A HYDROGEL NANOCOMPOSITE BASED ON POLYACRYLAMIDE (PAM) AND PREPARATION METHOD THEREOF

Abstract

The invention discloses a hydrogel nanocomposite based on polyacrylamide (PAM) and preparation method thereof. The hydrogel nanocomposite comprises polyacrylamide (PAM) as a basic material, with 0.1-0.5 wt. % of silver (Ag) and 0.1-0.5 wt. % hydroxylated boron nitride (BNOH). According to the preparation method, the initial phase involves synthesizing Ag nanoparticles, functionalizing hexagonal boron nitride (hBN), and preparing dimer hydrogel composites. Building on the Ag nanoparticles and hydroxylated boron nitride (BNOH), a hydrogel trimer composite is then created by considering the hydrophilicity, degradation behavior, and compressive strength of the dimer composites, while incorporating the desiccated mass of neat PAM hydrogel into a mixture containing the selected optimal concentrations of 0.1-0.5 wt. % of silver (Ag) and 0.1-0.5 wt. % boron nitride (BNOH). As a result, the hydrogel composite that has been fabricated is a biomaterial that is highly promising for the regeneration of soft tissues, particularly articular cartilage. This research paves the way for developing self-healing, intelligent nanocomposites with a wide range of applications in cartilage tissue engineering. The present invention developed self-healing, intelligent nanocomposites with a wide range of applications in cartilage tissue engineering.

Specification

Description:Field of the Invention
The invention relates to a preparation method of a composite hydrogel, and more particularly to a hydrogel nanocomposite based on polyacrylamide (PAM) and the preparation method thereof. The present invention developed self-healing, intelligent nanocomposites with a wide range of applications in cartilage tissue engineering.
Background of the Invention
The invention addresses the challenge of creating a singular material that possesses self-healing, strong adhesion, and high strength, all highly desirable for applications such as cartilage tissue engineering. Current materials frequently demonstrate exceptional performance in one or two of these attributes, but they encounter difficulty in integrating all three. The invention creates a hydrogel nanocomposite based on polyacrylamide (PAM) that resembles cartilage in terms of its properties. This material is highly bonded, durable, and self-healing. The material's mechanical properties and long-term durability are further improved by incorporating noncovalent bonds through computational modelling, rendering it a promising candidate for soft tissue regeneration.
To generate polyacrylamide (PAM)/chitosan hydrogels, Shi-Neng Li et al.(2019) implemented a cross-linking methodology. Hyperbranched polysiloxane (HSi) was employed to cross-link the hydrogel, resulting in a composite hydrogel with substantially improved mechanical properties. This composite hydrogel possesses a tensile strength of 302 kPa, an elongation at break of 2263%, and a toughness of 3.85 MJ/m³. The hydrogel matrix and the incorporated additives exhibited robust interactions responsible for these enhancements.
Olaret et al. (2022) synthesized a polyacrylamide hydrogel that was grafted with silver (Ag)-decorated carbon nanotube (CNT) nanoparticles for cartilage applications. The compression stress was increased to 630 kPa across all composites, and hysteresis was reduced from 13.25 to 7.65 as a result of the incorporation of Ag-decorated CNTs. Furthermore, the composite hydrogels exhibited substantial antibacterial activity, with a 98% efficacy against Gram-positive bacteria (Staphylococcus aureus) and a 95% efficacy against Gram-negative bacteria (Escherichia coli). The inclusion of Ag-decorated CNTs in the PAM matrix not only improved mechanical strength but also conferred antibacterial properties, rendering these composites viable contenders for bone tissue regeneration applications.
Isabel González-Sánchez and colleagues(2015) reported the development of methacrylate hydrogels incorporated with silver nanoparticles for tissue engineering, despite the relatively limited literature on silver-added PAM hydrogel composites. Silver nanoparticles were integrated into hydrogels through cross-linking, which led to their antimicrobial properties.
Xue et al. (2018) found that the self-healing and tensile properties of hydrogel composites were improved by the incorporation of functionalized boron nitride nanosheets (BNNS). BNNS was reinforced into poly(acrylic acid) hydrogels (PAA/BNNS-NH2) and modified with amine groups (BNNS-NH2). The concentration of 0.5 mg/L exhibited the most significant mechanical improvements among the various concentrations tested (0.1, 0.5, 0.8, and 1.0 mg/L). This concentration exhibited a fracture stress of 1311 kPa and a toughness of 4.7 MJ/m³, as well as excellent self-healing behavior, which was attributed to hydrogen bonding and metal coordination interactions
Duan et al. (2016) also observed that hexagonal boron nitride (hBN) outperformed graphene oxide in a polyacrylamide matrix. The strong interfacial interactions between hBN and the PAM matrix were responsible for the maximal compressive strength of 600 kPa, which was attained with 0.12 wt% hBN content in PAM.
In Chinese patent no. CN104189960A, describes a method for preparing a hydroxyapatite/chitosan/polyacrylamide cartilage substitute material having excellent mechanical properties. The preparation technology retains the advantages of both hydroxyapatite (HA) and chitosan (CS), and enhances the mechanical properties of the single material of the polyacrylamide polymer, broadens the application range of the composite material, and is expected to be in tissue engineering.
Research on the reinforcement of silver (Ag) and hexagonal boron nitride (hBN) in polyacrylamide (PAM) matrices is scarce in the current literature, despite the exceptional properties of these materials. The objective of the present invention is to address this lacuna by investigating the theoretical and experimental synergies that exist between reinforcing Ag and hydroxylated BN (BNOH) within a PAM matrix, a subject that has not been thoroughly examined. In this context, we have created a hydrogel composite (PAM-Ag-BN) that is self-healing, durable, and robust by utilizing the optimal concentrations of Ag and BNOH.
The long-term durability of these hydrogel samples was evaluated for the first time using electrochemical testing in simulated body fluid. We investigated the interfacial interactions between a single unit of the prepared hydrogel and its two essential components (Ag and BNOH) to further validate the experimental results and gain a more comprehensive understanding of the composite's stability.
