Description: Description:
TITLE: Bridging efficacy of biocompatible Nanofibrous Scaffold for Soft Tissue Regeneration

FIELD OF THE INVENTION
The present invention relates to the field of biomedical engineering, specifically to the development of multifunctional, nanofibrous scaffolds with enhanced biocompatibility, mechanical strength, and anticancer properties. The invention is particularly suitable for applications in soft tissue regeneration and wound healing.

BACKGROUND OF THE INVENTION

Soft tissue regeneration involves the repair or replacement of damaged skin, muscles, and connective tissues using biocompatible scaffolds that mimic the extracellular matrix (ECM). Conventional scaffolds suffer from poor mechanical properties, limited biocompatibility, inadequate bioavailability of therapeutic agents, and poor multifunctionality. There remains a significant need for advanced scaffolds that address these limitations.

Natural polymers such as gelatin and collagen offer bioactivity but lack mechanical integrity, while synthetic polymers like PVA provide strength but are biologically inert. Moreover, most current scaffolds are either single-functional or fail to provide sustained therapeutic benefits. Therefore, a composite scaffold integrating biocompatibility, mechanical durability, thermal stability, and therapeutic potential is of great importance in tissue engineering and cancer treatment.

In one of the prior art US patent application number US11497832B2 titled Biocompatible hydrogel compositions and uses thereof discloses biocompatible and/or biodegradable hydrogel compositions comprising native collagen and chondroitin sulfate, the collagen and chondroitin sulfate being chemically cross-linked thereby forming a matrix. The native collagen may comprise recombinant human collagen type I (rHCI), recombinant human collagen type III (rHCIII), or a combination thereof, for example. Methods and uses thereof for regeneration or repair of tissue, improvement of tissue function, mechanical stabilization of tissue, prevention of tissue damage, or prevention of tissue loss of function are described, particularly with respect to cardiac tissue and myocardial infarction events.

In yet another prior art PCT patent application number WO2008157608A1 titled Composite scaffolds for tissue regeneration discloses a porous multi-layer composite scaffold useful for tissue regeneration and a method of fabricating the same. The porous multi-layer composite scaffold comprises a first layer comprising crosslinked collagen and a polysaccharide; a second layer of crosslinked collagen and calcium based minerals, which is covalently bonded to the first layer; and a third layer of crosslinked collagen and a polysaccharide, which is covalently bonded to the second layer. Preferably, the second layer further comprises a polysaccharide. The ratio of collagen to polysaccharide in each of the three layers is from about 3:1 to about 1:1 by weight. The porous multi-layer composite scaffold may further comprises a biologically active agent.

Several prior art studies have reported scaffolds using components like gelatin, collagen, alginate, or hydroxyapatite for tissue regeneration, but they often suffer from poor mechanical strength, limited biocompatibility, lack of multifunctionality, and inadequate thermal stability. For example, existing systems such as gelatin–collagen or alginate–PVA–HAP scaffolds lack dual therapeutic capabilities or effective crosslinking strategies. The present invention overcomes these drawbacks by incorporating a synergistic blend of PVA, SA, gelatin, bovine collagen, and hydroxyapatite nanoparticles, crosslinked with glutaraldehyde, to deliver a scaffold with superior tensile strength, excellent biocompatibility, and both regenerative and anticancer functionality.

OBJECTIVE OF THE INVENTION
The principal object of the present invention is:
To fabricate a nanofibrous scaffold using sodium alginate (SA), polyvinyl alcohol (PVA), gelatin, bovine collagen, and hydroxyapatite nanoparticles (n-HAP), which exhibits enhanced mechanical, thermal, and biological properties.
To improve the tensile strength and stability of the scaffold through glutaraldehyde crosslinking.
To demonstarte the in vitro biocompatibility behaviour with fibroblast cells of MCF-10A and cytotoxicity against the MCF-7 Breast cancer cells.
To study the multifunctional potential of the developed scaffold in promoting the tissue regeneration applications.
SUMMARY OF THE INVENTION
It will be understood that this disclosure is not limited to the particular systems, and methodologies described, as there can be multiple possible embodiments of the present disclosure which are not expressly illustrated in the present disclosure. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present disclosure.

