items of the final graft. The present sterility testing results showed 0 CFU from the processed glycerol preserved dermal samples radiated with 25kGy gamma radiation.
BIOMECHANICAL TESTING:
An ideal acellular dermal graft should be capable of bearing anisotropic load as required in their clinical applications. The load bearing capacity of a dermal graft depends upon the integrity and flexibility of the collagen matrix. To assess the biomechanical properties, the ultimate tensile load was determined using the UTM-Universal Testing Machine (Tinius Olsen Universal Testing Machine with a calibrated load cell of 5KN). The biomechanical testing was focused on strength of the dermal samples and no control was used. Each dermal sample length, width and thickness were individually recorded using digital callipers. The measured specimens were pressure clamped onto the UTM grippers and were pulled at a rate of 100% strain per second until failure. The load to failure portion of the test graph was used to determine the ultimate tensile force. Irradiated glycerol preserved human acellular dermal graft (gly-HAD) sample with dimensions (length x width x thickness) 100x17x1.2 (mm) was clamped onto the UTM for testing [Figure 4(A). Irradiated freeze-dried human acellular dermal graft (FD-HAD) sample with dimensions 100x17x1.2 (mm) was removed from the sterile packaging and rehydrated with 0.9% saline at room temperature for 15 minutes before testing followed by clamping onto the UTM grippers. To estimate the suture retention strength, the materials were removed from their sterile packages at the time of

testing and cut into strips of 60 mm length and 15mm wide. A single vertical stitch of No. 2 FiberWire was passed through the dermal sample at a distance of 10 mm from one end (single end sutured gly-HAD) and clamped onto the UTM for suture pull-out strength [Figure 4(B)]. To stimulate clinically relevant configuration, another sample was tested where single vertical stitch of No. 2 FiberWire was passed through the dermal sample at a distance of 10 mm from both the ends (Double end sutured gly-HAD) as shown in Figure 4(C).
As shown in Figure 5, the irradiated glycerol preserved human acellular dermal graft (gly-HAD) sample exhibited ultimate tensile force of 210 N whereas the irradiated freeze-dried/lyophilized human acellular dermal graft (FD-HAD) sample failed at 124N. The results confirmed that freeze-dried/lyophilized human acellular dermis (FD-HAD) was brittle and had less structural integrity owing to the rearranged collagen network. In other words, the present gly-HAD sample clearly demonstrated higher mechanical strength and structural integrity.
Additionally, the suture retention test showed that the single ended sutured glycerol preserved human acellular dermis (gly-HAD) withstood suture strength until the suture was pulled through the graft material at 126 N. Further, the double ended sutured gly-HAD withstood suture strength from one end at 102 N (Figure 6). No suture breakage was observed among the gly-HAD samples. These results imply that gly-HAD offers sufficient mechanical integrity and effectiveness, which is essential for its successful use in various clinical scenarios.
Example 4:
Comparison of present process with conventional processes
The following Table 3 provides a comparison of the structure, properties and end practical use of the acellular tissue grafts preserved by the present process and conventional preservation processes (cryopreservation and freeze drying).

Table 3

Preservation process Inter- chain Water mediated Overall structure and use of the
for prepared hydrogen hydrogen bonds preserved acellular tissue graft
acellular tissue graft bonds
Cryopreservation/ Fully Fully preserved or - Expensive
deep freezing preserved partially lost - Cold-chain maintenance
depending on the required
cryoprotectant and - Cryoprotectant to be removed before use
freeze-thaw - Increased pre-operative
conditions preparation time
Freeze Partially Partially or fully lost - Preoperative rehydration
drying/Lyophilization preserved required - Microfractures leading to reduced biomechanical strength
Hygroscopic solvent, Fully Partially displaced by - Room temperature storage
e.g., glycerol (present preserved hygroscopic solvent - Retained mechanical
process) and fully preserved strength
- Reduced pre-operative
preparation time
- Cost-effective
To summarize, based on the above examples/experimental results, it was observed that mild chemical decellularization (hypertonic or hypotonic treatment along with detergent treatment) effectively removes nuclear and cellular debris. For instance, hypertonic solutions created an osmotic gradient that drove the epidermal cells to shrink and disassociate from the dermis. Hypertonic solution facilitated cytolysis and therefore showed excellent decellularization effects. Coupling with anionic or non-ionic detergents led to cell lysis and reduction in bioburden by solubilizing the peptidoglycan cell wall, phospholipid cell membrane and disrupting the nuclear material. The above results clearly show the decellularization of the dermal tissue by combination of hypertonic or hypotonic treatment with detergent treatment to be highly effective as confirmed by H&E staining and further preservation via hygroscopic solvent such as glycerol enabled long-term storage of graft in hydrated