None of the prior art indicated above either alone or in combination with one another discloses what the present invention has disclosed. The present invention discloses a hydrogel nanocomposite based on polyacrylamide (PAM) and the preparation method thereof.
Object of the Invention
The primary object of the present invention is to develop a hydrogel composite using polyacrylamide (PAM) as a basic material, with different amounts of silver (Ag) and boron nitride (BNOH).
Another object of the present invention is to create a singular material that possesses self-healing, strong adhesion, and high strength, all highly desirable for applications such as cartilage tissue engineering.
Summary of Invention
This summary is not a comprehensive overview of the disclosure and does not reflect the main/essential features of the establishment or specify the scope of the establishment. Its sole purpose is to present some of the concepts presented here in a simpler way as a precursor to more detailed.
Herein enclosed a method for synthesis a trimer hydrogel based on polyacrylamide (PAM) as a basic material, with optimal amounts of silver (Ag) and boron nitride (BNOH). The synthesis method comprising the steps of: the initial phase involves synthesizing Ag nanoparticles, functionalizing hexagonal boron nitride (hBN), and preparing dimer hydrogel composites. Building on the Ag nanoparticles and hydroxylated boron nitride (BNOH), a hydrogel trimer composite is then created by considering the hydrophilicity, degradation behavior, and compressive strength of the dimer composites, while incorporating the desiccated mass of neat PAM hydrogel into a mixture containing the selected optimal concentrations of 0.1-0.5 wt. % of silver (Ag) and 0.1-0.5 wt. % boron nitride (BNOH). Subsequently, the sample was rinsed and re-immersed in deionized water to eliminate any excess or unbonded ions. The surface morphology (using SEM), phase analysis (using XRD), and compressive testing of the resulting trimer hydrogel composite (PAM-Ag1-BN3) were subsequently performed as additional investigation.
The PAM-Ag-BNOH nanocomposite surpasses the performance of conventional hydrogel composites, demonstrating extraordinary mechanical strength, including a compressive strength of 0.31 MPa and a Young's modulus of 0.29 MPa. The material is particularly well-suited for load-bearing applications in tissue engineering due to its improved mechanical properties. The PAM-Ag-BNOH nanocomposite is synthesized using generally available materials, such as hydroxylated boron nitride and silver nanoparticles, to guarantee a cost-effective production process. This synthesis method's potential for commercial implementation is further bolstered by its scalable nature.
Brief Summary of the Figures
The illustrated embodiments of the subject matter will be understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description is intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and methods that are consistent with the subject matter as claimed herein, wherein:
Fig.1: The functionalized hBN particles exhibit a distinct layered structure and enhanced stacking dispersion performance. In comparison to PAM-BN1 (Figure 1A, d) and PAM-BN3 (Figure 1A, e), the concentration of BNOH in PAM-BN5 is substantially higher (0.5 wt. % of BNOH, Figure 1A, f). The presence of Ag nanoparticles and BNOH in a PAM matrix can be verified through the use of elemental mapping and energy-dispersive X-ray spectroscopy (EDS) or XRD spectrum. The elemental mapping (Figure 1B, b-e) and EDS plot (Figure 1B, f) can be used to observe the uniform distribution of Ag, as well as other existing elements such as C, O, and N, in PAM-Ag1. This is owing to the PAM hydrogel. The presence of B, N, C, and O elements (Figure 1C, b-e) and EDS (Figure 1C, f) in the PAM matrix was further confirmed by the PAM-BN3 sample.
Fig.2: The retention of phases in the synthesis of PAM-based hydrogel composites was verified using XRD (Figure 2). The presence of amorphous polymeric material, polyacrylamide44, was responsible for the broad humps in the samples. Additionally, the crystallographic planes of PAM-Ag1, PAM-Ag3, and PAM-Ag5 dimer hydrogels exhibit featured peaks of Ag at approximately 27.7º, 32.1º, 34.2º, 44.5º, 67.7º, and 77.4º, respectively. The intensity of metallic or ceramic peaks was diminished by amorphous bulk polyacrylamide 48. The phases of BNOH and PAM-BN-based hydrogels were further verified, and it was shown that the specific peak (002 crystalline phase) of BN is present at 26.7º.
Fig.3: FT-IR was employed to identify the functional group and bonding present in the dimer hydrogels after the surface morphology and phases of all Ag and BNOH-based PAM hydrogels were determined (Figure 3). The spectrum of PAM-Ag1, PAM-Ag3, and PAM-Ag5 is depicted in Figure 3. It is evident that the O-H bond is responsible for the strong vibration bond at approximately 3353 cm-1, while the amide group in PAM hydrogel induces the characteristic N-H stretching vibration at approximately 2936 cm-1. The absorption at approximately 1662 cm-1 illustrates the C=O stretching in the -CONH2 group, while the shoulder peak at approximately 1560 cm-1 is a result of NH2 bending. The stability of PAM under reactive conditions was also confirmed by these peaks. The bonding between Ag and oxygen is observed as Ag-O at 840 cm-1. Due to the minimal overlap between metal and non-metal, this bonding is regarded as relatively feeble. The least overlapping is caused by the presence of a larger d orbital in Ag. The complexation between the reinforcers and PAM matrix is exemplified by the shoulder peaks in all the composites at approximately 3184 cm-1. PAM-BN composites exhibit characteristic bands in addition to the PAM-Ag dimer composites (Figure 3). The peaks of PAM are comparable to those of PAM-Ag hydrogels. Peaks at approximately 3402 cm-1 are attributed to B-OH stretching, 1371 cm-1 to B-O in-plane bending, and 786 cm-1 to B-O···H out-of-plane bending in the BNOH powder. The O-H stretching peak in PAM-BN composites is characterized by an intense peak at approximately 3390 cm-1. The OH group's greater polarity than the NH group results in a more intense O-H peak. In composites the B-O in-plane peak and B-O···H out-of-plane bending are shifted from approximately 1371 cm-1 (BNOH) to approximately 1375-1406 cm-1 (PAM-BN1, PAM-BN3, and PAM-BN5), and from approximately 786 cm-1 (PAM) to approximately 795 cm-1. The hydrogel bonding between BNOH and PAM hydrogel is the cause of the shifting. Consequently, the number of OH bonding is greater in PAM-BN-based hydrogel composites than in PAM-Ag-based hydrogels. The computational section facilitates a comprehensive investigation of interfacial (bonding and nonbonding) interactions through computational experiments.
