Claims:
1. A hydrogel comprising polymer reinforced with combination of carbon nanotubes (CNT) and inorganic re-inforcement agent.

2. The hydrogel as claimed in claim 1, wherein the polymer selected from a group comprising polyacrylamide (PAM), polyhydroxyethyl methacrylate, poly(N-isopropylacrylamide), alginate, chitosan, agarose, cellulose, chitin, dextran, elastin, di/tri block peptide copolymers and hyaluronic acid or any combination thereof.

3. The hydrogel as claimed in claim 1, wherein the carbon nanotube is selected from armchair carbon nanotubes, zigzag carbon nanotubes, chiral carbon nanotubes, non-functionalized, functionalized, single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs).

4. The hydrogel as claimed in claim 3, wherein the carbon nanotube is a functionalized carbon nanotube; and wherein the functionalization is with carboxylic group, ketone group, alcohol group and ester group or any combination thereof.

5. The hydrogel as claimed in claim 1, wherein the inorganic re-inforcement agent is selected from a group comprising titanium dioxide (TiO2), hydroxyapatite, silica, Iron Oxide (Fe2O3), Silver (Ag), Gold (Au) and alumina or any combination thereof.

6. The hydrogel as claimed in claim 5, wherein the inorganic re-inforcement agent is in the form of nanoparticles.

7. The hydrogel as claimed in claim 1, wherein the hydrogel comprises a combination of PAM, carbon nanotube (CNTs) functionalized with carboxylic group and titanium dioxide (TiO2) nanoparticles.

8. The hydrogel as claimed in claim 1, wherein the hydrogel comprises about 90% to about 99 % of the polymer.

9. The hydrogel as claimed in claim 1, wherein the hydrogel comprises about 0.30% to about 0.45% of the carbon nanotube.

10. The hydrogel as claimed in claim 1, wherein the hydrogel comprises about 8.2% to about 10 % of the inorganic re-inforcement agent.

11. The hydrogel as claimed in claim 1, wherein compressive strength of the hydrogel ranges from about 0.42 MPa to about 0.5 MPa

12. The hydrogel as claimed in claim 1, wherein elastic modulus of the hydrogel at high strain ranges from about 2.1 MPa to about 3 MPa.

13. The hydrogel as claimed in claim 1, wherein total insertion force of the hydrogel ranges from about 0.01 N to about 0.035 N.

14. The hydrogel as claimed in claim 1, wherein interaction energy of the hydrogel ranges from about 65.5 kcal/molto about 90.9 kcal/mol.

15. The hydrogel as claimed in claim 1, wherein HLEG value of the hydrogel .ranges from about 1.767eV to about 1.812 eV.

16. The hydrogel as claimed in claim 1, wherein hardness value of the hydrogel ranges from about 0.882 to about 0.898.

17. A method of preparing the hydrogel as claimed in claim 1, said method comprising
- preparing a dispersion of the inorganic re-inforcement agent and the CNT;
- immersing a polymeric hydrogel in the dispersion for a period of about 24 hours to about 48 hours to obtain the composite polymer; and
- optionally washing the composite polymer with water.

18. The method as claimed in claim 17, wherein the dispersion of the inorganic re-inforcement agent and the CNT is prepared in a polar solvent; wherein the polar solvent is water, preferably distilled water.

19. The method as claimed in claim 17, wherein the dispersion of the inorganic re-inforcement agent and the CNT has a concentration of the inorganic re-inforcement agent ranging from about 8.2% to about10% and a concentration of the CNT ranging from about 0.30% to about 0.45 %.

20. The method as claimed in claim 17, wherein the dispersion further comprises surfactant(s) selected from a group comprising sodium dodecyl sulfate (SDS), dodecyl-benzene sodium sulfonate, alkylphenol polyoxyethylene ether, dodecyl trimethylammonium bromide or any combination thereof.

21. The hydrogel as claimed in claim 1, for use as a biomedical implant.

22. A method of inducing self-healing of bone or cartilage, comprising application of the hydrogel as claimed in claim 1 to the bone or cartilage.

23. An in-vitro method of tissue generation/re-generation comprising contacting tissue with the hydrogel as claimed in claim 1 in an environment favoring tissue growth.

24. Use of the hydrogel as claimed in claim 1, as a biomedical implant.

25. The hydrogel as claimed in c1aim 21 or the use as claimed in claim 22, wherein the biomedical implant is a cartilage implant.

26. A kit comprising the hydrogel as claimed in claim 1, optionally, a dressing and/or ointment, and instructions for application of the hydrogel.
, Description:TECHNICAL FIELD
The present disclosure relates to the fields of polymer science and biomedical implants. The present disclosure particularly provides reinforced polymeric hydrogels fit for use as regenerative implants. Examples of such implants include but are not limited to cartilage or bone implants. Further provided herein are methods of preparation and application of the said hydrogel of the present disclosure.

BACKGROUND OF THE DISCLOSURE
The advent of cartilage tissue engineering has improved the mobility and life of millions of patients globally. The main concept of cartilage tissue engineering is to facilitate a three dimensional cartilage scaffold, which can offer an ideal hydrophilic environment, have significant mechanical strength as well as provide biocompatibility, cell proliferation and cell attachment. Besides the advancement in the field of cartilage implants, the localized damage of cartilage, especially in the knee and hip prostheses, is appalling for young or middle-aged patients due to the limited self-recovery and load-bearing capacity of these prostheses.

Approaches for increasing the mechanical strength of the hydrogel by altering the crosslinking density, lowering the swelling ratio, adding the reinforcing agents and introducing the interpenetrating polymer network have been previously discussed, but there is still room for the improvement. Puncture resistance of the hydrogels is an important phenomenon to investigate the internal damage of the tissue. The structural, stability and electronic feature analyses and interfacial bonding characteristics such as H-bonding, other noncovalent interactions (NCIs) and van der Waals (vdW) type interactions are most important for polymeric matrix and reinforcing particles. Such types of connection between the experiment and theory form the existing lacunae in the art for cartilage applications.

Addressing the aforesaid need in the art, the present disclosure provides hydrogels having high mechanical strength wherein the mechanical strength and puncture resistance of the material has been further validated by the computational experiments.

SUMMARY OF THE DISCLOSURE
In view of the drawbacks in the art as described above, provided herein is a hydrogel comprising polymer reinforced with combination of carbon nanotubes (CNT) and inorganic re-inforcement agent.

In some embodiments, the polymer selected from a group comprising polyacrylamide (PAM), polyhydroxyethyl methacrylate, poly(N-isopropylacrylamide), alginate, chitosan, agarose, cellulose, chitin, dextran, elastin, di/tri block peptide copolymers and hyaluronic acid or any combination thereof. In some embodiments, the carbon nanotube is selected from armchair carbon nanotubes, zigzag carbon nanotubes, chiral carbon nanotubes, non-functionalized, functionalized, single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs). In some embodiments, the carbon nanotube is a functionalized carbon nanotube; and wherein the functionalization is with carboxylic group, ketone group, alcohol group and ester group or any combination thereof. In some embodiments, the inorganic re-inforcement agent is selected from a group comprising titanium dioxide (TiO2), hydroxyapatite, silica, Iron Oxide (Fe2O3), Silver (Ag), Gold (Au) and alumina or any combination thereof. In some embodiments, the inorganic re-inforcement agent is in the form of nanoparticles.

In an exemplary embodiment, the hydrogel comprises a combination of PAM, carbon nanotube (CNTs) functionalized with carboxylic group and titanium dioxide (TiO2) nanoparticles.

In some embodiments, the hydrogel comprises about 90% to about 99 % of the polymer.

In some embodiments, the hydrogel comprises about 0.30% to about 0.45% of the carbon nanotube.

In some embodiments, the hydrogel comprises about 8.2% to about 10 % of the inorganic re-inforcement agent.

In some embodiments, compressive strength of the hydrogel ranges from about 0.42 MPa to about 0.5 MPa

In some embodiments, elastic modulus of the hydrogel at high strain ranges from about 2.1 MPa to about 3 MPa.

In a non-limiting embodiment, compressive strength of the hydrogel ranges from about 0.42 MPa to about 0.5 MPa and elastic modulus of the hydrogel at high strain ranges from about 2.1 MPa to about 3 MPa.

In some embodiments, total insertion force of the hydrogel ranges from about 0.01 N to about 0.035 N.

In some embodiments, interaction energy of the hydrogel ranges from about 65.5 kcal/mol to about 90.9 kcal/mol.

In some embodiments, HLEG value of the hydrogel ranges from about 1.767 eV to about 1.812 eV.

In some embodiments, hardness value of the hydrogel ranges from about 0.882 to about 0.8980.

Further provided herein is a method of preparing the hydrogel as described above, said method comprising
preparing a dispersion of the inorganic re-inforcement agent and the CNT;
immersing a polymeric hydrogel in the dispersion for a period of about 24 hours to about 48 hours to obtain the composite polymer; and
optionally washing the composite polymer with water.

In some embodiments, the dispersion further comprises surfactant(s) selected from a group comprising sodium dodecyl sulfate (SDS), dodecyl-benzene sodium sulfonate, alkylphenol polyoxyethylene ether, dodecyl trimethylammonium bromide or any combination thereof.

