Description:Title of Invention
Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat

Field of invention
The present invention relates to the technical field of medical materials, more particular to the composition and method of preparation for haemostatic sponge containing gelatin as an absorbable material that quickly achieve haemostasis and stops bleeding.

Background of the Invention
Surgical bleeding presents a significant challenge in preventing wound-related fatalities, necessitating effective haemostatic control during surgical procedures. Over bleeding can lead to tissue damage, hypovolemic shock, and decreased organ perfusion, increasing patient morbidity and mortality. Traditional haemostatic techniques, including suturing and cautery, may be inadequate for intricate surgeries or delicate tissues, prompting the need for advanced absorbable haemostatic consumables that offer improved performance. Certain patients may be unsuitable for traditional methods due to medical conditions. Rapid and effective haemostatic intervention is critical in increasing survival rates, particularly in extreme wound environments where traditional materials like gauze and bandages prove ineffective.
Effective haemostatic agents are essential for promptly stopping bleeding while minimizing complications. Traditional gelatin sponges often degraded quickly, posing risks of infection and inflammation due to leftover fragments. To overcome this challenge, researchers introduced a chemically cross-linked gelatin sponge, strengthening its structure and durability. The process involved adding a chemical to the gelatin solution before moulding it into sponges, followed by treatment to remove excess cross-linker. These new sponges proved more stable, with extended shelf life and improved haemostatic capabilities compared to traditional versions. Various cross-linkers like glutaraldehyde, genipin, carbodiimides, and epoxy compounds offer different advantages and considerations in sponge preparation. Glutaraldehyde forms stable bonds but raises toxicity concerns, while genipin, though less toxic, demands longer manufacturing times. Carbodiimides and epoxy compounds provide stable cross-linking but may require longer processing. Additionally, enzymatic cross-linkers like transglutaminase offer biocompatible alternatives for creating stable gelatin networks, reflecting the diverse considerations in selecting cross-linking agents based on intended use and safety requirements. However, the major disadvantage of chemical crosslinking was potential toxic residues left in the sponges. Removal of toxic residues from sponges was time consuming and might disrupt the porous structure of Gelatin sponge.
Gelatin sponges, typically made through a foaming and drying process, have inherent limitations due to their solubility at temperatures above 30°C, making them unsuitable for in vivo use without cross-linking. Chemical agents like formaldehyde and glutaraldehyde or radiation cross-linking stabilize the sponge structure, preventing rapid degradation and ensuring sustained haemostasis. Despite being widely used in surgical procedures, absorbable Gelatin sponges pose potential clinical complications including bacterial infection, adhesion formation, allergic reactions, foreign body reactions, hematoma formation, and delayed wound healing, especially if not properly sterilized or left in place for extended periods. These complications underscore the importance of careful consideration and monitoring when using Gelatin sponges as haemostatic agents.
Healthcare providers should be vigilant about potential complications associated with absorbable Gelatin sponge as a haemostat. Steps to minimize risks include proper sterilization, precise sponge placement, and vigilant patient monitoring. While the exact mode of action of Gelatin is not fully understood, it is believed that its ability to absorb and retain blood platelets within its structure contributes to haemostasis by forming a plug and activating the coagulation cascade. Products like GELFOAM® and SURGIFOAM® can be applied directly to bleeding sites, either dry or moistened with sterile saline or thrombin. However, enhancing Gelatin's haemostatic properties often involves complex procedures, including soaking the sponge in diluted thrombin solution, which can introduce errors and require trained personnel. High concentrations of thrombin used in these procedures may also lead to local thrombotic events. Efforts to improve these processes are needed to ensure effective and safe haemostasis during surgeries.
An invention disclosed in patent application number CN116251227A discloses a preparation method of an absorbable haemostatic fluid gelatin material, which comprises the following steps: cooling and freezing a gelatin aqueous solution through a program, and freeze-drying to form a gelatin sponge; the preparation method comprises the following steps: performing vacuum high-temperature crosslinking on gelatin sponge, mechanically crushing and screening, mixing particulate matters with target particle size with a solution containing a radiation stabilizer to prepare fluid gelatin, and performing heat-preservation aging and radiation sterilization. According to the preparation method, high-freezing-strength gelatin is taken as a raw material, a fluid gelatin matrix with proper particle size and pore size is prepared by controlling the cooling and freezing process of a gelatin solution, forming an ice crystal template for pore forming and combining freeze drying, vacuum thermal crosslinking and mechanical pulverization and screening, and the finally prepared fluid gelatin finished product has the characteristics of low secondary imbibition expansion rate, high freezing strength and the like. And the mixing is fast before use. In addition, the fluid gelatin material obtained by the method does not use a chemical cross-linking agent, is good in biological safety, and is suitable for haemostasis in the orthopaedics department, the hepatobiliary surgery department and the neurosurgery department.