The present invention discloses a multicomponent nanofibrous scaffold comprising sodium alginate, polyvinyl alcohol, gelatin, and bovine tendon collagen, integrated with hydroxyapatite nanoparticles (n-HAP). The scaffold exhibits enhanced tensile strength (from 17.52 MPa to 39.89 MPa), porosity (85.6%), swelling percentage (69.4%), and biocompatibility (93% cell viability in fibroblasts). Crosslinking with glutaraldehyde further improves thermal stability (up to 550 °C). The scaffold shows cytotoxic efficacy by reducing MCF-7 cancer cell viability by approximately 89%, offering a dual-functionality platform for tissue regeneration and anticancer treatment. The invention provides a cost-effective, eco-friendly, and scalable scaffold suitable for clinical applications.
BRIEF DESCRIPTION OF DRAWING
Figure 1 illustrates the exemplary embodiment of Graphical representation of Fabricated scaffold for Skin tissue regeneration
Figure 2 illustrates the exemplary embodiment of graphical representation of FT-IR spectra of (a) Sodium Alginate Polyvinyl Alcohol–Gelatin–Collagen scaffold (SPGC), (b) Hydroxyapatite nanoparticles incorpoarted Sodium Alginate–Polyvinyl Alcohol–Gelatin–Collagen scaffold (nH-SPGC), and (c) Glutaraldehyde crosslinked with the hydroxyapatite nanoparticles incorpoarted Sodium Alginate–Polyvinyl Alcohol–Gelatin–Collagen scaffold (Gn-SPGC).
Figure 3 illustrates the exemplary embodiment of graphical representation of TGA-DTA Curve Analysis of (a) SPGC, (b) nH-SPGC, and (c) Gn-SPGC
Figure 4 illustrates the exemplary embodiment of graphical representation of (a) Porosity, (b) Swelling, and (c) Contact Angle
Figure 5 illustrates the exemplary embodiment of Scanning electron images of (a) SPGC (b) nH-SPGC and (c) Gn-SPGC scaffolds
Figure 6 illustrates the exemplary embodiment of In-vitro biocompatible microscopic images of Gn-SPGC scaffolds treated with MCF – 10A cells (a) control cells (b) 50 µg/ml (c) 250 µg/ml and (d) cell viability (%). The samples are analyzed with a standard deviation of (n=3, p*<0.05).
Figure 7 illustrates the exemplary embodiment of In-vitro cytotoxicity microscopic images of MCF-7 cells for the Gn-SPGC scaffold (a) control; (b) 10 µg/ml, (c) 50 µg/ml, (d) 150 µg/ml, (e) 250 µg/ml and (f) cell viability (%) of the scaffold. The samples are analyzed with a standard deviation of (n=3, p*<0.05).

DETAILED DESCRIPTION OF THE INVENTION
Material Composition and Fabrication Process (100):
Preparation of Sodium Alginate and Gelatin Solution (1):
Procedure: To prepare a combined solution of sodium alginate and gelatin, begin by weighing a 1 gram of Sodium Alginate (SA) and dissolved in 100 milliliters of distilled water, then it is allowed to stir the mixture continuously for 2 hours to ensure complete dissolution. Once the solution is clear, homogeneous, and slightly viscous, a gram of gelatin is added to the SA solution and stirred for an additional 1 hour. To facilitate proper dissolution of gelatin, the temperature is maintained in the range of 35 – 40 °C using a hot plate stirrer. The resulting sodium alginate–gelatin solution is stored for further use.

Preparation of Polyvinyl Alcohol Solution:
Procedure: In parallel, 10 g of polyvinyl alcohol (PVA) is dissolved in 100 mL of distilled water at 80 °C (2) with continuous stirring for 3 hours. A moderate stirring speed is maintained to prevent splashing and ensure uniform mixing. Following complete dissolution, to eliminate entrapped air bubbles formed during stirring, the solution is subjected to ultrasonic treatment (sonication) for 15 minutes using a bath sonicator. This step is important for achieving a bubble-free, homogeneous solution suitable for downstream applications such as blending with other biopolymers, casting into films, or scaffold fabrication.