condition preserving the strength and also reducing the pre-op graft preparation time in surgical applications.
Though freeze-drying/lyophilization serves as a conventional and widely practiced means of preservation of hard tissues; however, the above results confirm that freeze-drying distinctly affects the collagen ultrastructure and mechanical properties of the graft. Further, while cryopreservation or deep freezing at -80°C also serves as a potential preservation method; however, it is linked with crystal formation in the soft matrix structure that reduces the biomechanical strength over time with repetitive freeze-thaw cycles. In addition to that, cryopreservation/deep freezing leads to an exorbitant cost for maintenance, inconvenience in logistics with increased pre-operative preparation. An ideal process should not affect the structure and mechanical properties of the graft, so as to mimic the native biological tissue. Preservation of the graft at room temperature is an added advantage primarily from ease, transportation burden, and cost effectiveness perspective. Overall, the present process of preparing and preserving acellular tissue graft showed superiority in retaining structural and mechanical properties of the graft when compared with conventional preservation techniques such as freeze-drying and cryopreservation. Room temperature storage, reduced pre-operative preparation time and cost-effectiveness are additional and important advantages of the acellular tissue graft prepared and preserved by the process of present disclosure.
Advantages of the present invention:
- the present process of preparing and preserving acellular tissue graft employs mild chemical decellularization via. a combination of hypertonic or hypotonic solution treatment (e.g., 0.05M to 2M NaCl solution) and detergent treatment (e.g., 0.1% to 1% anionic detergent such as SDS), thereby providing effective removal of nuclear and cellular debris.
- the present process of preparing and preserving acellular tissue graft does not employ or require any enzymatic treatment step, for e.g., endonuclease (DNase or RNase) treatment. Avoiding such enzymatic treatment step

reduces the cost and time of the process along with eliminating the risks of tissue incompatibility in the host and the associated immunogenic reaction.
- the present process of preparing and preserving acellular tissue graft employs a hygroscopic solvent (e.g., 25% to 60% glycerol) in the preservation step to provide room temperature long-term storage of the prepared acellular tissue graft in hydrated condition, thereby preserving the strength while reducing the pre-op graft preparation time in subsequent surgical applications.
- the present process of preparing and preserving acellular tissue graft is simple and cost-effective.
- the acellular tissue graft prepared by the present process is highly biocompatible. Particularly, the present process improves the preparation and preservation of acellular tissue scaffold which optimally mimics the native tissue.
- intact collagen scaffold in the acellular tissue graft obtained by the present process provides high biomechanical strength to the grafts.
- the present acellular tissue graft can be stored at room temperature in hydrated form, thereby avoiding the need for pre-op hydration step and providing long term off the shelf availability when compared to conventional preservation processes such as free-drying/lyophilization and cryopreservation/deep freezing.
Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.
The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and

modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
Thus, while considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
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. Similarly, terms such as “include” or “have” or “contain” and all their variations are inclusive and 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.
The terms "about" or “approximately” are used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical value/range, it modifies that value/range by extending the boundaries above and below the numerical value(s) set forth. In general, the term

"about" is used herein to modify a numerical value(s) or a measurable value(s) such as a parameter, an amount, a temporal duration, and the like, above and below the stated value(s) by a variance of +/-20% or less, +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention, and achieves the desired results and/or advantages as disclosed in the present disclosure. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.
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. As used in this specification and the appended claims, the singular forms “a,” “an” and “the” includes both singular and plural references unless the content clearly dictates otherwise. 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. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.
As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3

reciting 3 alternatives G, H and I, it is to be understood that the specification unambaiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.
Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
All references, articles, publications, general disclosures etc. cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication etc. cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

WE CLAIM:
1. A process of preparation and preservation of an acellular tissue graft
comprising:
- subjecting a tissue to decellularization with:
a) first decellularization agent comprising a hypertonic solution or a hypotonic solution, and
b) a second decellularization agent comprising a detergent selected from anionic detergent, or non-ionic detergent, or a combination thereof, to prepare the acellular tissue graft;
and
- subjecting said acellular tissue graft to preservation with a preservant
comprising a hygroscopic solvent.
2. The process as claimed in claim 1, wherein the first decellularization agent
is at a concentration ranging from about 0M to about 2M;
wherein the hypertonic solution is at a concentration of above 0.15M to about 2M, or about 1M to about 2M; or
wherein the hypotonic solution is at a concentration of 0M to below 0.15M, or about 0.05M to about 0.07M.
3. The process as claimed in claim 1 or claim 2, wherein the hypertonic
solution is selected from a group comprising sodium chloride (NaCl)
solution, potassium chloride (KCl) solution, magnesium chloride (MgCl2)
solution, calcium chloride (CaCl2) solution, potassium phosphate (K3PO4)
solution, sodium phosphate (Na3PO4) solution, and magnesium phosphate
[Mg3(PO4)2] solution; and
wherein the concentration of the hypertonic solution ranges from about 1M to about 2M.
4. The process as claimed in any one of the claims 1 to 3, wherein the
hypotonic solution is selected from a group comprising (NaCl) solution,