An invention disclosed in patent application number CN117065083A discloses a gelatin material capable of absorbing fluid as well as a preparation method and application thereof. According to the preparation method provided by the invention, the specific surfactants are added in the bubbling stage, so that on one hand, the surfactants can be uniformly dispersed in the gelatin solution, and the stability of a gelatin foam structure generated by bubbling is favourably improved; and on the other hand, hydrogen bonds can be generated between the surfactants and water molecules, so that the surfactants and the water molecules have relatively strong affinity and hygroscopicity, the influence of water evaporation on foam is counteracted, and the risks of foam cracking, rupture and foam collapse are reduced. Therefore, the preparation method not only can improve the yield of the gelatin sponge, but also can improve the haemostatic performance of the product by improving the porosity of the gelatin sponge.
An invention disclosed in patent application number CN115584050A discloses a preparation method of gelatin sponge. The preparation method comprises the following steps: preparing a gelatin solution; foaming the gelatin solution to obtain gelatin foam; freeze-drying and forming the gelatin foam to obtain crude sponge; carrying out oxidation crosslinking on the crude sponge to obtain gelatin sponge; wherein the oxidation cross-linking comprises the following steps: heating the crude sponge, introducing compressed air or oxygen, and reacting to obtain the gelatin sponge. The invention also discloses the gelatin sponge prepared by the preparation method and an embolism material obtained by using the gelatin sponge. According to the gelatin sponge prepared by the invention, a thermal oxidation crosslinking process is adopted, and due to the fact that sterilization and partial removal of a heat source can be realized in a heating process, the gelatin sponge has low bacterial endotoxin, does not contain cross-linking agents such as formaldehyde and glutaraldehyde, and is good in biocompatibility; the prepared embolism material is controllable in expansion time and higher in clinical operability and controllability.
Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat pertains to the creation of a medical material composed of gelatin aimed at halting bleeding during surgical interventions using customized and validated lyophilization process followed by thermal crosslinking. Gelatin, a natural protein sourced from collagen, has long served as a haemostatic agent owing to its ability to induce clotting upon contact with blood. Constituting the primary element of the sponge, gelatin is pivotal for its haemostatic attributes. It is derived from collagen found in animal connective tissues like skin, bone, and cartilage. Gelatin is a water-soluble protein with hydrophilic properties, meaning it attracts and absorbs significant amounts of fluid. This characteristic enables the sponge to swiftly soak up blood and other fluids at the surgical site, effectively managing bleeding.

Objectives of Invention
? Principal objective of the present invention is to prepare absorbable gelatin sponge that quickly achieve haemostasis and stops bleeding during surgery and extensive blood loss.
? Further objective of the present invention is to provide novel haemostatic compositions.
? Another objective of the present invention is to provide a method for preparing the haemostatic composition.
? Further objective of the present invention is to prepare a haemostatic agent with natural composition, cost-effective, eco-friendly and biocompatible, thereby improving its efficacy in achieving rapid and efficient haemostasis.
? Another objective of the present invention is to provide good absorption capacity so that it can absorb more blood (about 50 times of its own weight).
? Another objective of the present invention is to provide haemostat that act as a good carrier of active components such as thrombin and anti-fibrotic drugs.
• Further objective of the present invention is to provide haemostatic composition that has excellent haemostatic effect, mechanical strength, and biodegradability at the same time.

Summary
In order to overcome the above stated problems, present novel invention provides novel haemostatic compositions comprising absorbable gelatin sponge prepared by customized and validated lyophilization process followed by thermal crosslinking. The process described involves the creation of an absorbable gelatin sponge for surgical use. Gelatin is extracted from collagen-rich animal tissues, purified, and then dissolved in water to form a solution. This solution is moulded into sponge shapes and subjected to a lyophilization process, creating a porous structure. Thermal crosslinking is then applied to stabilize the sponge, involving the removal of bound water from gelatin molecules and the formation of intermolecular crosslinks. The resulting sponge is biocompatible and biodegradable, making it safe for use in the body and suitable for surgical procedures requiring temporary haemostasis. The process also includes steps such as packaging and sterilization to ensure product safety. Control of temperature and gelatin concentration is crucial for consistent quality. The sponge's porous structure allows for rapid absorption of fluids, making it effective in controlling bleeding during surgeries.
List of Components
1. Gelatin
2. Foaming agent
3. Gelatin Sponge
4. Water
101. Preparation of Gelatin Solution
102. Thermal Dissolution
103. Aeration Process
104. Molding and Shaping
105. Customized lyophilization
106. Thawing and Drying
107. Thermal Cross-linking
108. Packaging and Sterilization
109. Quality Control

List of Drawings
Figure 1: Flow chart
Figure 2: Product performance test result’s comparision in graphical presentation

Detailed Description of the Invention:
Outlined in detail below are the procedural steps that comprise the formulation process of the present novel Composition and Method for Preparation of Absorbable Gelatin Sponge (3) as Haemostat.
PROCESS VARIANT 1:
Preparation of Gelatin Solution (101):
• Prepare 1 to 5% gelatin solution (1) in purified water (4).
• Employ a gentle stirring with glass rod for effective mixing to ensure uniform dispersion of gelatin (1) particles in the solvent.