Figure 4: (A) AFM topographies of the engineered hydrogel surfaces, (B) contact angle values and (C) connections between surface roughness and water contact angles measured through all designed Ag and BN-based hydrogel samples.
Figure 5: (a) Swelling ratio, (b) enlarged version (dotted circle of plot a) of the swelling ratio data during initial hours of immersion of the hydrogels and (c) degradation behaviour of PAM-Ag1, PAM-Ag3, PAM-Ag5, BNOH powder, PAM-BN1, PAM-BN3 and PAM-BN5 dimer hydrogels.
Figure 6 : Potentiodynamic polarization curve of PAM, PAM-Ag1, PAM-BN3 and PAM-Ag-BN hydrogels.
Figure 7: Microscopic images (40X) of Influence of chondrocytes C28/I2 cell viability on untreated (A), PAM-Ag1 (B), PAM-BN3 (C), PAM-Ag-BN (D) incubated for 3 days.
Figure 8: % Inhibition capacity of PAM-Ag1 (B), PAM-BN3 (C), PAM-Ag-BN hydrogel composites for E. coli bacteria.
Figure 9 : Optimized/Equilibrium Structures (top), 3D-Isosurafces (left), and 2D-Scatter Plots (right) of the PAM-Ag-BNOH Trimer Complex (Composite Model) at the B3LYP/6-31G Level of Theory.
Figure 10: Schematic representation of cartilage regeneration by PAM-Ag-BN hydrogel using various experimental and theoretical studies.
Detailed Description of the Invention
The detailed description of various exemplary embodiments of the disclosure is described herein with reference to the accompanying drawings. It should be noted that the embodiments are described herein in such details as to clearly communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure as defined by the appended claims.
It is also to be understood that various arrangements may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects, and embodiments of the present disclosure, as well as specific examples, are intended to encompass equivalents thereof.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments. As used herein, the singular forms "a"," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "comprising," "includes" and/or "including," when used herein, specify the presence of stated features, integers, steps, operations, elements and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups thereof.
It should also be noted that in some alternative implementations, the functions/acts noted may occur out of the order noted in the figures. For example, two figures shown in succession may, in fact, be executed concurrently or may sometimes be executed in the reverse order, depending upon the functionality/acts involved.
In addition, the descriptions of "first", "second", "third", and the like in the present invention are used for the purpose of description only, and are not to be construed as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may include at least one of the features, either explicitly or implicitly.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong.
It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The present invention involved the preparation of hydrogel composites using polyacrylamide (PAM) as a basic material, with different amounts of silver (Ag) and boron nitride (BNOH). This present invnetion examines the stability of Ag and BNOH-based novel PAM hydrogel samples in terms of their electrochemical performance in SBF for the first time. Furthermore, this invention establishes a connection between the empirical and theoretical investigations of hydrogels used in the process of cartilage regeneration. An attempt has been made to comprehend the durability of the manufactured hydrogel composites, which are connected by the interfacial bonding between nanoparticles and the matrix. This has been achieved by employing electronic structure calculations, molecular modelling, and density functional theory techniques. Consequently, it is deemed suitable for use as an ideal implant in cartilage repair applications.
The present invention developed a hydrogel composite for the cartilage regeneration. The hydrogel is a trimer composite comprises of: polyacrylamide (PAM) as a basic material, with 0.1-0.5 wt. % of silver (Ag) and 0.1-0.5 wt. % boron nitride (BNOH).
The invention provides a method for preparing a composite hydrogel by comprising the following steps:
Step 1: Synthesis of polyamide, Ag nanoparticles, and functionalization of hBN
a) A free radical polymerisation reaction was employed to synthesize polyacrylamide hydrogel (PAM). Ammonium persulfate (0.20 g) and tetramethylethylenediamine (610.14µL) employed as catalysts, while acrylamide (10 g) employed as the monomer. During the cross-linking reaction, N,N'-methylene bisacrylamide (0.266 g) was incorporated as a crosslinker.
b) A conventional chemical reduction process was employed to synthesize silver (Ag) nanoparticles from AgNO3. In particular, a cylinder within an ice chamber was used to contain 30 mL of a 0.002 M sodium borohydride (NaBH4) solution, which was stirred continuously. The frigid bath was employed to reduce the rate of sodium borohydride decomposition during the reaction. Afterward, 2 mL of a 0.001 M AgNO3 solution was dropwise added to the agitated mixture, followed by the addition of a few droplets of NaCl. This process resulted in a change in the colour of the suspension from yellowish to grey. The Ag nanoparticles were stabilised using poly(vinylpyrrolidone). Ag nanoparticles of approximately 30-50 nm in size were obtained after the precipitate was filtered and dried.
c) The hydrophilicity of hexagonal boron nitride (hBN) was improved by functionalising it with hydroxyl groups (-OH), resulting in hydroxylated boron nitride (BNOH). In order to accomplish this, 3 g of hBN was immersed in a mixture of 12 mL of concentrated H2SO4, 2.5 g of K2S2O8, and 2.5 g of P2O5 at 80ºC, with continuous agitation for 5 hours. Following this, the solution was chilled to room temperature, diluted with 500 mL of deionised water, filtered, washed, and subsequently added to 120 mL of concentrated H2SO4 in a cold bath. In order to expedite the oxidation reaction, 15 g of KMnO4 was introduced with vigorous agitation, and the mixture was maintained at 35ºC for 2 hours while being stirred. Subsequently, 250 mL of deionised water was gradually introduced while agitating continued for an additional 2 hours. The reaction was terminated by the addition of 700 mL of water and 20 mL of 30% hydrogen peroxide (H2O2). The white mixture that resulted was filtered, washed, and desiccated at 60ºC, resulting in the production of hydroxylated boron nitride with enhanced hydrophilicity. This property renders it appropriate for biomedical applications.