Further provided herein is the hydrogel as described above, for use as a biomedical implant.

Also envisaged herein is a method of inducing self-healing of bone or cartilage, comprising application of the hydrogel as described above to the bone or cartilage.

The present disclosure further provides an in-vitro method of tissue generation/re-generation comprising contacting tissue with the hydrogel as described above in an environment favoring tissue growth.

Further envisaged herein is use of the hydrogel as described above, as a biomedical implant.

In some embodiments, the biomedical implant is a cartilage implant.

The present disclosure further provides a kit comprising the hydrogel as described above, optionally, a dressing and/or ointment, and instructions for application of the hydrogel.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:

Figure 1 depicts scanning electron microscopic (SEM) images of raw powders used in the preparation of hydrogel composites (a) TiO2 and (b) CNT.
Figure 2 depicts SEM images on the cross-section of the (a) PAM, (b) PAM-TiO2, (c) PAM-CNT and (d) PAM-TiO2-CNT hydrogels.
Figure 3depicts (a) XRD pattern (b) FT-IR spectrum for PAM, PAM-TiO2, PAM-CNT and PAM-TiO2-CNT hydrogel composites.
Figure 4 depicts L929 mammalian cell viability on composite hydrogel samples after 72 hour culture.
Figure 5 depicts microscopic images (100 µm scale) of L929 cells cultured for 72  hourculture on (a) untreated (b) PAM (c) PAM-TiO2 (d) PAM-CNT and (e) PAM-TiO2-CNT.
Figure 6 depicts contact angle plot for PAM, PAM-TiO2, PAM-CNT and PAM-TiO2-CNT hydrogel composites.
Figure 7 depicts (a) Swelling ratio and (b) Degradation profile of PAM, PAM-TiO2, PAM-CNT and PAM-TiO2-CNT hydrogel composites.
Figure 8 depicts stress-strain curves for the PAM and PAM based composite hydrogels.
Figure 9depicts force-displacement curves for hydrogel samples during needle insertion test.
Figure 10 depicts 3D images of needle insertion hole after self-healing using surface profilometry for (a) PAM, (b) PAM-TiO2, (c) PAM-CNT and (d) PAM-TiO2-CNT hydrogel composites.
Figure 11 depicts top surface SEM images showing apatite formation in SBF for (a) PAM, (b) PAM-TiO2, (c) PAM-CNT and (d) PAM-TiO2-CNT hydrogel composites.
Figure 12 depicts XRD pattern of the hydrogel samples showing characterisctics apatite peaks.
Figure 13 depicts optimized/equilibrium structures of the CNT (top left), TiO2 (top right) and PAM (below left). Models at the B3LYP/6-31G level of theory where PAM 3D-Isosurface and 2D-Scatter Plot are shown at bottom middle and bottom right, respectively.
Figure 14 depicts optimized/equilibrium structures (left), 3D-Isosurafces (middle), and 2D-Scatter Plots (right) of the PAM-CNT (top) and PAM-TiO2 (bottom) dimer model complexes at the B3LYP/6-31G level of theory.
Figure 15 depicts optimized/equilibrium structures (top left), 2D-Scatter Plots (top right), and 3D-Isosurafces (bottom) of the PAM-TiO2-CNT at the B3LYP/6-31G level of theory.
Figure 16 depicts HOMO and LUMO Surface Plots of the Optimized/Equilibrium Structures of the Dimer (PAM-CNT and PAM-TiO2) and Trimer (PAM-TiO2-CNT) Model Complexes at the B3LYP/6-31G level of theory.
Figure 17 depicts MESP Plots of the Monomer (PAM and CNT) Units, Dimers (PAM-TiO2 and PAM-CNT) and Trimer (PAM-TiO2-CNT) Composites.
Figure 18 depicts schematic representation of the experiments in the present disclosure, leading to the observation of highest mechanical strength and strong interfacial bonding between TiO2 and CNT nanoparticles with PAM.

DETAILED DESCRIPTION OF THE INVENTION

General definitions
As used herein, the term “hydrogel” is a type of “gel,” and refers to a polymeric matrix, consisting of a three-dimensional network of macromolecules (e.g., hydrophilic polymers) hydrophobic polymers, blends thereof) held together by covalent or non-covalent crosslinks to form an elastic gel. The polymeric matrix may be formed of any suitable synthetic or naturally occurring polymer material.

The terms “hydrogel”, “polymer composite”, “polymer-based composite material”, “hybrid hydrogel”, “composite based hydrogel”, “dimer complex”, “trimer complex” and other obvious alternatives as derivable from the context of use of said terms have been used interchangeably in referring to the hydrogel of the present disclosure.

The following nomenclatures have been used throughout the present disclosure while referring to the hydrogels in order to identify the hydrogels by the respective components: “PAM-TiO2”, “PAM-CNT” (both generally having been referred to as “two component hydrogels”, “dimer complex” or “dimer composite”) and “PAM-TiO2-CNT” (also referred to as “three component hydrogel”, “trimer complex” or “timer composite”).

The definitions of “hydrophobic” and “hydrophilic” polymers are based on the amount of water vapor absorbed by polymers at 100% relative humidity. According to this classification, hydrophobic polymers absorb only up to 1% water at 100% relative humidity (“rh”), while moderately hydrophilic polymers absorb 1-10% water and hydrophilic polymers are capable of absorbing more than 10% of water.

The term “crosslinked” as used herein refers to a composition containing intramolecular and/or intermolecular crosslinks, whether arising through covalent or noncovalent bonding, and may be direct or include a cross-linker. “Noncovalent” bonding includes both hydrogen bonding and electrostatic (ionic) bonding.

The term “polymer” includes linear and branched polymer structures, and also encompasses crosslinked polymers as well as copolymers (which may or may not be crosslinked), thus including block copolymers, alternating copolymers, random copolymers, and the like. Those compounds referred to herein as “oligomers” are polymers having a molecular weight below about 1000 Da, preferably below about 800 Da. Polymers and oligomers may be naturally occurring or obtained from synthetic sources.

As used herein, the term ‘comprising’ when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and/or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The suffix ‘(s)’ at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.

As used in this specification and the appended claims, the singular forms ‘a’, ‘an’ and ‘the’ includes both singular and plural references unless the content clearly dictates otherwise. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms ‘a’ (or ‘an’), ‘one or more’, and ‘at least one’ can be used interchangeably herein.

Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

The terms ‘about’ or ‘approximately’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier ‘about’ or ‘approximately’ refers is itself also specifically, and preferably, disclosed.

As used herein, the terms ‘include’, ‘have’, ‘comprise’, ‘contain’ etc. or any form said terms such as ‘having’, ‘including’, ‘containing’, ‘comprising’ or ‘comprises’ are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Disclosure
The present disclosure provides a polymer-based composite material having improved mechanical strength, and more particularly, to a polymer composite which is reinforced with a combination of carbon nanotubes and inorganic re-inforcement agent.

Said composite material of the present disclosure is a hydrogel. In some embodiments, the hydrogel is a potential candidate for applications such as but not limited to cartilage replacement, bone repair, drug delivery, wound healing and biosensing.

Accordingly, in some embodiments, provided herein is a hydrogel comprising a polymer reinforced with combination of carbon nanotubes and inorganic re-inforcement agent.

In some embodiments, the hydrogel comprises polymer selected from a group comprising but not limited to polyacrylamide (PAM), polyhydroxyethyl methacrylate, poly(N-isopropylacrylamide), alginate, chitosan, agarose, cellulose, chitin, dextran, elastin, di/tri block peptide copolymers and hyaluronic acid or any combination thereof.

In some embodiments, further polysaccharides and polypeptides characterized by features of ease of synthesis, biocompatibility and biodegradability, rendering them suitable for tissue engineering applications may be employed in the preparation of hydrogels of the present disclosure.

In exemplary embodiments, the hydrogel comprises polyacrylamide (PAM) as the polymeric component.

In some embodiments, the carbon nanotube is selected from armchair carbon nanotubes, zigzag carbon nanotubes, chiral carbon nanotubes, non-functionalized, functionalized, single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs).

In some embodiments, the carbon nanotube is a functionalized carbon nanotube. In some embodiments, the functionalization is with carboxylic group, ketone group, alcohol group and ester group or any combination thereof.

In exemplary embodiments, the carbon nanotube is carbon nanotube functionalized with carboxylic group.

In some embodiments, the inorganic re-inforcement agent is selected from a group comprising titanium dioxide (TiO2),hydroxyapatite, silica, Iron Oxide (Fe2O3), Silver (Ag), Gold (Au) and alumina or any combination thereof.

In some embodiments, the inorganic re-inforcement agent is incorporated in the form of nanoparticles.

In exemplary embodiments, the inorganic re-inforcement agent is titanium dioxide (TiO2), more preferably TiO2 nanoparticles.