Combination of Solutions:
Procedure: To prepare the composite scaffold solution, equal volumes of the previously prepared sodium alginate/gelatin (SA/gelatin) solution and the polyvinyl alcohol (PVA) solution are measured and mixed in volumetric 1:1 ratio. (3). The combined solution is transferred to a clean beaker and subjected to continuous magnetic stirring for 3 hours at room temperature to ensure thorough mixing and uniform dispersion of the three polymer components. Following this, an optimized amount of bovine tendon collagen, a natural extracellular matrix protein known for its excellent biocompatibility are carefully weighed and added to the blended polymer solution. Stirring is continued until the collagen is fully dissolved and uniformly distributed within the mixture, producing a homogeneous, composite scaffold solution.

Formation of the Scaffold:
Procedure: A volume of 30 mL of the above scaffold solution is transferred to 250 ml conical flask and allowed to freeze dry at – 20 °C for overnight (4) and the sample is lyophilized. Finally, the dried scaffold is named as SPGC (Sodium Alginate–Polyvinyl Alcohol–Gelatin–Collagen).

Incorporation of Hydroxyapatite Nanoparticles (n-HAP):
To enhance the mechanical and biocompatible properties, an optimized quantity of hydroxyapatite nanoparticles (n-HAP) is added to the SPGC solution. The mixture is stirred thoroughly to promote uniform nanoparticle dispersion and facilitate surface interaction. By following the same procedure, the above solution is lyophilized and labeled as nH-SPGC (5).

Crosslinking with Glutaraldehyde (Gn-SPGC):
For chemical crosslinking, a diluted glutaraldehyde solution is freshly prepared using ethanol. Then, the nH-SPGC solution is allowed to crosslinked with desired amount of diluted glutaraldehyde solution (6) and allowed to stir for another 30 mins. Finally, the crosslinked scaffold is lyophilized by following the same procedure and labeled as Gn-SPGC (7).

Porosity test
Among the various porosity tests, the scaffold porosity is measured by employing the liquid displacement method. The scaffolds are cut into 1cm2 dimensions and immersed in a synthetic PBS solution. Before immersing the scaffold in solution, the initial known volume of the scaffold is noted as V1 by measuring the respective scaffold for calculation. The scaffold is soaked in the corresponding solution for 30 minutes, then the soaked scaffold is weighed and noted as V2. Furthermore, the remaining solution in the soaked scaffold is noted as V3. The porosity percentage of the respective samples is obtained by calculating the values of V1, V2, and V3 with the given below formula
Porosity %=((V_1-V_3))/((V_2-V_3)) X 100
Swelling Test
Observing the swelling behaviour of the scaffold material is important for understanding the biological interaction between the bio-fluids and tissues. For this, the initial mass of the cleaned dry scaffold is measured as Ms, and then the scaffold is immersed in 1 ml of PBS solution with pH 7.4. Subsequently, in 2 hours and 24 hours, the scaffold is taken from the PBS solution and the excess PBS on the surface of the scaffold is removed and its swollen mass is weighed and measured as M. Then, the swelling percentage of the scaffold is determined by the below equation
Swelling %=[(M-Ms)/M] × 100
Contact angle measurement
Contact angle measurement is an analysis used to determine the wettability of the material. The analysis is carried out by Contact Angle Goniometer (Ossila). The synthesized scaffold material is cut down to 10 mm2. Approximately, 2 μL droplet of water is placed on the material. The angle of contact between the material and droplets is measured by an image analyser and the photographs are captured by a digital camera

Tensile strength
INSTRON Universal Testing Machine E-3000 with a load cell of 4.5 kg under a cross-head speed of 0.1 mm/min. It is used to measure the tensile properties of the scaffolds. So, the scaffold specimens are cut into rectangular pieces (1cm×5cm) to test the tensile strength
Equation for tensile strength is used to calculate the elongation at break, where Li is the sample’s initial length and Lmax is the maximum length at the tear point.
Tensile strain at break (%)= L_max/L_i X 100
Below equation is used to calculate the ultimate tensile strength of the samples, at the sample tear point, Umax is the input load and the cross-section of the samples is C.
Tensile Strength (MPa)= U_max/C