Thermal Dissolution (102):
• Transfer the gelatin (1) solution to a temperature-controlled water bath maintained at 40-60°C.
• Add 0.03 to 0.05% of gelatin of Sodium Lauryl Sulfate (SLS), a foaming agent (2), to aid in the dissolution process.
• Monitor the solution over 10-15 minutes, periodically agitating to facilitate complete dissolution.
Aeration Process (103):
1. Apply continuous agitation to the solution for 20-30 minutes to introduce air bubbles and promote aeration.
Molding and Shaping (104):
• Pour the aerated gelatin (1) foam into molds of varying shapes and sizes designed according to specific sponge dimensions.
• Utilize precision pouring techniques to achieve accurate filling of molds without introducing air gaps.
Customized Lyophilization (105):
• Transfer the filled molds to a customized lyophilizer set to -5°C for a predetermined duration of 10-12 hours.
• Low temperature conditions promote the formation of a frozen gelatin sponge (3) with a porous structure conducive to rapid fluid absorption.
Thawing and Drying (106):
• Thaw the frozen gelatin sponge (3) samples gradually to room temperature up to 25°C to 30°C.
• Subject the thawed sponges to drying under controlled conditions (60°C to 80°C; not more than 10%RH) for 10-12 hours to remove excess moisture.
Thermal Cross-linking (107):
• Perform thermal cross-linking of the gelatin sponge (3) in an oven.
• Set the oven temperature within the range of 60-80°C to induce cross-linking reactions for 2 to 8 hours, enhancing the structural integrity of the sponges.
Packaging and Sterilization (108):
• Package the cross-linked gelatin sponge (3) in sterile barrier system in compliance with ISO 11607 series requirement under sterile conditions to prevent contamination.
• Employ gamma radiation for sterilization, ensuring the elimination of microbial contaminants while preserving the integrity of the sponge material.
Quality Control (109):
• Conduct thorough quality control assessments to verify the physical and chemical properties of the prepared gelatin sponge (3).
• Adhere to regulatory standards and guidelines governing the production of medical devices to ensure product safety and efficacy.

PROCESS VARIANT 2:
Preparation of Gelatin Solution (101):
• Prepare 1 to 5% gelatin solution (1) in purified water (4).
• Employ a gentle stirring with glass rod for effective mixing to ensure uniform dispersion of gelatin (1) particles in the solvent.
Thermal Dissolution (102):
• Transfer the gelatin (1) solution to a temperature-controlled water bath maintained at 20-40°C.
• Add 0.03 to 0.05% of gelatin of Sodium Lauryl Sulfate (SLS), a foaming agent (2), to aid in the dissolution process.
• Monitor the solution over 05-10 minutes, periodically agitating to facilitate complete dissolution.
Aeration Process (103):
2. Apply continuous agitation to the solution for 10-20 minutes to introduce air bubbles and promote aeration.
Molding and Shaping (104):
• Pour the aerated gelatin (1) solution into molds of varying shapes and sizes designed according to specific sponge dimensions.
• Utilize precision pouring techniques to achieve accurate filling of molds without introducing air gaps.
Customized Lyophilization (105):
• Transfer the filled molds to a customized lyophilizer set to -3°C for a predetermined duration of 02-08 hours.
• Low temperature promotes the formation of a sponge with a porous structure conducive to rapid fluid absorption.
Thawing and Drying (106):
• Thaw the frozen gelatin sponge (3) samples gradually to room temperature up to 25°C to 30°C.
• Subject the thawed sponges to drying under controlled conditions (40°C to 60°C; not more than 10%RH) for about 10-12 hours to remove excess moisture.
Thermal Cross-linking (107):
• Perform thermal cross-linking of the gelatin sponge (3) in an oven.
• Set the oven temperature within the range of 40°C-50°C to induce cross-linking reactions for 2 to 8 hours, enhancing the structural integrity of the sponges.
Packaging and Sterilization (108):
• Package the cross-linked gelatin sponges (3) in sterile barrier system in compliance with ISO 11607 series requirement under sterile conditions to prevent contamination.
• Employ gamma radiation for sterilization, ensuring the elimination of microbial contaminants while preserving the integrity of the sponge material.
Quality Control (109):
• Conduct thorough quality control assessments to verify the physical and chemical properties of the prepared gelatin sponge (3).
• Adhere to regulatory standards and guidelines governing the production of medical devices to ensure product safety and efficacy.

PROCESS VARIANT 3:
Preparation of Gelatin Solution (101):
• Prepare 1 to 5% gelatin solution (1) in purified water (4).
• Employ a gentle stirring with glass rod for effective mixing to ensure uniform dispersion of gelatin (1) particles in the solvent.
Thermal Dissolution (102):
• Transfer the gelatin (1) solution to a temperature-controlled water bath maintained at 20-40°C.
• Add 0.03 to 0.05% of gelatin of Sodium Lauryl Sulfate (SLS), a foaming agent (2), to aid in the dissolution process.