While all permutations and combinations of the polymeric component, the carbon nanotube and the inorganic re-inforcement agent as can be derived based on the aforesaid embodiments are envisaged herein, in exemplary embodiments, the hydrogel of the present disclosure comprises a combination of PAM, carbon nanotube (CNTs) functionalized with carboxylic group and titanium dioxide (TiO2) nanoparticles. The PAM, in said hydrogel is essentially reinforced with a combination of carbon nanotube (CNTs) and titanium dioxide (TiO2) nanoparticles.

In a non-limiting embodiment, the carbon nanotube (CNTs) and titanium dioxide (TiO2) nanoparticles are encapsulated by or embedded within the PAM matrix.

In some embodiments, the hydrogel comprises about 90% to about 99% of the polymer.

In some embodiments, the hydrogel comprises about 0.30% to about 0.45% of the carbon nanotube.

In some embodiments, the hydrogel comprises about 8.2% to about 10% of the inorganic re-inforcement agent.

In some embodiments, the hydrogel comprises the polymer, the carbon nanotube and the inorganic re-inforcement agent at a ratio of about 230:1:20 to about 220:1.5:25.

In an exemplary embodiment, the hydrogel comprises the polymer, the carbon nanotube and the inorganic re-inforcement agent at a ratio of about 220:1.5:25.

In preferred embodiments, the hydrogel comprises PAM, the carbon nanotube functionalized with carboxylic group and TiO2 at a ratio of about 230:1:20 to about 220:1.5:25, preferably about 220:1.5:25.

The hydrogel of the present disclosure is characterized by properties of high mechanical strength, strong bonding, self-healing, bioactivity and cytocompatibility. Said properties deem the composite fit to be utilized as a hydrogel for facilitating cartilage repair.

In some embodiments, the hydrogel of the present disclosure has a low contact angle of about 41º to about 47º with Phosphate-buffered saline (PBS) as measured in the assessment of hydrophilic behaviour of the hydrogel. The hydrophilicity of any material is an essential behaviour for biomedical implants as it can augment the protein adsorption and cellular responses in biological systems. The rate of bone formation can also be enhanced by the hydrophilic implants. Therefore, the hydrogels of the present disclosure were found to be highly suited for application as biomedical implants.

In some embodiments, the cytocompatibility of the hydrogel is derivable from the retention of about 90% to about 100% cell viability even upon prolonged exposure of cells to the hydogel.

In some embodiments, compressive strength of the hydrogel of the present disclosure ranges from about 0.42MPa to about 0.5 MPa.

In an exemplary embodiment, compressive strength of the hydrogel is about 0.44 at a maximum strain of about 96%.

While polymer-based hydrogel composites have been previously reported in literature, compressive strength of said hydrogels as previously reported are comparatively lower than that of the hydrogels of the present disclosure. Some examples are provided in Table 1 –

Table 1: Polymer based hydrogels and their compressive strength as reported in literature

Hydrogel composites Compressive strength
Polyacrylamide (PAM) with CNT Strength 0.04 MPa
PAM-Cellulose Nano Fibres-CNT Strength 0.30 MPa
PAM-LMWG octylaldonamide Strength 0.15 MPa
PAM methacrylated gelatin Strength 0.32 MPa
PAM/chitosan with crosslinked by hyperbranched polysiloxane Strength 0.30 MPa
Chitosan/cellulose nanocrystals/polyacrylamide Strength 0.25 MPa
Alginate-polyacryalmide hydrogel with Si Strength 0.18 MPa
Poly(N-isopropylacrylamide)/ZrO2 Strength 0.009 MPa

In some embodiments, the hydrogel of the present disclosure shows efficient self-healing performance, which can be attributed to the strong mechanical interaction and bonding between re-inforcement agents such as nanoparticles of inorganic re-inforcement agent and hydrogel (Eg. PAM) matrix. In a non-limiting embodiment, hydration triggers self-healing of the hydrogel.

Without intending to be limited by theory, the type, nature and strength of the interactions between two components depend mainly on the orientation of molecules as well as the dipole moment. If the orientation of the molecules is not favorable, it can result in weak bonding, which can be damaged easily by for example – wear and tear and/or mechanical force. In case of the composite based hydrogel of the present disclosure the strong bonding - metal-nonmetal and hydrogen bonding - between the polymer and the carbon nanotubes (CNT) and the inorganic re-inforcement agent reduces damage to the hydrogel and improves its self-healing property.

In a non-limiting embodiment, the composite based hydrogel of the present disclosure is a good cartilage substitute with ability to stimulate the natural healing in the surrounding tissue.

The bioactivity of the hydrogel for applicability as a cartilage implant can be examined by in vitro apatite formation ability in simulated body fluid (SBF). Apatite is essential for providing support to cartilage tissue and to promote osteogenic tissue formation and bone integration.

In a non-limiting embodiment, the hydrogel of the present disclosure shows formation of the apatite crystals on the hydrogel surface. Based on the same, it can be derived that the composite hydrogels have potential to show signs of osteoconduction after implantation along with the self-calcification in the body.

The high content of apatite in composite hydrogels (mainly in PAM-TiO2-CNT) can be attributed to the presence of TiO2 and functionalized CNTs. The Ti-O and –COOH groups present in the hydrogel can promote heterogeneous nucleation of amorphous calcium phosphate. Therefore, the designed PAM hydrogel with encapsulation of TiO2 and CNTs are found to be bioactive and have the ability to accelerate osseointegration once applied in vivo.

While materials such as PAM hydrogels have been previously identified as a potential candidate for cartilage replacement, their bio-applicability was found to be strictly hampered due to limited mechanical strength and low puncture resistance. In the polymer composite based hydrogel of the present disclosure, the mechanical strength of the hydrogel has been increased by altering the crosslinking density, lowering the swelling ratio, adding the reinforcing agents and introducing the interpenetrating polymer network.

In some embodiments, the hydrogel has elastic modulus at high strain ranging from about 2.1 MPa to about 3MPa.

In a non-limiting embodiment, compressive strength of the hydrogel ranges from about 0.42 MPa to about 0.5 MPa and elastic modulus of the hydrogel at high strain ranges from about 2.1 MPa to about 3 MPa.

In some embodiments, the total insertion force of the hydrogel of the present disclosure, as measured through a needle insertion test ranges from about 0.01 N to about 0.035 N. Said strength is responsible for lessening of the number of conical cracks and internal damage in hydrogel as a result of the lessening of friction force at the interface which lessens shearing of the gel at the interface.

In a non-limiting embodiment, puncture resistance of the hydrogel of the present disclosure is about 3 times to about 7 times higher than that of a corresponding non-reinforced hydrogel.

In another non-limiting embodiment, puncture resistance of the hydrogel of the present disclosure is about 5 times higher than that of a corresponding non-reinforced hydrogel.

In some embodiments, in swelling experiments, the hydrogel of the present disclosure showed non-swellable behaviour which is significant for applicability as cartilage implants. The hydrogel shows low swelling ratio due to the increased covalent crosslinking between PAM and embedded nanoparticles. The addition of re-inforcement agents such as TiO2 and CNT in a polymeric hydrogel such as PAM (PAM-TiO2-CNT) makes a condensed PAM network that restricts the swelling of hydrogel. This also confers rigidity to the hydrogel and reduces its degradation.

In a non-limiting embodiment, based on molecular modelling approach (MMA), it was found that the three monomer constituents of the hydrogel are bound together by the interaction between two dimer units of the polymer-CNT and the polymer-inorganic reinforcement agent.

In another non-limiting embodiment, in case of the PAM-TiO2-CNT hydrogel, the trimer is held together by the dimer interactions - PAM-TiO2 (Ti-O and Ti⋯N metal-nonmetal interactions (MNIs) as well as one N-H⋯O and two C-H⋯O weak H-bonding interactions) and PAM-CNT (one O-H⋯O and one N-H⋯O strong H-bonding interactions) - which binds all three monomer constituents together strongly and make it a highly stable composite.

In some embodiments, the hydrogel has interaction energy ranging from about 65.5 kcal/mol to about 90.9 kcal/mol.

In some embodiments, the hydrogel has HOMO–LUMO energy gap (HLEG) value ranging from about 1.767 eV to about 1.812 eV.

In some embodiments, the hydrogel has hardness value ranging from about 0.882 to about 0.898

The present disclosure further provides a method of preparing the hydrogel as described above, said method comprising
preparing a dispersion of the inorganic re-inforcement agent and the CNT;
immersing a polymeric hydrogel in the dispersion for a period of about 24 hours to about 48 hours to obtain the composite polymer; and
optionally washing the composite polymer with water.

In some embodiments, the dispersion of the inorganic re-inforcement agent and the CNT is prepared in solvent selected from a group comprising polar solvents such as but not limited to water, preferably distilled water.

In some embodiments, the dispersion of the inorganic re-inforcement agent and the CNT has a concentration of the inorganic re-inforcement agent ranging from about 8% to about 10% and a concentration of the CNT ranging from about 0.30% to about 0.45%.

In some embodiments, the dispersion further comprises surfactant(s) selected from a group comprising sodium dodecyl sulfate (SDS), dodecyl-benzene sodium sulfonate, Alkylphenol polyoxyethylene ether and Dodecyl trimethylammonium bromide or any combination thereof.

In some embodiments, the concentration of surfactant in the dispersion ranges from about 0.15% to about 0.2%.