Step 2: Preparation of Dimer
? The aqueous solutions of variable concentrations of Ag (0.1, 0.3, and 0.5 wt%) and BNOH (0.1, 0.3, and 0.5 wt%) were prepared separately to accomplish the reinforcement of silver (Ag) and hydroxylated boron nitride (BNOH), which is approximately 1 µm in size.
? The polyacrylamide (PAM) hydrogel was immersed in these solutions separately after being desiccated. Subsequently, the dimer composites were submerged in deionised water for 24 hours to eliminate surplus bound ions from the surface of the PAM hydrogel composites.
? Consequently, six dimer composite hydrogels were synthesised, which were referred to as PAM-Ag1 (0.1 wt% Ag), PAM-Ag3 (0.3 wt% Ag), PAM-Ag5 (0.5 wt% Ag), PAM-BN1 (0.1 wt% BNOH), PAM-BN3 (0.3 wt% BNOH), and PAM-BN5 (0.5 wt% BNOH). Subsequently, the optimal concentrations of Ag and BNOH were employed to generate a trimer composite hydrogel for subsequent investigations.
Step 3: Investigation of Dimer composite hydrogels sample prepared in Step 2
• The hydrophilicity, degradation behavior, and compressive strength of the six dimer composite hydrogels referred to as PAM-Ag1 (0.1 wt% Ag), PAM-Ag3 (0.3 wt% Ag), PAM-Ag5 (0.5 wt% Ag), PAM-BN1 (0.1 wt% BNOH), PAM-BN3 (0.3 wt% BNOH), and PAM-BN5 (0.5 wt% BNOH) investigated.
Step 4: Preparation of trimer
• the desiccated mass of neat PAM hydrogel was submerged in the mixture that contained the selected optimal concentrations of Ag and BNOH. The optimal concentrations of Ag and BNOH were obtained from the ranges of 0.1 wt.%, 0.3 wt.%, and 0.5 wt.% after consideration of Step 3 results
• Subsequently, the sample was rinsed and re-immersed in deionized water to eliminate any excess or unbonded ions.
In the preferred embodiment, the surface morphology (using SEM), phase analysis (using XRD), and compressive testing of the resulting trimer hydrogel composite (PAM-Ag1-BN3) were subsequently investigated.
Characterization of Synthesized Dimer and Trimer:
1. The surface morphology was observed using the cross sections of the dimer hydrogels (PAM-Ag1, PAM-Ag3, PAM-Ag5, and PAM-BN1, PAM-BN3, PAM-BN5). The dispersion of Ag nanoparticles in the PAM matrix is evident in the Ag-based composite hydrogels. Simultaneously, PAM-Ag5 exhibits the maximum levels of Ag (0.5 wt. % of Ag), which suggests that Ag is agglomerated in PAM. BNOH particles are composed of large aggregates of smaller, brittle particles with irregularly formed margins, each measuring approximately 120 nm in thickness. The functionalized hBN particles exhibit a distinct layered structure and enhanced stacking dispersion performance. In comparison to PAM-BN1 (Figure 1A, d) and PAM-BN3 (Figure 1A, e), the concentration of BNOH in PAM-BN5 is substantially higher (0.5 wt. % of BNOH, Figure 1A, f). The presence of Ag nanoparticles and BNOH in a PAM matrix can be verified through the use of elemental mapping and energy-dispersive X-ray spectroscopy (EDS) or XRD spectrum. The elemental mapping (Figure 1B, b-e) and EDS plot (Figure 1B, f) can be used to observe the uniform distribution of Ag, as well as other existing elements such as C, O, and N, in PAM-Ag1. This is owing to the PAM hydrogel. The presence of B, N, C, and O elements (Figure 1C, b-e) and EDS (Figure 1C, f) in the PAM matrix was further confirmed by the PAM-BN3 sample. Other researchers have observed a comparable pattern of Ag nanoparticles and BNOH particles in a hydrogel matrix.
2. The retention of phases in the synthesis of PAM-based hydrogel composites was verified using XRD (Figure 2). The presence of amorphous polymeric material, polyacrylamide44, was responsible for the broad humps in the samples. Additionally, the crystallographic planes of PAM-Ag1, PAM-Ag3, and PAM-Ag5 dimer hydrogels exhibit featured peaks of Ag at approximately 27.7º, 32.1º, 34.2º, 44.5º, 67.7º, and 77.4º, respectively. The intensity of metallic or ceramic peaks was diminished by amorphous bulk polyacrylamide 48. The phases of BNOH and PAM-BN-based hydrogels were further verified, and it was shown that the specific peak (002 crystalline phase) of BN is present at 26.7º.
3. FT-IR was employed to identify the functional group and bonding present in the dimer hydrogels after the surface morphology and phases of all Ag and BNOH-based PAM hydrogels were determined (Figure 3). The spectrum of PAM-Ag1, PAM-Ag3, and PAM-Ag5 is depicted in Figure 3. It is evident that the O-H bond is responsible for the strong vibration bond at approximately 3353 cm-1, while the amide group in PAM hydrogel induces the characteristic N-H stretching vibration at approximately 2936 cm-1. The absorption at approximately 1662 cm-1 illustrates the C=O stretching in the -CONH2 group, while the shoulder peak at approximately 1560 cm-1 is a result of NH2 bending. The stability of PAM under reactive conditions was also confirmed by these peaks. The bonding between Ag and oxygen is observed as Ag-O at 840 cm-1. Due to the minimal overlap between metal and non-metal, this bonding is regarded as relatively feeble. The least overlapping is caused by the presence of a larger d orbital in Ag. The complexation between the reinforcers and PAM matrix is exemplified by the shoulder peaks in all the composites at approximately 3184 cm-1. PAM-BN composites exhibit characteristic bands in addition to the PAM-Ag dimer composites (Figure 3). The peaks of PAM are comparable to those of PAM-Ag hydrogels. Peaks at approximately 3402 cm-1 are attributed to B-OH stretching, 1371 cm-1 to B-O in-plane bending, and 786 cm-1 to B-O···H out-of-plane bending in the BNOH powder. The O-H stretching peak in PAM-BN composites is characterized by an intense peak at approximately 3390 cm-1. The OH group's greater polarity than the NH group results in a more intense O-H peak. In composites the B-O in-plane peak and B-O···H out-of-plane bending are shifted from approximately 1371 cm-1 (BNOH) to approximately 1375-1406 cm-1 (PAM-BN1, PAM-BN3, and PAM-BN5), and from approximately 786 cm-1 (PAM) to approximately 795 cm-1. The hydrogel bonding between BNOH and PAM hydrogel is the cause of the shifting. Consequently, the number of OH bonding is greater in PAM-BN-based hydrogel composites than in PAM-Ag-based hydrogels. The computational section facilitates a comprehensive investigation of interfacial (bonding and nonbonding) interactions through computational experiments.