• Monitor the solution over 20-25 minutes, periodically agitating to facilitate complete dissolution.
Aeration Process (103):
3. Apply continuous agitation to the solution for 40-50 minutes to introduce air bubbles and promote aeration.
Molding and Shaping (104):
• Pour the aerated gelatin (1) solution into molds of varying shapes and sizes designed according to specific sponge dimensions.
• Utilize precision pouring techniques to achieve accurate filling of molds without introducing air gaps.
Customized Lyophilization (105):
• Transfer the filled molds to a customized lyophilizer set to -8°C for a predetermined duration of 15-20 hours.
• Low temperature conditions promote the formation of a sponge with a porous structure conducive to rapid fluid absorption.
Thawing and Drying (106):
• Thaw the frozen gelatin sponge (3) samples gradually to room temperature up to 25°C to 30°C.
• Subject the thawed sponges to drying under controlled conditions (60°C to 80°C; not more than 10%RH) for 10-12 hours to remove excess moisture.
Thermal Cross-linking (107):
• Perform thermal cross-linking of the gelatin sponges (3) in an oven.
• Set the oven temperature within the range of 80-100°C to induce cross-linking reactions for 2 to 8 hours, enhancing the structural integrity of the sponges.
• Further optionally one or more of an Anti-fibrotic drug like Pirfenidone and Nintedanib can be soaked in thermally cross-linked sponge followed by air dried at room temperature.
• Further optionally one or more of Blood clotting components like Factor X, Prothrombin, thrombin, Fibrinogen, Thromboplastin can be injected in thermally cross-linked sponge using syringe.
Packaging and Sterilization (108):
• Package the cross-linked gelatin sponges (3) in sterile barrier system in compliance with ISO 11607 series requirement under sterile conditions to prevent contamination.
• Employ gamma radiation for sterilization, ensuring the elimination of microbial contaminants while preserving the integrity of the sponge material.
Quality Control (109):
• Conduct thorough quality control assessments to verify the physical and chemical properties of the prepared gelatin sponges (3).
• Adhere to regulatory standards and guidelines governing the production of medical devices to ensure product safety and efficacy.
In the above novel process, the foaming agent (2) used is either alone or in combination of surfactant from the group of Ionic, anionic and cationic agents preferably Sodium Lauryl Sulfate (SLS), sodium laureth sulfate (SLES), sodium coco sulfate (SCS), and sodium lauroyl sarcosinate (SLSA). It is to be used in the concentrations of 0.03% to 0.05% of main ingredient.
Sr. No. Tests Limit Result
Process Variant 1 Process Variant 2 Process Variant 3
1. Color Off white to slight yellow Slight Yellow Slight Yellow Slight Yellow
2. Odor Odorless Odorless Odorless Odorless
3. Solubility in Water Insoluble Insoluble Insoluble Insoluble
4. Residue on ignition Not more than 2.0% 1.10 0.94 1.89
5. Digestibility Not more than 75 minutes 40 15 42
6. Water absorption Not less than 35 times its weight of water 50 29 34
TABLE 1: Product performance Test Results of In-House Manufactured Gelatin Sponge

For a comprehensive assessment, the following compositions are outlined for comparison with the present novel formulation.
EXPERIMENT 1:
In this example, Absorbable Gelatin Sponge (3) crosslinking with ionization radiations such as gamma radiations, e-beam, UV radiations to cross-link gelatin sponge (3). These radiations can induce crosslinking of the gelatin (1) molecules through the formation of reactive intermediates. In this embodiment UV radiations were used for Crosslinking. Wavelength range: UV radiation is categorized into three main regions based on wavelength: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm). For crosslinking applications, UVA and UVB radiation are typically used, as they are more effective in initiating crosslinking reactions. UV radiation induces crosslinking through a process called photochemical reaction. When UV photons interact with a photosensitive material, they are absorbed by specific molecules or photo initiators present in the material. This absorption leads to the generation of highly reactive species (e.g., free radicals) that can initiate crosslinking by reacting with polymer chains. Photo initiators are compounds that absorb UV radiation and subsequently generate reactive species. They play a crucial role in initiating and controlling the crosslinking process. Commonly used photo initiators include benzoin methyl ether (BME), benzoin ethyl ether (BEE), benzoin isobutyl ether (BIB), and benzoin benzyl ether (BBE). The effectiveness of UV crosslinking depends on the intensity of UV radiation (irradiance) and the exposure time. Higher irradiance levels generally lead to faster crosslinking, but they must be carefully optimized to prevent overheating or damage to the material. Exposure time is determined based on the desired degree of crosslinking and the thickness of the material being treated. The crosslinking process can be controlled by adjusting various parameters, such as the concentration of photo initiators, UV exposure intensity, and exposure time. The selection of photo initiators and their concentration can be tailored to achieve specific crosslinking characteristics, such as curing speed, depth of cure, and material properties.