In exemplary embodiments, the dispersion comprises sodium dodecyl sulfate (SDS) as the surfactant.

In a non-limiting embodiment, the dispersion is maintained at room temperature.

In some embodiments, the polymeric hydrogel employed in the above process is a pre-prepared, commercially available hydrogel or is a hydrogel prepared prior to the aforesaid the method in-situ. In some embodiments, the polymeric hydrogel is a PAM hydrogel prepared by a free radical polymerization reaction with acrylamide monomer.

In some embodiments, the step of washing may be performed by dipping the composite in water for about 10 hours to about 24hours, preferably about 24 hours. Said washing step ensures exclusion of extra bounded ions from the surface of polyacrylamide hydrogel.

In some embodiments, the method of preparing the hydrogel of the present disclosure comprises
preparing a dispersion of the inorganic re-inforcement agent and the CNT; and
immersing the polymeric hydrogel in the dispersion for a period of about 24 hours to about 48 hours to obtain the composite polymer.

In some embodiments, the method of preparing the hydrogel of the present disclosure comprises
preparing a dispersion of the inorganic re-inforcement agent and the CNT;
immersing the polymeric hydrogel in the dispersion for a period of about 24 hours to about 48 hours to obtain the composite polymer; and
washing the composite polymer with water.

In an exemplary embodiment, the method of preparing the hydrogel of the present disclosure comprises
preparing a dispersion of TiO2 nanoparticles and carbon nanotube functionalized with carboxylic group; and
immersing PAM hydrogel in the dispersion for a period of about 24 hours to about 48 hours to obtain the composite polymer.

In an exemplary embodiment, the method of preparing the hydrogel of the present disclosure comprises
preparing a dispersion of TiO2 nanoparticles and carbon nanotube functionalized with carboxylic group;
immersing PAM hydrogel in the dispersion for a period of about 24 hours to about 48 hours to obtain the composite polymer; and
washing the composite polymer with water.

The present disclosure further envisages applications of the hydrogel as described above. Said applications include but are not limited to biomedical implants such as cartilage implants, biosensors, drug delivery and wound healing.

The present disclosure provides the hydrogel as described above for use as a biomedical implant such as but not limited to a cartilage implant.

Further provided herein is the use of the hydrogel as described above as a biomedical implant such as but not limited to a cartilage implant.

Also provided in the present disclosure is a method of inducing self-healing of bone or cartilage, comprising application of the hydrogel as described above to the bone or cartilage.

In some embodiments, said application is facilitated by way of implantation.
In some embodiments, in the above-described method, the hydrogel is applied to an injured or damaged bone or cartilage. Said injury or damage may be due to age related wear and tear or caused due to any accident or any other medical condition.

In a non-limiting embodiment, the biomedical implant serves as a scaffold for the formation of new tissue. In some embodiments, said scaffold may additionally comprise a layer or fabric or mesh as support.

Further provided in the present disclosure is an in-vitro method of tissue generation/re-generation comprising application of the hydrogel as a scaffold for tissue-regeneration.

In some embodiments, the aforesaid in-vitro method comprises contacting tissue with the hydrogel of the present disclosure in an environment favoring tissue growth.

In a non-limiting embodiment, such an environment favoring tissue growth includes but is not limited to suitable culture/growth medium, favorable temperature, favorable humidity level, favorable light conditions for a suitable period of time.

In some embodiments, in the aforesaid in-vitro method, the tissue and scaffold are maintained at a temperature of about 37°C, for about 24hours to about 48hours.

Once regenerated, the prepared hydrogel may be applied for medical or non-medical applications. In some embodiments, medical applications include but are not limited to use as a biomedical implant. In a non-limiting embodiment, non-medical applications include but are not limited to use of the hydrogel for the purposes of research.

Further provided herein is a kit comprising the hydrogel of the invention, optionally, a dressing and/or ointment, and instructions for application of the hydrogel.

It is to be understood that the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

Descriptions of well-known/conventional methods/steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES:

EXAMPLE 1: Preparation of the composite polymer-based hydrogel
For the purposes of comparison three different composites were prepared –comprising PAM-TiO2, PAM-CNT and PAM- TiO2-CNT. Post their preparation, a comparison in terms of properties and performance was conducted between the two component (dimer) and three component (trimer) composites.

Initially, a polyacrylamide hydrogel (PAM) was prepared by the free radical polymerization reaction with acrylamide monomer. Ammonium persulfate and tetramethylethylenediamine were used as catalysts while N,N’-methylene bisacrylamide served as a crosslinker for the reaction. Polyacrylamide hydrogel (PAM) was prepared by first making the monomer solution and then adding the cross-linking agent to it. The monomer solution was prepared by adding about 10 g of acrylamide, about 0.266 g of N,N’-methylene bisacrylamide and about 0.20 g of ammonium persulfate to distilled water to prepare about 100 ml of solution. Further, about 610.14 µl of tetramethylethylenediamine was mixed to cure the solution. Concentration of all components used in the preparation of the hydrogel is given in Table 2 along with the nomenclature of the samples.

In order to prepare the CNTs for addition to the polymer composite, about 400mg of pristine CNTs was dispersed in 100ml of a solution of sulphuric acid and nitric acid (3:1) and subjected to sonication for about 7 hours. The resultant precipitate was then filtered, washed with deionized water and dried at about 80℃ for one day. The CNTs were thereafter grafted with carboxylic group to prepare functionalized CNTs.

Separate solutions of TiO2 and CNT in water, at room temperature, were prepared comprising about 10g/L of TiO2 and about 0.5g/L of CNT, respectively.

For the preparation of PAM-TiO2 and PAM-CNT composites, sections of PAM hydrogel as prepared above were dried and then immersed in about 100 ml of the TiO2 and CNT solution, separately for about 48 hours.

A combined solution of TiO2 and CNT comprising about 10g/L of TiO2 and about 0.5g/L of CNT was also prepared for the preparation of the PAM-TiO2-CNT composites. Sections of PAM hydrogel were dried and then immersed in about 100ml of the combined solution for about 48 hours for the mutual encapsulation of TiO2 and CNT in the PAM matrix.

The composite hydrogels obtained were dipped in distilled water for about 24 hours for the exclusion of extra bounded ions from the surface of polyacrylamide hydrogel. Thus, four types of samples were prepared - pure PAM, PAM-TiO2, PAM-CNT and PAM-TiO2-CNT (Table 1).

Table 2: Nomenclature and chemical concentration of the fabrication hydrogel composites

Description of samples Nomenclature Chemical composition
Polyacrylamide hydrogel PAM For PAM hydrogel:
Acrylamide 10 g
N,N’-methylene bisacrylamide 0.266 g
ammonium persulfate 0.20 g
Polyacrylamide hydrogel with TiO2 nanoparticles PAM- TiO2 tetramethylethylenediamine610.14 µl
Polyacrylamide hydrogel with carbon nanotubes PAM-CNT For Encapsulation of TiO2 and/or CNT:

TiO2 10 g/l
CNTs 0.5 g/l
For CNT dispersion
Sodium dodecyl sulphate (SDS) 2 g/l
Polyacrylamide hydrogel with TiO2 and carbon nanotubes
PAM-TiO2-CNT

EXAMPLE 2: Physiochemical characterizations of hydrogels

SEM analysis
Scanning electron microscope (FE-SEM model Ultra 55 Karl Zeiss) was used to observe the top surface morphology of the hydrogels prepared in Example 1.

The SEM images of raw powders of TiO2 and CNT used in the preparation of hydrogel composites are shown in Figure 1a and Figure 1b, respectively.

In the analysis of the hydrogels described in Example 1, the top surface morphology on the cross sections of the hydrogels revealed a smooth surface (without any specific feature) for pure PAM (Figure 2a) while well dispersed TiO2 nanoparticles could be observed for PAM-TiO2 sample (Figure 2b). In PAM-CNT composite gel, CNTs were embedded within the PAM matrix (Figure 2c). The combination of TiO2, as well as CNTs in the PAM matrix, was clearly visible in PAM-TiO2-CNT hydrogel, wherein both TiO2 and CNTs were encapsulated in the PAM matrix (Figure 2d).

XRD and FTIR analysis
Subsequent to the confirmation of nanoparticles encapsulation by PAM gel, the retention of phases in polymer composite based hydrogels was confirmed from X-Ray Diffraction (XRD) (Figure 3a).

The pure polyacrylamide hydrogel (PAM) showed broad humps, indicating the amorphous nature of PAM. Furthermore, characteristics TiO2 peaks were visible in PAM-TiO2 and PAM-TiO2-CNT hydrogels. The anatase TiO2crystallographicplanes are presented as (101), (110), (105), (211) and (204) at ~23º, 29º, 55º, 57º, 64º 2θ values. Since the bulk of the composition comprised of polyacrylamide as compared to a smaller amount ofTiO2, the peaks related to TiO2 showed less intensity. Nonetheless, the CNT peaks were not visible in the XRD spectrum of PAM-CNT hydrogel due to less reflectivity of carbon in XRD (Figure 3a).