In vitro cell line study on the prepared scaffolds
According to the Mosmann (1983 by using (3‐ (4, 5‐dimethyl thiazol‐2yl) ‐ 2, 5-5-diphenyl tetrazolium bromide (MTT), the in-vitro cell viability assay was carried out. Initially, 1 mg of sample was mixed with sterile complete media and further diluted for desired concentrations, similarly MTT was diluted in a phosphate buffered solution of 5 mg/ml concentration. The cell viability study was examined on human breast fibroblast cells of MCF-10A, and on the human breast cancerous cells of MCF-7 which were brought from the NCCS, Pune. Then, the cells were cultured with the DMEM, a high glucose medium which contains 1% antibiotic–antimycotic solution and 10% fetal bovine serum, also it was incubated in the presence of 5 % CO2 at 37 ℃. Using EDTA the cells were trypsinized during the formation of monolayer which is obtained in the cultured flask. Through the trypsin treatment, the cells were detached and inactivated by adding 3 to 5 ml of serum containing media. And, the cells were collected by centrifuging at 1500 rpm and stored, then the supernatant was discarded. Furthermore, the collected cells were seeded to a 96 well plates and after 24 hours of cells incubation, various concentration of the samples were treated with the cells followed by 12 - 24 hours of incubation. Finally, the serum containing media was removed and prepared MTT solution was added to the plates and left for 4 hours of incubation at room temperature. During this incubation period of 4 hours, a purple formazan crystals were formed which are dissolved in 100 µl of DMSO solution. The absorbance of each concentrated plates was recorded with microplate reader at 570 nm. Thus, MTT is cleaved by mitochondrial Succinate dehydrogenase and reductase of viable cells, the yield cells are quantified with respect to the formation of purple formazan. This formazan production is directly proportional to the number of viable cells and its production is inversely proportional to the degree of cytotoxicity. From this study, the percentage of cell viability on MCF-10A and MCF-7 were calculated by below equation:
Cell viability (%)= ((Total number of viable cells )/(Total number of cells))*100
Result and Discussion
Fourier Transform Infrared Spectroscopy (200)
The structural and functional groups present in the scaffold material are analysed by the FTIR spectrum and each spectrum of SPGC, nH-SPGC, and Gn-SPGC are displayed in Figure 2. As the result of multicomponent present in the scaffolds material, it is quite difficult to analyse each individual function group present in the material due to the overlapping of bands. But the characteristic peak of each material is analysed and discussed. The characteristic peak of PVA observed at 842 cm-1, 1109 cm-1, and 1110 cm-1, are due to the C-H rocking, and C-O stretching vibration, in addition to this, 1349 cm-1 is observed due to the presence of CH-OH bending vibration. The major characteristics peaks of phosphate group present in the synthesized n-HAP are observed at 562 cm-1, and 616 cm-1 are attributed to the PO4-3 (ν4) symmetric bending, and 1022 cm-1 is attained due to the presence of PO4-3 (ν4) asymmetric stretching band. The peaks appeared at 1415 cm-1 and 1634 cm-1 are attributed to the symmetric and asymmetric stretching group of COO due to the presence of sodium alginate group. The small peak obtained at 1249 cm-1 represents the N-H group of amide III which proved the existence of gelatin and the collagen fibrils are also overlapped to the SA peak of 1634 cm-1 which is the main characteristic peaks of collagen (β-sheet) of the Amide I group and N-H group. Then, the IR absorption occurs at 2854 cm-1 and 2855 cm-1 are due to the occurrence of glutaraldehyde crosslinking agent. Intresetingly, after crosslinking the Gn-SPGC scaffold material, the phosphate groups are barely visible from the FTIR data. From the Figure 2, the fabricated scaffold of nH-SPGC and Gn-SPGC does not change any major peak shifting but the increase in peak intensity is observed. From the obtained FTIR results, the functional groups of SA and PVA reveals the hydroxyl and carboxyl group which contributes to the hydrophilicity and biocompatibility of the scaffold material. The gelatin and collagen, indicate the amide group that facilitates the interactions of cells and tissue regeneration. Moreover, the presence of hydroxyapatite in scaffold would provide a major bioactive substrate for mineralization and bound with connective tissue, promoting cell adhesion and proliferation.