In this embodiment, gelatin sponges were cross-linked via UV radiation exposure instead of thermal cross-linking at stage 107, using UVA light (? = 365 nm) for 60 minutes. They were then sterilized using gamma radiation for subsequent use in surgical applications. Finally, the gelatin sponges were obtained through UV radiation treatment. To evaluate the product performance characteristics of the prepared sponge; samples are analyzed for the tests as mentioned in the USP.
Sr. No. Tests Limit Result
1. Color Off white to slight yellow Slight Yellow
2. Odor Odorless Odorless
3. Solubility in Water Insoluble Insoluble
4. Residue on ignition Not more than 2.0% 1.09
5. Digestibility Not more than 75 minutes 36
6. Water absorption Not less than 35 times its weight of water 42
TABLE 4A: Product performance Test Results of In-House Manufactured Gelatin Sponge with UV Radiations
EXPERIMENT 2:
In this example, gelatin sponge (3) was prepared in combination with thrombin or Factor X to enhance its haemostatic properties. The gelatin (1) has a protective and stabilizing effect on the thrombin with which it is impregnated. In this embodiment, the cross-linked gelatin sponges were injected with Lyophilized thrombin (100 IU) solution using syringe and immediately pack in a primary packaging. Moreover, the sponges were sterilized using gamma radiation for further use in surgical applications. Finally, the gelatin sponges (3) were obtained with combination of thrombin.
To evaluate the product performance characteristics of the prepared sponge; samples are analyzed for the tests as mentioned in the USP.
Sr. No. Tests Limit Result
1. Color Off white to slight yellow Slight Yellow
2. Odor Odorless Odorless
3. Solubility in Water Insoluble Insoluble
4. Residue on ignition Not more than 2.0% 1.13
5. Digestibility Not more than 75 minutes 35
6. Water absorption Not less than 35 times its weight of water 46
TABLE 5A: Product performance Test Results of In-House Manufactured Gelatin Sponge with thrombin
EXPERIMENT 3:
The efficacy of thermally cross-linked gelatin sponge (3) as mentioned in Example 1 was compared with chemically cross-linked gelatin sponge (3). The preparation of chemically cross-linked gelatin sponge (3) is described below;
In this embodiment chemical cross linker i.e. 0.8 mL of glutaraldehyde was added as a chemical cross-linking agent instead of foaming agent (2). Finally, the gelatin sponges (3) were obtained with combination of aldehyde group crosslinking agents.
To evaluate the product performance characteristics of the prepared sponge; samples are analyzed for the tests as mentioned in the USP.
Sr. No. Tests Limit Result
Chemically cross-linked Gelatin sponge Thermally cross-linked Gelatin sponge
1. Color Off white to slight yellow Slight Yellow Slight Yellow
2. Odor Odorless Odorless Odorless
3. Solubility in Water Insoluble Insoluble Insoluble
4. Residue on ignition Not more than 2.0% 1.18 1.10
5. Digestibility Not more than 75 minutes 35 40
6. Water absorption Not less than 35 times its weight of water 41 50
TABLE 6A: Comparison of Product performance test results of thermally cross-linked Gelatin sponge with chemically cross-linked Gelatin sponge
EXPERIMENT 4:
Absorbable Gelatin Sponge (3) loaded with Anti-fibrotic drugs such as Pirfenidone (1000 µg/kg), Nintedanib (500 µg/Kg) of gelatin (1) can be used in surgical procedures to prevent the formation of scar tissue and fibrosis. In this embodiment, the cross-linked sponges are soaked in Pirfenidone (1000 µg/kg) solution followed by air dried at room temperature. Moreover, the sponges were packed and sterilized using gamma radiation for further use in surgical applications. Finally, the gelatin sponges (3) were obtained with combination of anti-fibrotic drugs.
To evaluate the product performance characteristics of the prepared sponge; samples are analyzed for the tests as mentioned in the USP.
Sr. No. Tests Limit Result
1. Color Off white to slight yellow Slight Yellow
2. Odor Odorless Odorless
3. Solubility in Water Insoluble Insoluble
4. Residue on ignition Not more than 2.0% 1.14
5. Digestibility Not more than 75 minutes 33
6. Water absorption Not less than 35 times its weight of water 39
TABLE 7A: Product performance Test Results of In-House Manufactured Gelatin Sponge with Anti-fibrotic drugs

Figure 2 illustrates a comprehensive comparison of performance test results for the absorbable gelatin sponge (3). Notably, among the variants tested, the novel process variant 1 showcased exceptional superiority in water absorption, digestibility, and residue on ignition. This remarkable performance underscores the substantial potential of novel process variant 1, positioning it as a standout candidate for advancing the product's overall efficacy and market competitiveness.