4. The hydrophilicity/hydrophobicity of any material is denoted by the contact angle (CA) values. The hydrophilicity or hydrophobicity of the materials is contingent upon their surface irregularity. Additional evidence of surface irregularity can be obtained by distinguishing topographical bright and edged areas from the sublayer using AFM (Figure 4A). The hydrophilic behaviour of PAM-Ag1 (29º) was the most pronounced in Ag-based PAM composites, as opposed to PAM-Ag3 (45º) and PAM-Ag5 (62º). The lower content of Ag in PAM renders PAM-Ag1 more hydrophilic than other Ag-based hydrogels, as the Ag nanoparticles are hydrophobic and PAM is hydrophilic. Additionally, the hBN is classified as hydrophobic; however, the hydrophobic nature of the hBN is converted to hydrophilic through the functionalization of the OH group 62. Consequently, the highest hydrophilicity was achieved by PAM-BN5 with a BN content of 0.5 wt. %, which also had the lowest CA (22º) among the other PAM-BN compositions (Figure 4B). The CA of PAM-BN5 is lower than that of PAM-Ag1 as a result of the hydrophilic nature of BNOH and PAM, while the hydrophobic nature of Ag exhibits a comparatively higher CA.
5. The hydrogels' swelling behaviour is assessed by immersing the samples in PBS and weighing them at various time intervals. The swelling rate of the materials can be influenced by the compact network of the hydrogel, surface area, and particle size, among other factors. The hydrogel samples exhibited rapid swelling during the initial hours of immersion. The dotted circle in Figure 5a illustrates this rapid enlargement, while Figure 5b illustrates the enlarged view of the dotted portion. The edema ratio for all samples was nearly doubled, from 0.5 hours to 1.0 hours to 1.5 hours (Figure 5b). PAM-Ag and PAM-BN-based composites are classified as stable biomaterials due to their non-swelling behaviour (after equilibrium), which is a significant characteristic for cartilage repair applications. The increased content of Ag and BNOH in PAM-Ag5 and PAM-BN5, respectively, resulted in a reduced extent of swelling (Figure 5a). This was attributed to the increased number of covalent crosslinks between PAM and Ag, as well as with BNOH, which created a compact structure of PAM-Ag5 and PAM-BN5 and restricted the swelling in PBS. Nevertheless, the swelling ratio of PAM-BN5 was the lowest, as a result of the greater number of bonds formed between BNOH and PAM than between PAM and Ag (as illustrated in the FTIR).
6. In vitro, degradation of the hydrogel samples was assessed by calculating the percentage of remaining gel after 0, 5, 10, 15, and 20 days in PBS. Figure 5c indicates that the hydrogels experience a substantial weight loss during the initial week of immersion, while the gels maintain a constant weight in PBS after 10 days. After 20 days of immersion in PBS, the dimer hydrogels that were prepared demonstrated an optimal percentage of remaining gel (approximately 75%) for cartilage applications. The rigid structure of the samples resulted in reduced damage in materials with the highest percentage of remaining gels, approximately 90%, as a result of the higher content of Ag and BN (PAM-Ag5 and PAM-BN5) (Figure 5c). Enhanced hydrolysis was induced by the reduced percentage of remaining polymer in PAM-Ag1, which was a result of the highest absorption of water during the swelling process.
According to the investigation, the robust mechanical strength is an essential characteristic of any bone implant. The compressive strength of PAM-Ag1 was found to be higher (~0.21 MPa) in Ag-based PAM hydrogel composites when compared to the other hydrogel samples through a compression test. The concentration of Ag in PAM-Ag1 was the lowest, and the distribution of Ag nanoparticles within the PAM matrix was sufficient. This results in a substantial interfacial bonding between Ag and PAM, facilitated by an Ag-O bond. As the concentration of Ag increased from 0.1 wt.% to 0.5 wt.%, the compressive strength began to decrease (0.19 MPa for 0.3 wt. % and 0.15 MPa for 0.5 wt. %). Agglomeration was the consequence of the increased concentration of Ag nanoparticles in PAM, which in turn led to a decrease in strength. Additionally, the mechanical strength of PAM was enhanced in comparison to PAM-Ag1 by the incorporation of BNOH. The micron-sized BNOH particles demonstrated the highest strength of 0.25 MPa with a 0.3 wt. % of BNOH in PAM (PAM-BN3), which was attributed to the effective dispersion of BNOH in PAM. The PAM-BN1 with a lower concentration (0.1 wt. %) is unable to make a significant contribution to the strength, whereas the excess concentration of BNOH (0.5 wt. %) results in the extraction of BNOH micron particles from the PAM matrix during compression. The intense interfacial interaction of BN with PAM was the reason for the improved mechanical performance of BNOH over Ag. BN was functionalized, and BNOH formed numerous hydrogen bonds with the PAM matrix. The compressive strength was increased as a consequence of the robust B-O···H bonding between BNOH and PAM (a comprehensive discussion is provided in the computational analysis section).