Thermogravimetric Analysis (300)
The thermal behaviour of the fabricated scaffold is examined by the Thermogravimetric analysis and the TGA-DTA curve of SPGC, nH-SPGC, and Gn-SPGC are displayed in Figure 3. Initially, the analysis is observed in the range of 0 – 550 ℃. While heating the sample, different weight-loss stages are observed from each scaffold material. Here the plot is taken against the temperature and the weight loss of the scaffold. From the obtained result, when the mass decreases steadily to the increase of temperature, the first stage of the weight loss is occurred between 0 – 150 ℃, moreover the decrease in the mass after 100 ℃ may be due to the loss of water molecules in the scaffold material. The secondary weight loss that occurred between 150 – 350 ℃, is due to the partial dissociation of the major polymers into monomers and the abrupt decreases which represent the breakdown of the SA, and PVA side chains and its 65 % loss takes place in the range of 325 ℃ to 450 ℃. Further decomposition of the scaffold material occurs in the range of 350 - 550, which may be attributed to the broader degradation profile caused by crosslinking, possibly due to the presence of glutaraldehyde. As the temperature increases in the Gn-SPGC scaffold material, the decomposition rate decreases, indicating that the scaffold becomes more thermally stable and sustainable. The residual mass percentage of the scaffold is 10.33 % at 499.9 ℃; 9.21 % at 499.9 ℃; and 25.13 % at 549.7 ℃ for the respective samples. Hence, the incorporation of n-HAP in the fabricated scaffold increases the thermal ability and its bioavailability decreases the toxicity of glutaraldehyde which makes the scaffold material more biocompatible in tissue regenerative applications.

Porosity and Swelling Functions of the Scaffold (400)
The porosity study examined the presence of pores or void spaces in the structure of the fabricated scaffold. Figure 4a shows the bar graph of the porosity percentage of SPGC, nH-SPGC, and Gn-SPGC samples. The SPGC material shows a porosity value of 87.6 % , relatively the nH-SPGC, and Gn-SPGC material show a slight difference in the percentages of 89.7 % and 93.3 % . After the n-HAP is loaded and crosslinked, an increase in the porosity value is observed in both nH-SPGC and Gn-SPGC scaffolds. Considering the n-HAP’s higher porosity, it gradually increases the void spaces or pores in the SPGC [Bensalah et al., 2018]. Hence, the attained highest porous network material results in the integration of tissues, promoting cell migration and proliferation, controlled release of bioactive molecules, and promoting a greater way for several biomedical applications.

On the other hand, the swelling behaviour of the fabricated scaffold such as SPGC, nH-SPGC, and Gn-SPGC is analyzed by calculating the swelling ratio. In 2010, Hima Bindu reported that essential properties for tailoring the mechanical and biological performance in implants or tissue engineering include understanding and controlling the swelling behaviour of the fabricated scaffold. The swelling percentage of SPGC, nH-SPGC, and Gn-SPGC are 78.4 %, 73.4 %, and 69.4 %. From Figure 4b, it is obvious that the addition of hydroxyapatite nanoparticles decreased the swelling percentage of the scaffold material. Generally, the decrease in the swelling percentage might be influenced by several factors such as pH condition, temperature, the concentration of the polymer used, density of the crosslinking agent, etc. For the nH-SPGC scaffold, the swelling percentage is decreased from 78.4% to 73.4%. After crosslinking with glutaraldehyde, the Gn-SPGC is also slightly decreased to 69.4%. Furthermore, the decrease in the swelling percentage is because of crosslinking the apatite material to the hydroxy and carboxylic groups of fabricated scaffolds. Interestingly, the key parameter to characterize the scaffold's ability is to retain or absorb water, especially in the context of polymer-based scaffolds for regenerative medicine or tissue engineering. In this way, the fabricated scaffold shows a significant swelling property.