To assess the safety and efficacy of the present novel Absorbable Gelatin Sponge (3) produced via presently disclosed novel process variant 1, a comprehensive series of biocompatibility assessments has been conducted, encompassing the following methodologies:
1. Intra-cutaneous Reactivity Test
Intracutaneous reactivity test of absorbable gelatin sponge (3) was conducted in New Zealand white rabbits. Polar (physiological saline) and non-polar (cottonseed oil) extracts were prepared by extracting 0.1 g of Absorbable Gelatin Sponge (3) per milliliter of solvent at 37°C for 72 h. Solvent controls were also subjected to the same temperature and time conditions. Three rabbits were treated intracutaneously with polar and non-polar extracts/solvent controls, respectively. The skin reactions were visually scored approximately at 24 h, 48 h and 72 h following the Absorbable Gelatin Sponge (3) extracts administration according to the scoring system mentioned in ISO 10993-10:2010(E). The animals were observed for 3 consecutive days for morbidity, mortality and abnormal clinical signs and symptoms following the Absorbable Gelatin Sponge (3) extract administration. The results indicated that animals treated with the Absorbable Gelatin Sponge (3) extracts did not show any skin reactions. Hence, it is concluded that the given Absorbable Gelatin Sponge (3), for surgical use, supplied by Aegis Lifesciences, meets the requirements of ISO 10993-10:2010(E) under the conditions of the present study.
2. Skin Sensitization Study
Skin sensitization potential of absorbable gelatin sponge (3) was evaluated using the guinea pig maximization test as per ISO 10993-10:2010(E), Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization. Polar (physiological saline) and non-polar (sunflower oil) extracts were prepared by extracting 0.1 g of Absorbable Gelatin Sponge (3) per milliliter of solvent at 37 °C for 72 h. Solvent controls were also subjected to the same temperature and time conditions. Induction of sensitization was a two-stage procedure with intradermal injections initially administered, followed by a closed topical patch exposure on day 7. Two weeks following the topical patch induction, the challenge exposure was administered for 24 h. Skin reaction grading was performed using a Magnusson and Kligman scale at 24 h and 48 h after removing the challenge patch. A comparison of the skin reactions elicited in terms of incidence and severity were made to determine whether the Absorbable Gelatin Sponge (3) induces sensitization. The susceptibility of these strains of guinea pigs to known sensitizing agent, a-Hexy1cinnamaldehyde has been established at GLR Laboratories and confirmed every six months. Therefore, the assay was considered valid. The results indicated that animals treated with the Absorbable Gelatin Sponge (3) extracts did not show any sensitization reactions. Hence, it is concluded that the given Absorbable Gelatin Sponge (3), for surgical use supplied by Aegis Lifesciences, is a non-sensitizer under the conditions of the present study.
3. Acute Systemic Toxicity
Acute systemic toxicity of absorbable gelatin sponge (3) was evaluated in Swiss albino mice. Polar (physiological saline) and non-polar (sunflower oil) extracts were prepared by extracting 0.1 g of Absorbable Gelatin Sponge (3) per milliliter of solvent at 37°C for 72 h. Solvent controls were also subjected to the same temperature and time conditions. Four groups of mice, each comprising of five males were treated intravenously and intraperitoneal, with polar and non-polar extracts/solvent controls, respectively. The animals were observed for 3 consecutive days for morbidity, mortality, and abnormal clinical signs and symptoms following the Absorbable Gelatin Sponge (3) extract administration. The results indicated that animals treated with the Absorbable Gelatin Sponge (3) extracts did not show any systemic toxicity. Hence, it is concluded that the given Absorbable Gelatin Sponge (3), supplied by Aegis Lifesciences, meets the requirements of ISO 10993-11 :2006 (E) under the conditions of the present study.
4. 28 Days Subcutaneous Implantation Test
Absorbable gelatin sponge (3) (test item) was evaluated the safety and living tissue reaction including final integration, resorption or degradation when implanted subcutaneously in New Zealand White rabbits over a period of 28 days under the test conditions as described in ISO 10993-6:2007(E). The Absorbable Gelatin Sponge (3) and negative control items were subcutaneously implanted to each animal to yield a total of 12 Absorbable Gelatin Sponge (3) and 12 negatives controls items respectively for a period of 28-day implantation period. All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality throughout the experimental period. Body weight was recorded at receipt, on the day of implantation (Day I) and at termination of the observation period.
Result - No treatment related clinical signs of toxicity and mortality were observed during the experimental period. No treatment related changes were observed in body weight and percent change in body weight with respect to day I. No macroscopic changes were noticed in implantation sites in any of the animal. Microscopically, the Absorbable Gelatin Sponge (3) at implanted sites completely degraded and reabsorbed when compared to negative control implanted sites.
Conclusion -Under the experimental conditions employed in the study, the biological response of living tissue to Absorbable gelatin sponge (3) implanted in subcutaneous tissue of New Zealand White Rabbits over a period of 28 days under test conditions, it can be concluded that the Absorbable Gelatin Sponge (3) was non-irritant and absorbable in 28 days when compare with the control.
5. In Vitro Cytotoxicity Study
Absorbable gelatin sponge (3) was evaluated for in vitro cytotoxicity in compliance with the International Standard ISO 10993-05:2009 by elution method in L-929 mouse fibroblast cells. The growth medium in each well was replaced with extraction media of Absorbable Gelatin Sponge (3), negative control (High Density Polyethylene) and positive control (Polyurethane) in triplicates. Blank wells were treated with extraction medium maintained similar to Absorbable Gelatin Sponge (3) extraction without any treatment. Approximately post 48 hours of incubation, the treatment wells were examined microscopically for any abnormal cell morphology and cell lysis.