In the preferred embodiment, PAM-Ag1, PAM-BN3 sample selected to compare the performance of PAM-Ag-BN trimer.
The performance of the PAM-Ag-BN trimer were characterised by:
A. Electrochemical analysis of the hydrogel samples
Implants that are predominantly composed of polymers may begin to dissolve in bodily fluids after a specific amount of time. Additionally, to cells and tissues, human body fluid contains a variety of inorganic constituents, such as phosphates, sugar, and proteins, which have the potential to cause corrosion or degradation of any polymeric or metal alloy implants. Consequently, it is imperative to assess the impact of these components on implant material through electrochemical experiments in synthetic body fluid (SBF), which has an ionic composition that is comparable to that of human plasma. The titanium alloy coated with four hydrogels (PAM, PAM-Ag1, PAM-BN3, and PAM-Ag-BN) was stabilized with constant potential values using an open circuit potential. Subsequently, potentiodynamic polarisation studies were conducted, and the surface morphology of the samples was observed after corrosion.
The samples underwent potentiodynamic polarisation, and Figure 6 illustrates the Tafel polarisation curve. The electrochemical parameters, including the anodic and cathodic Tafel, slopes ßa and ßc, corrosion potential (Ecorr), and corrosion current density (icorr), were obtained by extrapolating the Tafel polarisation curve.
PAM-Ag-BN exhibited the greatest polarisation resistance (55067 O cm2) among the hydrogel composites. PAM-BN3 exhibited superior corrosion resistance in comparison to PAM-Ag1 among dimer composites. The strong interlocking of BN particles with the PAM matrix through hydrogen bonding was the reason for the enhanced corrosion resistance of BN over Ag. The maximum corrosive resistance behaviour of the trimer composite was established by the synergistic effect of Ag nanoparticles and BN micro-particles, which was achieved through the combined bonding of Ag and BNOH with PAM.
B. Biomineralization and Self-healing of the hydrogel composites in vitro
The bioactivity of the implant material is confirmed by the ability of cartilage implants to form calcium-phosphate crystals or apatite. Apatite, which also facilitates bone integration and the development of osteogenic tissue, provides support for the cartilage tissue. The advanced biocompatibility of the trimer composite hydrogel, which contains both bioactive Ag and BNOH particles, resulted in a higher formation of apatite crystals. The hydrogel's self-healing was verified by severing the trimer sample. The Gibbs-Donnan effect explains that the swelling and expansion of the polymer network occur through osmosis, and the fractured sample is hydrated by the addition of water droplets. The hydrogel's compact structure is achieved by the capillary action, which causes the pores to contract. The hydrogel sample's fractured surface exhibited substantial curing when the healed sample was stretched by hand. The healed sample underwent a compression test, and the measured compressive strength was 0.11 MPa. Therefore, the hydrogel sample with amended self-healing properties has the potential to serve as a cartilage implant that can expedite the natural curative process in the adjacent tissue.
C. Cytotoxicity assessment on chondrocyte cells
Figure 7 illustrates the impact of PAM-Ag1, PAM-BN3, and PAM-Ag-BN on the cytotoxicity of C28/I2 chondrocytes. The cell viability of PAM-Ag1 was 95.8%, 95.5%, and 94.5% for 3, 5, and 7 days, respectively, while PAM-BN3 was 97.4%, 97.1%, and 96.4%. The functionalization of hexagonal boron nitride with the OH group was the reason for the comparatively increased call viability in PAM-BN3. The cell viability of PAM-Ag-BN was 98.5 %, 98 %, and 97.3 % for 3, 5, and 7 days, respectively. For the PAM-Ag-BN trimer composite, the synergistic effect of Ag and functionalized BN led to a higher percentage of cell viability. The utilization of biocompatible components-conjugated nanoparticles is becoming an appealing approach for the prevention of a variety of pathogens and the enhancement of cell viability. The biocompatibility and non-toxicity of all the samples are demonstrated by the minor difference in cell viability and inhibition capacity between PAM-Ag1, PAM-BN3, and PAM-Ag-BN. The synergistic effect of Ag and functionalized BN was responsible for the minor increase in the cell viability of PAM-Ag-BN. This was because Ag nanoparticles are well-known biomaterials, and hydroxylated BN can increase the water solubility of BN, further enhancing its bioactivity. As a method for enhancing biocompatibility, numerous studies have successfully implemented a combination of two or more therapy components. This approach aims to either enhance the antibacterial activity and cell viability or generate a synergistic effect.
D. Antimicrobial analysis of hydrogels
The quantitative antimicrobial activity of PAM-Ag1, PAM-BN3, and PAM-Ag-BN against E. coli bacteria was assessed using the MTT assay. The MTT assay results indicated that bacterial growth was inhibited by over 80% in all of the samples (Figure 8). The PAM-Ag-BN exhibited the highest bacterial inhibition of 85% against E. coli, while PAM-BN3 and PAM-Ag1 exhibited bacterial inhibition of 83.3 % and 81.8%, respectively. These findings indicate that PAM-Ag1, PAM-BN3, and PAM-Ag-BN exhibit potent antibacterial properties and may be employed in cartilage repair tissue engineering.
E. Computational analysis
In achieving profound insights into structural, bonding (metal-metal, metal-nonmetal) and nonbonding interaction(s) [like H-bonds (HBs) and vdW], and electronic features of all five moieties (two components and three composite models), three techniques [two based on the NCI-plot (3D-isosurface map and 2D-scatter plot) and one based on the QTAIM tool] were used and a variety of interesting intramolecular H-bonding and vdW interactions have been observed which self-stabilize the parent entities (particularly, the PAM and BNOH). Regarding the NCI plots, here it is important to mention that the dark blue colour as displayed in both the 3D-isosurface map and 2D-scatter plot shows interactions (either moderate metal-metal or metal-nonmetal or very strong H-bonding interaction) whereas the light blue (or bluish-green) colour indicates the moderate (or weak) H-bonding interactions. Moreover, the green colour gives the impression of the extremely weak H-bonding and vdW type of interaction. The occurrence of steric effect is exposed by the low-gradient spikes appearing at the positive side (0 to 0.03 au). Such effect as expressed by the red ellipsoid describes the electron density depletion which is due to the electrostatic repulsion.