Furthermore, the bonding between the composite hydrogels was confirmed by Fourier-transform infrared (FT-IR) spectrum (Figure 3b). A strong vibration bond at ~3337 cm-1was observed due to O-H bond while the characteristic N-H stretching vibration was observed at ~ 2953 cm-1 due to the presence of amide group in PAM hydrogel. The carbonyl group stretching vibration band was observed at ~1654 cm-1. The presence of shoulder peaks at ~3188 cm-1 in PAM-TiO2, at ~3186 cm-1 in PAM-CNT and at 3185 cm-1 in PAM-TiO2-CNT composite hydrogels, was reflective of some complexation between PAM and nanoparticles by hydrogen bonding. Furthermore, the shifting in peaks from ~3337 cm-1 to 3286 cm-1 and a broader band from 2953 cm-1 to 2925 cm-1 in PAM-CNT indicated the formation of hydrogen bonding between functionalized CNT and PAM. The TiO2 bonded with PAM through C-O-H deformation and Ti-O-C vibrations, as confirmed by absorption peaks at ~1008 cm-1 and 1310 cm-1. In the case of the PAM-TiO2-CNT, the composite exhibited strong metal-nonmetal bonding of the PAM with the TiO2 (Ti-O-C) as well as H-bonding with functionalized CNTs (-O-OH).

The PAM-TiO2-CNT was concluded to be a stable and strong composite in view of the presence of metal-nonmetal bonding and H-bonding.

EXAMPLE 3: Cytotoxicity measurement

The in vitro cytotoxity of the nanocomposite hydrogels towards L929 mouse fibroblast cells was measured by MTT (3-dimethylthiazol-2, 5-diphenyltetrazolium bromide) assay.

The L929 fibroblast cells were cultured using Dulbecco's Modified Eagle Medium (DMEM) -high glucose media withabout10% fetal bovine serum (FBS), about 1% penicillin and streptomycin and incubated at about 37℃ in about 5% CO2 and about 80-90% humidity. The cells were then seeded (about 200 µL of cell suspension containing 20,000 cells/well) in a 96-well plate without the test agent and allowed to grow for about 12-16 hours. The hydrogels of about 10 mm diameter, about 5 mm thickness sized were sterilized in ethanol (about 70 %) for about 5 minutes and UV light for about 15 minutes. Afterwards, the sterilized samples were made into thin slices and kept over the cells and incubated again for about 72 hours. About 20 µL of MTT reagent was added to the cells at a final concentration of about 0.5 mg/mL of total volume and incubated for about 3 hours. The MTT reagent was then removed and about 100 µL DMSO was added and the optical density (OD) of the media was observed at 570 nm for the calculation of cell viability.

% cell viability was calculated by the following formula –

Cell viability (%)=(〖OD〗_test-〖OD〗_blank)/(〖OD〗_control-〖OD〗_blank )×100

Where 〖OD〗_test, 〖OD〗_blank and 〖OD〗_control are the optical densities of cells incubated with hydrogels, only media without cells and media with cells, respectively.

The % mammalian cell viability of the samples is reported in Figure 4. The prepared hydrogels exhibited cell viability of more than about 98%. The highest cell viability (100 %) was observed for PAM-TiO2-CNT composite due to the synergistic effect of biomaterials TiO2 and functionalized CNTs. The ISO standard for biocompatibility assessment reveals that the composite hydrogels allowing cell viability more than about 70% are not cytotoxic to L929 mouse fibroblast cells.

The microscopic images in Figure 5 display the cellular response to hydrogels. The L929 mouse fibroblast cells revealed a fusiform morphology and the cells proliferated and spread very well on the culture plate. The results indicated that the prepared hydrogels (PAM, PAM-TiO2, PAM-CNT and PAM-TiO2-CNT) exhibited no apparent cytotoxicity to L929 mammalian cells with incubation of about 72 hours and could be a potential biomaterials for cartilage applications.

EXAMPLE 4: Hydrophilicity of hydrogels

The hydrophilic behaviour of the prepared hydrogels was determined by measuring contact angle between a drop of fluid and the hydrogel surface (Figure 6). Phosphate buffer saline (PBS, pH 7.4) was used to measure the contact angle on the samples with a 5 µl drop.

The measured contact angle was ~70º for pure PAM. The contact angle of PAM-TiO2 (~54º) and PAM-CNT (~44º) composites showed enhanced hydrophilic behaviour of composites due to the presence of TiO2 and hydrophilic –COOH functionalized CNTs in the samples, respectively. However, the combination of TiO2 and CNTs in PAM-TiO2-CNT hydrogel was found to create a more hydrophilic environment and thus a further lowered contact angle (~40º, Figure 6).

The hydrophilicity of any material is an essential behaviour for biomedical implants as it can augment the protein adsorption and cellular responses in biological systems. The rate of bone formation can also be enhanced by the hydrophilic implants.Therefore, the composite polymer-based hydrogels of the present disclosure were found to behighly suited for application as biomedical implants.

EXAMPLE 5: Swelling kinetics and degradation performance of hydrogels

Swelling

The swelling behaviour of pure PAM and PAM based composite hydrogels were characterized by weighing the dried mass of the samples followed by the dipping of the hydrogels in about 100 ml PBS (pH 7.4) at about 37℃ for about 24 hours until the swelling equilibrium was reached. The swollen hydrogels were weighed after different time intervals (2, 5, 10, 15, 20, 25 h). The swelling ratio (SR) was calculated using the equation:

SR=(W_s-W_i)/W_i (1)
Where, W_s and W_i are the weights of the hydrogel at equilibrium swelling and the initial state, respectively.

The mass of hydrogels after immersing in PBS showed the swelling ratio of the samples (Figure 7a). The water absorption rate depends on several factors like specific surface area, molecular and surface structure and particle size. The PAM and PAM based hybrid hydrogel exhibited an equilibrium swelling after about 10 hours of immersion (Figure 7a; Table 3). The stable volume of PAM-TiO2-CNT hydrogel even after about 25 hours, represented non-swellable behaviour of the material, which is a significant aspect for cartilage regenerative implants. The lower swelling ratio in composite hydrogels was due to the increased covalent crosslinking between PAM and embedded nanoparticles. The addition of TiO2 and CNT in PAM (PAM-TiO2-CNT) makes a condensed PAM network that restricts the swelling of hydrogel.

Table 3: Swelling ratio of the samples

Time (h) PAM PAM-TiO2 PAM-CNT PAM-TiO2-CNT
2 10.26194 7.872879 9.968301 5.154979
5 15.05122 10.30452 11.39169 6.834674
10 18.94342 11.13708 11.90973 7.311983
15 19.19764 11.50455 12.28628 7.552044
20 19.95918 11.94923 12.98574 7.94118
25 20.21198 12.51266 13.06077 8.120508

Degradation behaviour
To determine the degradation behaviour of the hydrogels, the mass of the hydrogels was noted after immersion for about 48 hours in PBS. The degradation started after an equilibrium swelling and the remaining mass of the hydrogels was recorded at the selected time intervals (up to 20 days). The mass remaining (in %) was calculated by the following equation.

Remaining gel (%)=(W_0-W_t)/W_0 ×100

Where, W_0 and W_tare the weights of hydrogel after 48 hours and after time t, respectively.

Figure 7b clearly shows that there was no effective mass loss after about 15 days of immersion of hydrogels in PBS. The least damage was observed for PAM-TiO2-CNT hydrogel with highest fraction of remaining gel (~90%) after about 20 days when compared to that of PAM-TiO2 (remaining gel ~80%), PAM-CNT (remaining gel ~83%) and pure PAM (remaining gel ~75%). The hydrogel having highest water absorption exhibited enhanced hydrolysis and thus degradation, while increased rigidity in the polymer based composite hydrogels of the present disclosure (with less amount of water) was found to facilitate lower degradation.

Table4: Degradation rate of the samples (% weight remaining)

Time (days) PAM PAM-TiO2 PAM-CNT PAM-TiO2-CNT
1 88.88±1.9 93.48±0.7 93.17±0.1 96.21±0.5
3 85.19±0.5 88.27±1.8 91.09±0.9 94.22±0.8
5 83.23±0.9 85.36±2.7 86.65±2.1 91.65±0.0
7 78.09±0.8 82.53±1.4 84.27±3.0 89.58±0.7
15 73.62±1.9 79.96±0.8 82.28±1.5 88.50±0.4
20 72.24±2.6 78.73±0.7 81.38±1.3 88.23±0.5

EXAMPLE 6: Compression experiment analysis

Mechanical properties of a material are important for load-bearing applications. In order to analyze the mechanical strength of the hydrogels, the hydrogels of Example 1 were subjected to a compression experiment. The compression test was performed on the hydrogels with a diameter 4.5 mm and height of 6 mm using a universal machine (Magnum Pvt. Ltd, India). The constant rate of compression was 3.33 mm/min.

The stress-strain curve (Figure 8) prepared based on the observations from these experiments shows that the pure PAM exhibited lower compressive strength (0.15 MPa) and was fractured at ~61 % strain. However, the addition of nanoparticles into the gel enhanced the strength of the hydrogel (Table 5). For instance, the TiO2 and CNT added composites (PAM-TiO2 and PAM-CNT) displayed high strength (~0.37 MPa) without rupturing even at ~95 % and 65 % strain (Figure 8), respectively while the highest strength was shown by PAM-TiO2-CNT composite, sustaining the highest strain (more than ~0.43 MPa) without rupturing.