Result - No cytotoxic reactivity was observed in L-929 mouse fibroblast cell lines treated with the Absorbable Gelatin Sponge (3) extract (Discrete intracytoplasmic granules with no cell lysis, Grade 0 reactivity). Similarly, cells in the blank wells and negative control showed discrete intracytoplasmic granules with no cell lysis (Grade 0), whereas the positive control resulted in nearly complete destruction of the cell monolayer (Grade 4).
Conclusion - Since there was no reactivity observed for Absorbable Gelatin Sponge (3) and as the reactivity grade was not greater than 2, Absorbable Gelatin Sponge (3), is considered to be non-cytotoxic to the sub confluent monolayer of L-929 mouse fibroblast cells.
6. Genotoxicity Test Study:
The results of the Bacterial Reverse Mutation test with a method I (Plate incorporation) and method II (Pre incubation) indicate that under the tested conditions, the Absorbable Gelatin Sponge (3) Polar and Non-Polar Extracts at highest concentration 100% did not cause a positive increase in the mean number of revertant colonies per plate with any of the tester strains either in the presence or absence of metabolic activation (S9). Hence, Absorbable Gelatin Sponge (3) Polar and non-polar extracts (100%) showed no potential mutagenic effect over any tester strains, both in presence (+S9) and absence of metabolic activation (-S9)
7. Sub chronic toxicity study:
Under the condition of this study, the intravenous administration of polar extract and intraperitoneal administration of non-polar extract of Absorbable Gelatin Sponge (3) well tolerated by the animals and did not show any adverse toxicity.
8. Material Mediated Pyrogen Test:
Based on the results of the experiment, it is concluded that the extract of Absorbable Gelatin Sponge (3) in New Zealand White Rabbits is non-pyrogenic as it meets the requirements of pyrogen test as per U.S. Pharmacopoeia, General Chapters: <151> Pyrogen Test.
9. In-vivo mammalian Erythrocyte micro-nucleus test:
Based on the results obtained, it is concluded that the extract of Absorbable Gelatin Sponge (3) is non-genotoxic at the limit dose of 100% polar and non-polar Absorbable Gelatin Sponge (3) extract under the conditions employed during the experiment.


Type of Study/Test Article Species and Strain Method of Administration Test duration Reference GLP Compliance Testing Facility Study Number Result
Intracutaneous reactivity test New Zealand white rabbits Intracutaneously 3 days ISO 10993-10:2010(E) Yes GLR Laboratories Private Limited 158/001 Non-Irritant
Skin sensitization study Guinea pig Two-stage procedure with intradermal injections initially administered, followed by a closed topical patch exposure on day 7 21 days ISO 10993-10:2010(E) Yes GLR Laboratories Private Limited 158/002 Non-Sensitizer
Acute systemic toxicity Swiss albino mice Intravenously and intraperitoneal 3 days ISO 10993-11:2006(E) Yes GLR Laboratories Private Limited 158/003 Non-Toxic
28 Days Subcutaneous Implantation Test New Zealand white rabbits Subcutaneously 28 days ISO 10993-6:2007(E). Yes Bioneeds India Private Limited BIO-TX 1400 Non-Irritant and absorbable in 28 days
In Vitro Cytotoxicity Study L-929 mouse fibroblast cells In vitro 2 days ISO 10993-5:2009 Yes Bioneeds India Private Limited BIO-GT 268 Non-Cytotoxic
Genotoxicity Test for Absorbable gelatin sponge Bacterial reverse mutation Plate incorporation
Pre-incubation method 3 days ISO 10993-3:2014 (E) Yes Liveon biolabs private limited LBPL/IS/GT-165 Non-Genotoxic
Sub chronic toxicity study of Absorbable gelatin sponge Sprague- Dawley rats Intravenously and intraperitoneal 17 days ISO 10993-11:2017(E) Yes Bioneeds India Private Limited BIO-CTX 012 Non-Toxic
Material Mediated Pyrogen test New Zealand white rabbits Intravenous route 50 days ISO 10993-11:2017(E) and <151> USP-NF Pyrogen test Yes Bioneeds India Private Limited BIO-ATX 1201 Non-Pyrogenic
In-vivo mammalian Erythrocyte micro-nucleus test Swiss-Albino mice Intravenously and intraperitoneal 100 days ISO 10993-3:2014 (E), ISO 10993-33:2015 (E) Yes Bioneeds India Private Limited BIO-GNT 572 Non-Genotoxic
Table 1: Summary of biocompatibility tests for the safety and efficacy of novel Absorbable gelatin sponge
10. In-vitro blood coagulation test:
Coagulation methods are based on the use of native (fresh, non-anticoagulated) whole blood, anticoagulated whole blood (usually citrated), platelet-rich plasma or platelet-poor plasma. Collect and transfer 2.5 ml of whole blood into each of the two Test Tubes/Vacutainer Tubes. Transfer the samples of novel absorbable gelatin sponge (3) into Test Tubes/Vacutainer Tubes and set them aside for 6-7 minutes. Similarly, collect and transfer 2.5 ml of whole blood into a separate Test Tube/Vacutainer Tube without a sponge sample and set it aside for 6- 7 minutes. This will serve as the control sample for the testing. Test the above samples for PT, APTT, Fibrinogen, FDP, TT, D-Dimer as per the standard laboratory procedure and record the test results.