In the preferred embodiment, The molecular modelling approach was followed by the assembly of a trimer composite model (PAM-Ag-BNOH) using a 3D three-body with two-sided interfacial interactions (one between the PAM and Ag components and the other between the PAM and BNOH components) in this report, which is highly intriguing and crucial in addressing the experimental facets. The QTAIM tool facilitated a total of four types of interfacial interactions in the PAM-Ag-BNOH composition, to gain new insights and comprehend the binding features (i.e., interfacial interactions). The first type comprises one interface side of the PAM-Ag dimer constituent, while the second, third, and fourth types each comprise another interface side of the PAM-BNOH dimer segment. The PAM unit and Ag assembly of the PAM-Ag fragment are moderately bound together by the first type of interfacial interaction, which involves two MNIs (Ag-O and Ag?H) between the two components. The distance and QTAIM-based BPL between interacting Ag and O atoms of the Ag-O are detected to be 2.398 Å and 2.401 Å, respectively. Conversely, the Ag-H MNI's respective distance and BPL are 3.168 Å and 3.240 Å, which moderately embrace the PAM and Ag assembly and stabilize the one-side interface.
The second type of interfacial interaction is the B-O (covalent bond) interaction (CBL: 1.558 Å, BPL: 1.562 Å) between the O atom of the PAM and the B atom of the BNOH. This interaction very tightly stabilizes both the PAM and BNOH and strongly reinforces the other side-occurring interfacial interactions. The B-O bond distance is indicative of the fact that it was the most robust binding interaction among all bonding and nonbonding interactions in the PAM-Ag-BNOH trimer composite model. An analogous instance of the first form of interfacial interaction (B-O covalent bonding interaction) was also detected in the PAM-Ag dimer composite, as previously mentioned in the previous paragraph. Currently, the third type of interfacial interactions (i.e., NCIs) that are involved in the PAM-Ag-BNOH model are also the second type of interfacial interactions that were present in the PAM-BNOH dimer composite. A total of seven NCIs (weak to moderate HBs) were identified between the binding sites of the PAM and BNOH components of the PAM-Ag-BNOH composite, including two O-H?O, one N-H?O, two C-H?O, and two C-H?N, similar to the PAM-BNOH species. The structural parameters (HBD, BPL, bond angle) of both O-H?O bonds in the trimer complex have been analyzed as (1.722 Å, 1.750 Å, 174.3°) and (1.912 Å, 1.978 Å, 136.4°). The former O-H?O HBD is increased by 0.012 Å, while the latter O-H?O HBD is decreased by 0.012 Å when compared to the PAM-BNOH dimer complex. The QTAIM-based topological parameters and the structure/geometry-based H-bond strength quantifying criteria (Figure 9) both confirm that the strength of the former O-H?O interaction is reduced, whereas the latter one is enhanced. These structural changes are a clear demonstration of this. Additionally, the trimer composite contained only one NBP (one O?O), whereas the PAM-BNOH dimer complex contained two NBPs (one O?O and one N?N). This discrepancy may be attributed to the modification of component orientation to achieve a minimum energy (optimized/equilibrium) structure. It is crucial to note that the NCI plot and the QTAIM tool were instrumental in the promotion of these findings, as the structural parameters of the trimer composite model exhibit the highest degree of stability among all three.
Consequently, the aforementioned discussion demonstrates that the incorporation of the optimal concentration of Ag and BNOH into the PAM matrix can provide the following benefits: enhanced mechanical strength, long-term durability of the material in SBF (or corrosion resistance), restricted toxicity, good anti-microbial activity, and strong interfacial interactions between reinforced particles and PAM. The schematic representation of current research, Figure 10, illustrates that the prepared trimer hydrogel composite PAM-Ag-BN was subjected to a compression test to assess the long-term stability of the hydrogel in a simulated body fluid through electrochemical investigations. Additionally, the material's bioactivity was verified through biomineralization, self-healing, cytocompatibility, and antibacterial activity. The wet lab experiment was substantiated by the computational experiments, which demonstrated that the Ag assembly and functionalized BN (BNOH) acted as reactive sites in the PAM-Ag-BNOH composite hydrogel. This resulted in the formation of strong (B-O, H-bond), moderate (Ag-O, H-bond), and feeble (Ag?H, H-bond) and NBPs (O?O).
The developed hydrogel trimer composite has the following unique advantages:
• The PAM-Ag-BNOH nanocomposite surpasses the performance of conventional hydrogel composites, demonstrating extraordinary mechanical strength, including a compressive strength of 0.31 MPa and a Young's modulus of 0.29 MPa. The material is particularly well-suited for load-bearing applications in tissue engineering due to its improved mechanical properties.
• With a low corrosion current density (icorr) of 2.65 × 10?5 A/cm², the trimer hydrogel composite exhibits exceptional long-term durability and corrosion resistance in simulated body fluids. This improved stability extends the lifespan of implants, thereby reducing the necessity for frequent replacements and reducing healthcare expenses.
• The invention effectively integrates experimental and in-silico methodologies to create nanocomposites with exceptional self-healing, bonding, and strength properties, thereby bridging a substantial divide. This dual approach provides a comprehensive framework for the prediction and optimization of material performance before experimental validation, thereby minimizing the time and cost associated with material development.
• The nanocomposite's supramolecular cross-linked assembly facilitates bone formation and self-healing, while also demonstrating effective antimicrobial activity and minimal cytotoxicity. These characteristics are essential for biomedical applications, particularly in the engineering of cartilage tissue, where bioactivity and biocompatibility are of the utmost importance.
• The PAM-Ag-BNOH nanocomposite is synthesized using generally available materials, such as hydroxylated boron nitride and silver nanoparticles, to guarantee a cost-effective production process. This synthesis method's potential for commercial implementation is further bolstered by its scalable nature.