Contact Angle Measurement
A water droplet on contact angle measurements reveals the hydrophilicity or hydrophobicity nature of the SPGC, nH-SPGS, and Gn-SPGC scaffold materials. The contact angle measurements of each sample are displayed in Figure 4c. Generally, n-HAP and SPGC are hydrophilic materials, which completely rely on the increase in the polymeric solution and n-HAP material. This hydrophilicity property of the scaffold interrupts the interfacial polymerization progression, which leads to deviations in the arrangement of polymers in the resulting mixed layer surfaces. The contact angle measurements of SPGC, nH-SPGC, and Gn-SPGC are 60.22°, 57.27°, and 43.02°. These deviations in hydrophilicity, which reflected in the observed water contact angles on the surface of the scaffold materials. From the obtained result, it is known that the fabricated scaffold materials are hydrophilic.
Tensile strength
Table 1: Typical mechanical stability of the fabricated scaffolds.
SI.NO Specimen label Tensile stress at
Tensile strength
[MPa] Tensile strain
(Displacement) at
Break (Standard)
[%]
1. SPGC 17.52 103.15
2. nH-SPGC 27.43 105.17
3. Gn-SPGC 39.89 108.23

The strain-stress curve is employed to determine the tensile strength (MPa) of the scaffold material. The obtained tensile strain-stress is tabulated in Table 1. Usually, the mechanical properties of hydroxyapatite-based polymer scaffolds are completely constructed on the incorporation of polymers and hydroxyapatite structures. The addition of n-HAP increases the strength of tensile towards the fabricated material and it increases the affinity and flexibility of the fabricated scaffold, this might be due to the well-dispersed n-HAP samples in the polymeric solution, and evaluated by the presence of both hydroxy functional groups of n-HAP and carboxylic group of polysaccharides hydrogen and hydrogen bonds. The obtained mechanical results shows that the fabricated scaffold material holds a great attention in tissue regeneration applications.

Morphological Study (SEM) (500)
The scanning electron microscopic image of the scaffolds is displayed in Figure 5. From Figure 5(a), shows the morphology of the SPGC scaffold is spread uniformly with a smooth surface and with slight pores on the scaffold structures. Then from Figure 5(b), it is obvious that nH-SPGC material has a significant pores network on the scaffold surface due to the presence of hydroxyapatite nanoparticles in the scaffold. From Figure 5(c), the obtained pores in nH-SPGC are not disturbed and it is evenly distributed while adding the crosslinking agent of the Glutaraldehyde solution. The attained morphology is comparable with the previous report of Sadaf Batool et al., 2024 and it is in good agreement. And, it is notable that the incorporation of n-HAP does not form any agglomeration in the scaffold structure, indicates the nanoscale level of synthesized n-HAP. Hence, the blending of hydroxyapatite in sodium alginate, PVA, Gelatin, and collagen solution attained a soft surface with filamentous pores network on the scaffold. Furthermore, the achieved pores network is below 100 µm, which provides a larger surface area for providing a greater cell attachment and contributes to a superior cell proliferation and formation of new connective tissues.

In-vitro Cell line study on MCF10a and MCF-7cells (600)
It is essential to evaluate the biocompatibility and cytotoxicity of the scaffold materials for carrying out many biomedical applications. Initially, an in-vitro biocompatible study is performed on the MCF-10A cells for the fabricated Gn-SPGC scaffold by varying the concentration (50, 100, 150, 200, and 250 µg/ml) of the scaffold solution. The microscopic image of cell viability of the fabricated biocompatible scaffolds treated on MCF-10A cells are displayed in Figure 6. Obviously, with an increase in the concentration of the sample, the increased cell proliferation is experimentally observed from the microscopic images. The increase in the cell growth deliberately shows that the fabricated scaffold is non-toxic and biocompatible. The Bar graph for the percentage of cell viability is presented in the Figure 7d. Pointedly, the IC50 value of the fabricated Gn-SPGC scaffold is found to be 102.29 µg/ml. Interestingly, after the addition of n-HAP, and crosslinker, the biocompatibility of the sample exhibits a greater cell growth. The concentration of both cells and scaffold solution is in good agreement with the cell growth evaluation. So, it could be a strong evidence that the fabricated scaffold which shows great cell growth and moreover the scaffold’s porosity and swelling behaviour significantly supports the enhancement of cell viability.