The curves of the composite hydrogel, as presented in Figure 8, is a typical ‘J’ shape, which are specified for natural tendons and ligaments.

The elastic modulus was calculated in two regimes in the curve: the first elastic modulus was calculated at low strain (Elow, 0-10 %) and the second modulus was calculated at high strain values (Ehigh, 85-90 %). The elastic modulus was significantly higher for PAM-TiO2-CNT composite at high (2.340 MPa) as well as low (0.027 MPa) strain than that of other composites (Table 5).

Table 5: Elastic modulus at low and high strain, compressive strength of the PAM, PAM-TiO2, PAM-CNT and PAM-TiO2-CNT hydrogel composites.
Samples Elow(MPa) Ehigh (MPa) Compressive strength (MPa) Maximum strain (%)
PAM 0.011±0.01 0.091±0.02 0.148 61
PAM-TiO2 0.024±0.00 1.599±0.1 >0.37 95
PAM-CNT 0.013±0.01 1.503±0.2 >0.31 65
PAM-TiO2-CNT 0.027±0.01 2.340±0.4 >0.43 96

EXAMPLE 7: Needle insertion test

A displacement-controlled drive was used for needle insertion experiments with a hypodermic needle. The insertion force was measured with a force sensor (1 mN resolution) while the needle displacement was recorded by a linear variable differential transformer, connected to a linear reciprocation system. The setup was made by mixing the samples on a shaft along with a camera to record the insertion video. The needle insertion speed was kept constant at 0.33 mm/s and the distance of the needle from the surface of hydrogels was 15 mm.

The hydrogels prepared in Example 1 were subjected to the needle insertion test. The force versus displacement plots for needle piercing through different gel revealed that the insertion force acts as the function of time (or displacement). The force changes in steps due to conical crack formation resulting in a sharp fall in insertion force on the needle.

In the case of pure PAM, the total insertion force reached about 0.16 N to 0.25 N before it sharply fell to about 0.06 N (Figure 9). However, for PAM-TiO2-CNT sample, after the same insertion, depth of ~12.5 mm, the highest force was only up to ~0.035 N force and remained ~0.02 N throughout the rest of the displacement. This indicated that over the same length of displacement, the occurrence of conical cracks is much less than that for PAM (7 cracks in 12.5 mm displacement).

Higher insertion force (in case of PAM) is indicative of higher stress in the region of the gel around needle thus, higher damage to the gel. The first step of force represents the resistance provided by the material surface. Since, for PAM-TiO2-CNT based samples, the occurrence of conical cracks reduced significantly (1 shallow conical crack in about 12.5 mm displacement), this showed that the internal damage in the hydrogel of the present disclosure was the least, exhibiting similarity to tissue repair or least damage in cartilage tissue.

The maximum net force was observed for pure PAM gel, which showed maximum resistance forces generated in the gel along the needle. This shows once the tip of the needle was inserted, it was hardest to move through pure PAM, which also internally cracked the most. For PAM-TiO2 and PAM-CNT samples, the conical cracks were reduced by almost three times when compared to that of PAM.

The cutting force is proportional to displacement. Net force for the first crack in pure PAM hydrogel (Figure 9) was highest (about 0.16 N, crack 1) which was ~4 times that for PAM-TiO2-CNT samples (about 0.04 N), ~1.6timesthat for PAM-CNT (about 0.10 N) and ~5 times that for PAM-TiO2 (about 0.030 N). This was also responsible for the lessening of the number of conical cracks and internal damage in PAM-TiO2-CNT samples as the lessening of friction force at the interface lessens shearing of the gel at the interface.

Therefore, from needle insertion experiments, it was clear that a significantly lower friction force on insertion of needle was obtained for all three composites compared to that of pure gel, which can sustain much higher stress without failure, making it a viable replacement for human tissues or cartilage.

EXAMPLE 8: Analysis of Self-healing and biomineralization properties

The self-healing experiments were conducted by using surface profilometry after the needle insertion test. Self-healing is an essential performance for any bone/cartilage implant. The testing of self-healing was demonstrated by using surface profilometry test on the hole formed on needle inserted samples (after about 24 hours of insertion) and the self-recovered depth was thereafter observed. The self-healing in the hydrogel was also assessed by cutting the sample (PAM-TiO2-CNT sample) into two halves and then the two parts were placed in a Petri dish. Few drops of water were poured on the fractured surface of the hydrogel. The compression test was further performed on the healed sample.

The bioactivity of the bone implant materials is generally specified by the capacity to induce calcification that can endorse nucleation and proliferation of calcium phosphate crystals (apatite). This characteristic of prepared hydrogel composites was explored by immersing the samples in simulated body fluid (SBF) at about 37℃ for about 48 hours. The formation of apatite crystals was observed by SEM and XRD.

The initial depth (total displacement during needle insertion experiments) of the created hole was about 5 mm (Figure 10) for all the samples.

To measure the self-healing performance of the samples, the depth of the hole was measured after pouring about 2-3 drops of deionised water and it was found that the samples were healed automatically, and the initial depth was recovered (Figure 10). The depth was observed to be about 115μm for PAM (Figure 10a), while it was much reduced (Figure 10b, c) for two-component polymeric composite hydrogels (about 70 μm for PAM-TiO2 and about 75 μm for PAM-CNT composites). The recovery of depth/self-healing performance for the PAM-TiO2-CNT hydrogel was found to be maximum, wherein the depth was reduced to about 25 μm. (Figure 10d), which can be attributed to the strong mechanical interaction and bonding between nanoparticles and PAM matrix.

The self-healing property was further confirmed by cutting the self-healed PAM-TiO2-CNT hydrogel and performing compression test on the sample after healing. The self-healed hydrogel exhibited considerable compressive strength (>0.258 MPa), which was significantly higher than that observed for the pure PAM hydrogel (about 0.148 MPa, Table 5). Moreover, the elastic modulus of the self-healed PAM-TiO2-CNT hydrogel at lower and higher strain was calculated to be about 0.011 MPa and about 0.24 MPa, respectively.

Hydration can alter the surface morphology, triggering self-healing of the material. After pouring water on the damaged surface, the network of polymeric structure swells and expands by osmosis as per the Gibbs-Donnan effect. The shrinking of the pores happens through capillary action, making a compact hydrogel structure. The more severe damage results in less recovery by self-healing. Thus, PAM-TiO2-CNT hydrogel, having least damage during needle insertion, showed maximum recovery (showing the reduced depth of insertion about 25 μm). The re-inforced polymer composite based hydrogel of the present disclosure was therefore found to be a good cartilage substitute with ability to stimulate the natural healing in the surrounding tissue.

In addition to the above, the bioactivity of a specific cartilage implant was examined by in vitro apatite formation ability in simulated body fluid (SBF). The apatite is an essential part for providing support to cartilage tissue and to promote osteogenic tissue formation and bone integration. The apatite growth on the hydrogels occurs in two steps: in the first step, the hydrogels supplemented with calcium and phosphate in SBF until reached with super-saturation. In the next step, the apatite crystals started depositing on the hydrogel surface from the super-saturated fluid. Figure 11 shows the apatite crystals within the hydrogel surface. The apatite was higher in concentration for composite hydrogels (Figure 11 b,c,d) than that of PAM (Figure 11 a). The peak of apatite in XRD (Figure 12) further confirmed the formation of apatite in the hydrogel samples.

Strong apatite peaks were clearly visible for the PAM-TiO2-CNT composite, referring to formation of larger amount of apatite in the hydrogel of the present disclosure. This may be attributed to the synergistic effect of TiO2 and functionalized CNTs. Thus, the composite hydrogels were found to have potential to show signs of osteoconduction after implantation along with the self-calcification in the body. The high content of apatite in composites hydrogels (mainly in PAM-TiO2-CNT) was due to the presence of TiO2 and functionalized CNTs. The Ti-O and –COOH groups present in the hydrogel can promote heterogeneous nucleation of amorphous calcium phosphate. Therefore, the designed PAM hydrogel with encapsulation of TiO2 and CNTs was found to be bioactive and have the potential to accelerate osteointegration once applied in vivo.

EXAMPLE 9: Computational analysis

In order to understand the interfacial interaction of the TiO2 with functionalized CNT in the PAM based hydrogel (i.e. PAM-TiO2-CNT hybrid hydrogel), computational studies, particularly, the DFT approach was implemented and the hydrophilicity behavior was studied comprehensively. Further studies on structural stability, interaction energy (IE), inter- and intramolecular NCIs, and electronic feature analyses, systematic computational experiments were also carried out.