• The invention leverages the synergistic interactions between nano- and micron-sized particles, which are facilitated by hydrogen bonding. The material's overall performance is considerably improved by this robust interfacial interlocking, which renders it a promising candidate for a diverse array of applications beyond tissue engineering, such as wear-resistant coatings and soft robotics.
, Claims:1. A hydrogel nanocomposite comprises of: polyacrylamide (PAM) as a basic material, with 0.1-0.5 wt. % of silver (Ag) and 0.1-0.5 wt. % hydroxylated boron nitride (BNOH).
2. A method for synthesis of the hydrogel nanocomposite as claimed in the claim 1, wherein method comprising the steps of:
• Step 1: Synthesis of polyacrylamide, Ag nanoparticles, and functionalization of hBN
a) Synthesis of polyacrylamide comprising of: a free radical polymerisation reaction was employed to synthesize polyacrylamide hydrogel (PAM). Ammonium persulfate (0.20 g) and tetramethylethylenediamine (610.14µL) employed as catalysts, while acrylamide (10 g) employed as the monomer. During the cross-linking reaction, N,N'-methylene bisacrylamide (0.266 g) was incorporated as a crosslinker.
b) Synthesis of Ag nanoparticles comprising of: a cylinder within an ice chamber was used to contain 30 mL of a 0.002 M sodium borohydride (NaBH4) solution, which was stirred continuously. The frigid bath was employed to reduce the rate of sodium borohydride decomposition during the reaction. Afterward, 2 mL of a 0.001 M AgNO3 solution was dropwise added to the agitated mixture, followed by the addition of a few droplets of NaCl. This process resulted in a change in the colour of the suspension from yellowish to grey. The Ag nanoparticles were stabilised using poly(vinylpyrrolidone). Ag nanoparticles of approximately 30-50 nm in size were obtained after the precipitate was filtered and dried.
c) Functionalization of hBN comprising of: The hydrophilicity of hexagonal boron nitride (hBN) improved by functionalising it with hydroxyl groups (-OH), resulting in hydroxylated boron nitride (BNOH). 3 g of hBN was immersed in a mixture of 12 mL of concentrated H2SO4, 2.5 g of K2S2O8, and 2.5 g of P2O5 at 80ºC, with continuous agitation for 5 hours. Following this, the solution was chilled to room temperature, diluted with 500 mL of deionised water, filtered, washed, and subsequently added to 120 mL of concentrated H2SO4 in a cold bath. In order to expedite the oxidation reaction, 15 g of KMnO4 was introduced with vigorous agitation, and the mixture was maintained at 35ºC for 2 hours while being stirred. Subsequently, 250 mL of deionised water was gradually introduced while agitating continued for an additional 2 hours. Then the reaction was terminated by addition of 700 mL of water and 20 mL of 30% hydrogen peroxide (H2O2). Followed by white mixture precipitated, filtered, washed, and desiccated at 60ºC, resulting in the production of hydroxylated boron nitride with enhanced hydrophilicity.
• Step 2: Preparing of polyacrylamide (PAM) hydrogel dimer
? The aqueous solutions of variable concentrations of Ag (0.1, 0.3, and 0.5 wt%) and BNOH (0.1, 0.3, and 0.5 wt%) repared separately to accomplish the reinforcement of silver (Ag) and hydroxylated boron nitride (BNOH), which is approximately 1 µm in size.
? Followed by, the polyacrylamide (PAM) hydrogel was immersed in these solutions separately after being desiccated. Subsequently, the dimer composites were submerged in deionised water for 24 hours to eliminate surplus bound ions from the surface of the PAM hydrogel composites.
? Consequently, six dimer composite hydrogels were synthesised, which were referred to as PAM-Ag1 (0.1 wt% Ag), PAM-Ag3 (0.3 wt% Ag), PAM-Ag5 (0.5 wt% Ag), PAM-BN1 (0.1 wt% BNOH), PAM-BN3 (0.3 wt% BNOH), and PAM-BN5 (0.5 wt% BNOH). Subsequently, the optimal concentrations of Ag and BNOH were employed to generate a trimer composite hydrogel for subsequent investigations.
• Step 3: Investigating of Dimer composite hydrogels sample prepared in Step 2
? The hydrophilicity, degradation behavior, and compressive strength of the six dimer composite hydrogels referred to as PAM-Ag1 (0.1 wt% Ag), PAM-Ag3 (0.3 wt% Ag), PAM-Ag5 (0.5 wt% Ag), PAM-BN1 (0.1 wt% BNOH), PAM-BN3 (0.3 wt% BNOH), and PAM-BN5 (0.5 wt% BNOH) investigated.
• Step 4: Preparing of polyacrylamide (PAM) hydrogel trimer
? the desiccated mass of neat PAM hydrogel was submerged in the mixture that contained the selected optimal concentrations of Ag and BNOH. The optimal concentrations of Ag and BNOH were obtained from the ranges of 0.1 wt.%, 0.3 wt.%, and 0.5 wt.% after consideration of Step 3 results
? Subsequently, the sample was rinsed and re-immersed in deionized water to eliminate any excess or unbonded ions.
3. The method for synthesis of the hydrogel nanocomposite as claimed in the claims 1-2, wherein preparation of trimer hydrogel composite (PAM-Ag1-BN3) contained 0.1 wt% Ag and 0.3 wt% BNOH.
4. The method for synthesis of the hydrogel nanocomposite as claimed in the claim 1-3, wherein the characterisation of the hydrogel nanocomposite PAM-Ag-BN by:
? PAM-Ag-BN exhibited the greatest polarisation resistance of 55067 O cm2;
? PAM-Ag-BN exhibits a compressive strength of 0.31 MPa;
? The cell viability of PAM-Ag-BN was 98.5 %, 98 %, and 97.3 % for 3, 5, and 7 days, respectively; and
5. The method for synthesis of the hydrogel nanocomposite as claimed in claim 1-3, wherein PAM-Ag-BN exhibited the highest bacterial inhibition of 85% against E. coli, while PAM-BN3 and PAM-Ag1 exhibited bacterial inhibition of 83.3 % and 81.8%, respectively.

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