On the other hand, examining the cell proliferation is equally important to evaluate the cytotoxicity behaviour of cancer cells on the scaffold (700). The scaffold’s cell viability (%) on MCF-7 cells is examined for 24 hours and the microscope image of Gn-SPGC scaffold is depicted in Figure 7. By observing the cell viability from the figure 7, it is obvious that increasing the concentration of the scaffold solution, the cell viability of the cancer cell is decreased. And, the cell viability is significantly decreased from 100 to 11.68 % concerning the concentration. The IC50 value of the Gn-SPGC scaffold material is 6.09 µg/ml. However, an increase in the sample concentrations showed a less cytotoxicity effect and it is observed in the results. The report of Sorour Jadbabaei et al., 2021, has recently cited that polymers such as PVA, SA, Gelatin, or a combination of polymers are more biocompatible. To date, no toxicity is reported for the polysaccharide material of pure SA which is highly biocompatible [Y. Zhu et al., 2019]. Then in the case of pure PVA polymer slight toxicity is observed from the previous study which could result in irradiating the tissues surrounded by the Pure PVA. But this irradiation could be overcome by assimilating with other biocompatible materials like Sodium alginate, Collagen, Gelatin, Chitosan, and so on [S. Khalaji et al., 2020]. In our case, along with SA, Collagen, and gelatin, we have integrated with hydroxyapatite biomaterial which has several properties in all different fields. Hence, from the experimental results of in-vitro study revealed that the fabricated scaffolds are biocompatible, non-toxic and could act as an anticancer agent. Thus, the fabricated scaffold would gain a lot of attention in the upcoming days for its significant properties in the biomedical applications.

, Claims:We Claim

1. A method for preparing a biocompatible nanofibrous scaffold for soft tissue regeneration, comprising the steps of:
a) dissolving sodium alginate in distilled water to form a homogeneous solution (1);
b) adding gelatin to the sodium alginate solution and stirring at 35–40 °C to obtain a blended SA-gelatin solution (2);
c) preparing a separate polyvinyl alcohol (PVA) solution (3) by dissolving PVA in water at 80 °C and sonicating to remove air bubbles;
d) mixing the SA-gelatin solution and PVA solution in equal volumes under continuous stirring to form a composite polymer solution; and
e) incorporating bovine tendon collagen into the composite solution to enhance extracellular matrix mimicry and biocompatibility.
wherein, dispersing hydroxyapatite nanoparticles (n-HAP) into the polymer-collagen solution before freeze-drying to enhance mechanical strength and physiochemical properties.
wherein the hydroxyapatite nanoparticles are uniformly mixed by stirring to ensure homogeneous distribution in the scaffold matrix;
wherein the polymer solution is freeze-dried at −20 °C followed by lyophilization (4) to form a porous nanofibrous scaffold (5);
wherein, chemical crosslinking of the n-HAP incorportaed polymerics solution (nH-SPGC) with diluted glutaraldehyde solution under the magnetic reflux for 30 minutes (6) whereby, the crosslinker improves the scaffold's mechanical and thermal stability.
2. The method as claimed in claim 1, wherein the resulting scaffold (7) exhibits:
a) tensile strength ranging from 17.52 MPa to 39.89 MPa;
b) porosity of approximately 93.32%;
c) swelling percentage of 69.4%;
d) thermal stability up to 550 °C; and
e) residual mass of 25.13% at high temperature.
3. A method as claimed in claim 1, wherein the scaffold exhibits in vitro biocompatibility with over 90% fibroblast cell viability on MCF-10A and cytotoxicity activity by reducing MCF-7 cells by controlling the growth and reduced the cell viability by approximately 89%.
4. A method as claimed in claim 1, wherein the scaffold is fabricated using an eco-friendly, cost-effective, and scalable process comprising only water-based solvents and non-toxic materials.