Quantum computation was deployed by considering three starting models (monomers as PAM, TiO2, and CNT), two dimers (PAM-TiO2 and PAM-CNT) constructing bonding and nonbonding interfacial interactions and a trimer complex (PAM-TiO2-CNT) which was found to form much stronger interfacial interactions as compared to both the dimer complexes containing inter- as well as intramolecular NCIs. All model complexes were optimized in the framework of the DFT using Gaussian 09 electronic structure calculations package. The quantum chemical calculations (QCCs)were employed using the B3LYP/6-31G level approach. For all model composites, all frequencies were observed positive for all of the minima in the potential energy surface. NCI-plots prepared on the basis of the studies provide insight on the kind, nature and strength of interactions involved in the hydrogels. Additionally, hydrogen bond strength based interaction coordinate (HBSBIC) was also relied upon while quantifying the strength of H-bonding interactions probed in all species – i.e. monomers and dimeric and trimeric complexes.

At first, using the molecular modelling approach (MMA) (i.e. a theoretical model) and QCCs, two dimer complexes PAM-TiO2 and PAM-CNT were chosen and optimized where consideration of the first interfacial interactions in the former was taken between the PAM and TiO2 monomer units and the second interfacial interactions were analyzed between the PAM and CNT monomer units in PAM-CNT. Such modeled PAM hydrogel in combination with the TiO2 (i.e. PAM-TiO2) as well as CNT (i.e. PAM-CNT) were found to be stabilized by the intermolecular noncovalent interactions (NCIs) whereas the PAM hydrogel model species was found to be self-stabilized because of having intramolecular N-H⋯O and C-H⋯O NCIs as well as weak vdW interactions.

Analysis of the binding features of the PAM-TiO2-CNT composite by the molecular modelling approach (MMA)showed subtle details about the formation of several NCIs between the PAM and TiO2 monomer units as well as between the PAM and CNT monomer units.

Analysis of geometric and electronic features
Figures 13, 14 and 15 show analysis for the monomer units (CNT, TiO2 and PAM), dimer (PAM-CNT and PAM-TiO2), and trimer PAM-TiO2-CNT compositions, respectively with proper atomic labeling.

Some geometrical parameters related to the H-bonded three atoms D-H⋯A (where D is the proton donor and A is the proton acceptor) fragments of all three model composites like the covalent bond distances (CBD), hydrogen bond distances (HBD), and bond angles are provided in Table 6.

In order to maximize the number of NCIs involved between both connecting fragments (first one is the interaction(s) between the PAM and TiO2 and the second one is the interaction(s) between the PAM and CNT), a careful modeling was done in the parent geometrical structures of all three monomer units followed by the optimization and frequency calculations showing all minima in the potential energy surface. In attaining an appropriate as well as the most favorable binding interactions involved in the dimer and trimer assemblies, the MMA was employed from a single side of the TiO2 and CNT monomer units however, it was done from both sides for the PAM (Figure 15) keeping in mind various experimental facets.

The H-bonded structural parameters showed that the dimer complex PAM-CNT contains two types of interactions N-H⋯O (N-H CBD: 1.027 Å; O⋯H HBD: 1.85 Å; ∠NHO: 171.3°) and O-H⋯O (O-H CBD: 1.064 Å; O⋯H HBD: 1.447 Å; ∠OHO: 177.6°) occurring between the PAM and CNT constituents. Said analysis revealed that the O-H⋯O H-bonding was stronger than the N-H⋯O H-bonding interaction. The H-bond associated N-H CBD of the PAM monomer (i.e. associated with the CNT) and O-H CBD of the CNT monomer (i.e. associated with the PAM) were calculated as 1.013 Å and 0.983 Å, respectively which are smaller than their corresponding CBDs (N-H of the PAM: 1.027 Å; O-H of the CNT: 1.064 Å) found in the dimer complexes. Two strong metal-nonmetal interactions (MNIs) (Ti-O and Ti⋯N) (where Ti atom is the member of the TiO2 constituent and the O and N atoms are the parts of the PAM constituent) were seen in the case of PAM-TiO2 dimer complex and these interactions appear to have a prime role in stabilizing this complex. The Ti-O and Ti⋯N bond lengths were computed as 2.044 Å and 2.303 Å, respectively where Ti-O bonding interaction seemed to demonstrate higher strength than the Ti⋯N MNI. Three NCIs like one N-H⋯O (N-H CBD: 1.023 Å; O⋯H: 2.314 Å; bond angle: 105.1°) and two C-H⋯O (C-H CBDs: 1.091 Å and 1.1 Å; O⋯H HBDs: 2.175 Å and 1.166 Å; bond angles: 159.6° and 158.7°) weak H-bonding interactions were also seen in the PAM-TiO2 dimer complex which play a role to stabilize it further.

Table 6: Parameters of all the hydrogel species optimized at B3LYP/6-31G Level of Theory
Parameter Dimer Complex Trimer Complex
PAM-CNT PAM-TiO2 PAM-TiO2-CNT
Intermolecular H-bond Associated Covalent
Bond N-H (1.027Å)
O-H (1.064 Å) N-H (1.023 Å),
C-H (1.091 Å, 1.1 Å)

Metal-Nonmetal Bond
Ti-O (2.044 Å), Ti⋯N (2.303 Å) PAM-CNT Dimer Unit
N-H (1.028 Å), O-H (1.051 Å)
PAM-TiO2 Dimer Unit
C-H (1.092 Å, 1.099 Å),
N-H (1.022 Å)

Intermolecular H-Bond N-H⋯O (1.85 Å)
O-H⋯O (1.447 Å) N-H⋯O (2.314 Å),
C-H⋯O (2.175 Å, 2.166 Å)
PAM-CNT Dimer Unit
N-H⋯O (1.82 Å)
O-H⋯O (1.486 Å)
PAM-TiO2 Dimer Unit
N-H⋯O ( 2.31 Å)
C-H⋯O (2.155 Å, 2.186 Å)
Metal-Nonmetal Bond
Ti-O (2.035 Å), Ti⋯N (2.306
Å)
Bond Angle ∠NHO (171.3°)
∠OHO (177.6°) ∠NHO (105.1°),
∠CHO (159.6°, 158.7°) PAM-CNT Dimer Unit
∠NHO (170.2°), ∠OHO (177°)
PAM-TiO2 Dimer Unit
∠NHO (104.2°),
∠CHO (163.2°, 153.2°)
Hydrogen Bond Strength (HBSBIC) N-H⋯O (0.279)
O-H⋯O
(0.516) N-H⋯O (0.017),
C-H⋯O (0.242, 0.192) PAM-CNT Dimer Unit
N-H⋯O (0.282),
O-H⋯O ( 0.468)
PAM-TiO2 Dimer Unit
N-H⋯O (0.018),
C-H⋯O (0.255, 0.168)
Monomer
PAM CNT TiO2
CNT Associated N-H Bond (1.013 Å); TiO2 Associated C-H (1.091 Å, 1.095 Å) and N-H (1.009 Å) CBDs O-H (0.983 Å) Ti-O (1.804 Å -1.866 Å),
Ti-Ti (2.683 Å -2.741 Å),
∠TiOTi (94.2°-95.3°),
∠OTiO (83.9°-87.4°)

The H-bonded structural parameters shown in Table 6for the PAM-TiO2-CNT trimer complex speak well about the interfacial interactions found in both the PAM-TiO2 (Ti-O and Ti⋯N MNIs as well as one N-H⋯O and two C-H⋯O weak H-bonding interactions) and the PAM-CNT (one O-H⋯O and one N-H⋯O strong H-bonding interactions) dimer units which bind all three monomer constituents together strongly and make it the most stable composite. As compared to the Ti-O bond length (2.044 Å) of the PAM-TiO2 dimer complex, the Ti-O bond length (2.035 Å) in the trimer complex (PAM-TiO2-CNT) was found to be decreased and showing its stronger binding characteristics. Reduction in the intermolecular N-H⋯O HBD (1.82 Å) of the PAM-TiO2-CNT composite model (i.e. the HB existing between PAM and CNT dimer fragment) was observed in comparison to that of the PAM-CNT dimer complex (1.85 Å), demonstrating a stronger interaction in the former one. The HBSBIC values of the N-H⋯O interactions probed in PAM-CNT and PAM-TiO2-CNTwere calculated to be 0.279 and 0.282, respectively which strongly support the geometry/structure criteria based quantification of the hydrogen bond strength.

A marginal decrease in the N-H⋯O HBD (2.31 Å) (HB lying between the PAM and TiO2 dimer segment) of the trimer composite (PAM-TiO2-CNT) was found which was slightly lower than that (2.314 Å) of the PAM-TiO2 dimer complex. Such slight difference in HBDs (0.004 Å) of the N-H⋯O detected in the dimer unit of the trimer and dimer complex also gave a minor change (0.001) in the HBSBIC values - 0.018 and 0.017, respectively. The PAM-TiO2 associated N-H⋯O interactions in both (PAM-TiO2 dimer complex and PAM-TiO2 dimer unit of the trimer) show much weaker interactions which could be due to their quite longer bond distances (2.31 Å and 2.314 Å) as well as very small bond angles (104.2°, 105.1°). Such interactions are much weaker than the N-H⋯O HBs found in the PAM-CNT dimer complex as well as the PAM-CNT dimer unit of the trimer complex. Along with the above highlighted comparison, a few other H-bonded structural parameters like two weak C-H⋯O HBs acquired in the trimer complex also demonstrated its enhanced stability (see Table 6).