purified water, tris-HCl , potassium chloride (KCl) solution, magnesium chloride (MgCl2) solution, calcium chloride (CaCl2) solution, potassium phosphate (K3PO4) solution, sodium phosphate (Na3PO4) solution, magnesium phosphate [Mg3(PO4)2] solution; and
wherein the concentration of the hypotonic solution is about 0.05M to about 0.07M.
5. The process as claimed in any one of the claims 1 to 4, wherein the hypertonic solution is sodium chloride solution; or wherein the hypertonic solution is sodium chloride in water or a buffer.
6. The process as claimed in any one of the claims 1 to 5, wherein the detergent is at a concentration of about 0.1% to about 1%.
7. The process as claimed in any one of the claims 1 to 6, wherein the anionic detergent is selected from a group comprising sodium dodecyl sulfate (SDS), sodium deoxycholate, ammonium lauryl sulfate (ALS), sodium myreth sulfate and combinations thereof.
8. The process as claimed in any one of the claims 1 to 7, wherein the non-ionic detergent is selected from a group comprising 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, polyoxyethylene (20) sorbitan monolaurate, cetyltrimethyl ammonium bromide (CTAB) and combinations thereof.
9. The process as claimed in any one of the claims 1 to 8, wherein the hygroscopic solvent is at a concentration of about 25% to about 60%.
10. The process as claimed in any one of the claims 1 to 9, wherein the hygroscopic solvent is selected from a group comprising glycerol, sorbitol,

sucrose, glucose, mannitol, ethylene glycol, D-galactose and combinations thereof.
11. The process as claimed in any one of the claims 1 to 10, wherein subjecting
the tissue with the first decellularization agent is carried out for a duration
of about 24 hours to about 28 hours at a temperature ranging from about
36.2°C to about 37.2°C;
and wherein subjecting the tissue with the second decellularization agent is carried out for a duration of about 0.5 hours to about 2 hours at a temperature ranging from about 25°C to about 37.2°C.
12. The process as claimed in any one of the claims 1 to 11, wherein subjecting the acellular tissue graft to preservation with the preservant is carried out for a duration ranging from about 0.5 hours to about 72 hours at a temperature ranging from about 25°C to about 37.2°C.
13. The process as claimed in any one of the claims 1 to 12, wherein the process comprises rinsing the tissue with a rinsing solution after subjecting the tissue to decellularization with the first decellularization agent, or the second decellularization agent, or both;
wherein the rinsing solution is selected from a group comprising NaCl solution, phosphate buffered saline (PBS), Hanks balanced salt solution and combinations thereof.
14. The process as claimed in any one of the claims 1 to 13, wherein the
acellular tissue graft is disinfected before subjecting said acellular tissue
graft to preservation;
and wherein the disinfection is carried out for a duration of about 30 minutes to 120 minutes by a disinfectant selected from a group comprising isopropyl alcohol, hydrogen peroxide, peracetic acid and combinations thereof.

15. The process as claimed in any one of the claims 1 to 14, wherein the tissue is a human tissue, animal tissue or plant tissue.
16. The process as claimed in any one of the claims 1 to 15, wherein the tissue is selected from a group comprising skin or dermis, blood vessels, pericardium, heart valves, tendons, ligaments and bones.
17. The process as claimed in any one of the claims 1 to 16, wherein the tissue is skin tissue or dermis or human full thickness skin graft (FTSG); and wherein the prepared and preserved acellular tissue graft is a human acellular dermal graft.
18. The process as claimed in any one of the claims 1 to 17, wherein the process comprises:
- subjecting a dermal tissue to decellularization with:
a) about 0.05M to about 2M NaCl solution, and
b) about 0.1% to about 1% SDS, to prepare the acellular dermal
graft;
and
- subjecting the acellular dermal graft to preservation with about 25% to
60% glycerol.
19. The process as claimed in any one of the claims 1 to 18, wherein the process
comprises:
- subjecting a dermal tissue to decellularization with:
a) hypertonic solution of about 1M to about 2M NaCl solution or hypotonic solution of about 0M to below 0.15M NaCl solution, and
b) about 0.1% to about 1% SDS, to prepare the acellular dermal graft;
and

- subjecting the acellular dermal graft to preservation with 25% to 35% glycerol.
20. A kit for preparing and preserving an acellular tissue graft comprising:
- a first decellularization agent comprising a hypertonic solution or a hypotonic solution;
- a second decellularization agent comprising a detergent selected from one or more of anionic detergent, non-ionic detergent and a combination thereof;
- a preservant comprising a hygroscopic solvent; and
- a package insert or label comprising instructions for preparing and preserving the acellular tissue graft by the process as claimed in any one of the claims 1 to 19.

21. The kit as claimed in claim 20, wherein the first decellularization agent is about 0.05M to about 2M NaCl solution, the second decellularization agent is about 0.1% to about 1% SDS, and the preservant is about 25% to about 60% glycerol.
22. An acellular tissue graft prepared according to the process as claimed in any one of the claims 1 to 19.
23. The acellular tissue graft as claimed in claim 22, wherein the acellular tissue graft is a human acellular dermal graft.
24. The acellular tissue graft as claimed in claim 22 or claim 23, wherein said acellular tissue graft is employed for reconstructive orthopaedics, craniofacial or maxillofacial surgery, dentistry, plastic surgery and wound care.