Test Control Novel Absorbable Gelatin Sponge Range
(As per ISO 10993-4)
Prothrombin Time (PT) (s) 13.1 11.8 11.3-14.7
Activated Partial Thromboplastin Time (APTT) (s) 32.2 31.9 21.9-33.6
Fibrinogen (mg/dl) 272 262 200-400
Fibrinogen degradation product (FDP) (µg/ml) <5 <5 0-5
Thrombin time (TT) (s) 16.9 16.1 11.8-17.6
D-Dimer (ng/ml) 108 116 0.00-500
Table 2: In-vitro blood coagulation test

Most gelatin sponges in the market are made using chemical crosslinking methods, which can be toxic. However, the present novel invention emphasizes a new approach to manufacturing a novel absorbable gelatin sponge. It utilizes a customized lyophilization process followed by thermal crosslinking, eliminating the need for any chemical crosslinking agents. Based on its performance and biocompatibility, the novel thermally cross-linked gelatin sponge from the present invention is deemed safe, non-toxic, and offers superior product performance.
, Claims:Claims:
I claim,
1. A Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat which is Non-Irritant, Non-Sensitizer, Non-Toxic, Non-Cytotoxic, Non-Genotoxic and Non-Pyrogenic, wherein
prepare a gelatin solution (101) by dissolving a gelatin (1) powder in purified water (4), followed by thermal dissolution (102) in a temperature-controlled water bath, along with the addition of a Foaming agent (2) to aid dissolution;
wherein further the said solution undergoes aeration process (103) to introduce air bubbles and is further poured into molds;
wherein the filled molds further undergo customized lyophilization (105) to create a porous frozen gelatin sponge (3);
wherein further the said frozen gelatin sponge (3) samples are thawed and dries (106) gradually to room temperature and then dried under controlled conditions respectively;
wherein further for the Thermal cross-linking (107) of the said gelatin sponge (3) is performed in an oven;
wherein optionally for the cross-linking (107), of the said gelatin sponge (3) is performed with UV radiation exposure using UVA light;
wherein further optionally one or more of an anti-fibrotic drugs preferably Pirfenidone or Nintedanib can be soaked in the said thermal cross-linked gelatin sponge (3) followed by air dried at room temperature;
wherein further optionally one or more of blood clotting components from the group of Factor X, Prothrombin, thrombin, Fibrinogen, Thromboplastin can be injected in to the said thermal cross-linked gelatin sponge (3) using a syringe; and
wherein for the Packaging and Sterilization (108), the said thermal cross-linked gelatin sponge (3) is packed in sterile barrier system and further sterilized.
2. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein for the preparation of gelatin solution (101), take 1 to 5% gelatin (1) powder in purified water (4) to create a gelatin (1) solution with gentle stirring.
3. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein further for the Thermal Dissolution (102) process, the said gelatin (1) solution is transferred to a temperature-controlled water bath maintained at 20-60°C, and further add the foaming agent (2) in the range of 0.03 to 0.05% of the taken gelatin (1) powder.
4. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein further for the Aeration Process (103) continuous agitation of the said gelatin (1) solution having the foaming agent (2) is done for 10-50 minutes to introduce air bubbles, and is further poured into molds.
4. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein further for the Customized Lyophilization (105), the said filled molds are subjected to lyophilizer having temperature of -3°C to -8°C for 02-20 hours.
5. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein further for the Thawing and Drying (106) process, the said frozen gelatin sponge (3) is gradually brought to room temperature up to 25°C to 30°C for thawing; and further the said thawed sponges are subjected to drying at 40°C to 80°C along with not more than 10%RH, for 10-12 hours.
6. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein further for the Thermal Cross-linking (107), the said thawed and dried gelatin sponge (3) is placed in an oven with a temperature range of 40-100°C for 2 to 8 hours.
7. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein for the Packaging and Sterilization (108) process, the said thermally cross-linked gelatin sponge (3) is packed in sterile barrier system under sterile conditions and further subjected for sterilization.
8. The Composition and Method of Preparation for Absorbable Gelatin Sponge as Haemostat as claimed in claim 1, wherein the foaming agent (2) is selected either alone or in combination from a group of Sodium Lauryl Sulfate (SLS), sodium laureth sulfate (SLES), sodium coco sulfate (SCS), and sodium lauroyl sarcosinate (SLSA).