Some select energy-based parameters are displayed in Table 6.In order to achieve better insights into the stability pattern, the interaction energy (IE)was analyzed using the density functional theory (DFT) approach. First, the IEs of the dimer complexes PAM-TiO2 and PAM-CNT were calculated to be -46.5 kcal/mol and -31.9 kcal/mol, respectively where the IE of the former was found to be about 1.5 times greater than the latter. Said finding shows the higher stability of the PAM-TiO2 dimer complex as compared to the PAM-CNT dimer complex. This appears to be due to stronger Ti-O and Ti⋯N MNIs as well as a few weak H-bonding (one N-H⋯O and two C-H⋯O) interactions involved in the PAM-TiO2 complex whereas only two H-bonding interactions (one O-H⋯O and one N-H⋯O) have been detected in the case of PAM-CNT dimer complex.

Further, IE value of the trimer complex (PAM-TiO2-CNT) was calculated to be about -81.9 kcal/mol (highest – therefore indicating the most stable complex) which was much larger than both the dimer complexes (PAM-TiO2 and PAM-CNT). Said high IE value of the trimer complex results from favourable orientations such as strong metal-nonmetal and hydrogen bonding in the complex. By taking into consideration all the above highlighted three analyzed IE outcomes, the trimer complex (PAM-TiO2-CNT) was found to be the most stable species.

NCI-RDG Isosurface Plots
Recognition and graphical visualization of the NCIs regions were produced by means of the NCI-reduced density gradient (NCI-RDG) approach in association with the nature and strength involved therein, which permits an inclusive description of hydrogen bonds (HBs), vdW interactions, and steric repulsion in all model complexes. Usually, the localized blue lentils describe the strong attractive interactions (i.e. hydrogen bond) (represented by arrow in Figure 14) and thin as well as delocalized green regions illustrate the vdW (extremely weak) (indicated by arrow) interactions. H-bonded as well as vdW interactions involved in all figures (2D scattered plots and 3D isosurface) have been represented by black arrows in the figure.The red isosurface(lying right side of zero value) exposes the steric clashes engaged in the species (for example: see Figure 14, right).The RDG isosurface displayed on horizontal axis is 0.5 (ranging from -0.05 to +0.05). The Ω(r) values ranging from -0.035 atomic unit (au) to +0.02 au are shown on vertical axis (Figure 14, right). The detailed description of the NCI tool clarifies that the higher density values (Ω(r) < 0) show the stronger attractive interactions while the very low-density values (Ω(r) > 0) indicate the repulsive interactions. Moreover, for example, in the case of H-bonded PAM monomer, blue color spike (values lying between -0.024 and -0.03 au in the scatter map two-dimensional (2D) plot) and blue colored disc-shaped NCI isosurface 3D representation showing two N-H⋯O interactions where O atom acts as proton acceptor and N atom acts as proton donor signifying the attractive interaction give a clear indication of presence of intramolecular HBs (N-H⋯O) (stabilizing interaction). The green color spikes (values ranging from -0.005 au to -0.015 au) in the 2D scatter plot and green color disc-shaped NCI 3D isosurface demonstrate a variety of vdW interactions involved in the PAM monomer. The presence of steric effect is evidently shown by the low-gradient spikes appearing at positive side (+0.005 to +0.015) (see Figure 14, right). This effect as shown by the red ellipsoid depicts the electron density depletion which is because of the electrostatic repulsion.

The NCI 2D scatter plots and 3D isosurface maps of the dimer and trimer complexes clearly showed the kind of proton acceptors and proton donors they are (see Figures 13forPAMmonomer, 14for the dimer and 15for the trimer complexes). In the case of PAM monomer, two intramolecular N-H⋯O H-bonding interactions could be seen (see 3D-isosurface of Figure 13). Some vdW type interactions (isosurface in green color scheme) are shown in the same Figure. Figure 14 explains the interactions detected in the PAM-TiO2 dimer where mainly five interactions were seen in which two strong MNIs as Ti-O (where Ti atom is the part of the TiO2 monomer unit and O atom is the part of the PAM monomer unit) and Ti⋯N (where Ti atom belongs to the TiO2 and N atom belongs to the PAM) as well as three weak (one N-H⋯O and two C-H⋯O HBs (where the proton donor C-H bond belong to the PAM monomer unit and the proton acceptor O atom belong to the TiO2 monomer unit)) were observed.

Further, NCI 3D isosurface and 2D scatter plots of the dimer complexes PAM-CNT and PAM-TiO2 were generated. In the case of the combination of the PAM and CNT (PAM-CNT) species, one N-H⋯O and one O-H⋯O HBs (in blue color disc) can be seen in Figure 14 (top middle) between the PAM and CNT monomer units using the NCI plot tool (3D isosurface) which are responsible for the construction of the dimer complex and make it stable. Moreover, the NCI 2D scatter plot also confirmed the above said N-H⋯O HBs (blue color spikes) for the values range between -0.02 au and -0.03 au. Apart from that, two intramolecular HBs (two N-H⋯O) (in greenish-blue color discs),vdW type interactions (in green color isosurface) were probed in the PAM fragment of the PAM-CNT dimer complex. Such intramolecular type interactions found in the PAM segment were further verified by the 2D scatter plot where the green color peaks ranging from -0.005 au to -0.02 au clearly show the vdW type interactions.

As shown in Figure 14(bottom), in the case of PAM-TiO2 dimer complex, two strong MNIs (Ti-O and Ti⋯N), one moderate N-H⋯O, and two weak C-H⋯O HBs were observed using the NCI 3D isosurface and 2D scatter plot analyses. In Figure 14(bottom), a strong Ti-O MNI can fairly be affirmed by the NCI 3D isosurface (middle, solid bond) and NCI 2D scatter plot (right, dark blue color peak lying close to -0.045 au) diagrams. Also, the Ti atom of the TiO2 monomer unit and O atom of the PAM monomer are strongly connected to each other as shown in solid line. Similarly, a strong Ti⋯N MNI was also detected from the NCI 3D isosurface (blue color disc) and 2D scatter plot (blue color spike lying around -0.03 au) where the Ti atom of the TiO2 fragment and N atom of the PAM segment are linked to each other. Three more weak HBs (one N-H⋯O and two C-H⋯O) are also shown in the NCI plot (3D isosurface) (three green color discs represented by black dashed lines) and NCI (2D) scatter plot (three greenish-blue color spikes ranging from -0.015 au to -0.025). An evidence of existence of vdW type interactions was detected as shown in the NCI 3D isosurface (multiple green color isosurface) and 2D scatter plot (green color peaks ranging from -0.005 au to -0.012 au) analyses. The NCIs observed in the above two dimer complexes showed that the interfacial NCIs analyzed in the PAM-TiO2 complex make it more stable than the PAM-CNT complex (vide-infra).

Finally, to further analyze the interfacial NCIs engaged in the PAM-TiO2-CNT trimer complex consisting of two interfaces (see Figure 15), an MMA based theory was adopted where the PAM monomer unit exists between the TiO2 and CNT constituents, forming a variety of the bonding and nonbonding interactions. From Figure 15, one can note that the NCI-plots provided a relevant information about the PAM constituent bridged between two other constituents TiO2 and CNT monomer units where the PAM-CNT dimer unit was found to be connected by the two monomer units PAM and CNT via two moderate N-H⋯O and O-H⋯O interfacial H-bonding interactions and the PAM-TiO2 dimer unit was found to be linked by the two strong Ti-O and Ti⋯N MNIs, one N-H⋯O, as well as two weak C-H⋯O HBs.

Looking into NCI (2D) scatter plot (top right in Figure 15) of the trimer complex (PAM-TiO2-CNT), the blue color spikes confirm the presence of strong Ti-O and Ti⋯N MNIs (ranging between -0.035 au to -0.05 au) as well as one moderate N-H⋯O and two weak C-H⋯O NCIs (greenish-blue spikes lying between -0.015 au and -0.025 au) existing between the PAM and TiO2 constituents. Similarly, existence of strong O-H⋯O and N-H⋯O HBs between the PAM and CNT constituents was also confirmed by the 2D scatter plot (greenish-blue spikes lying between -0.018 au and -0.024 au) as well as the 3D isosurface (blue color discs). Hence the Ti-O and Ti⋯N MNIs as well as the O-H⋯O and N-H⋯O HBs were found to play a prime role in strongly stabilizing the composite (trimer complex) which is constructed between the metal and nonmetal positions (PAM-TiO2 dimer unit) as well as the HB donor and acceptor positions of two dimer units (one N-H⋯O and one O-H⋯O for the PAM-CNT and one N-H⋯O for the PAM-TiO2 dimer units). Some vdW interactions were also found to act as supplementary sites in stabilizing the trimer complex.

The NCI 2D and 3D plot-based images and related graphical envisions provide detailed information about the nature and kind of NCIs taking part therein both the dimer as well as trimer composite models. The probed NCIs including vdW interactions showed that the trimer composite is more stable than both dimer composites. Such findings are also supported by the HOMO-LUMO gap values reported below.

Frontier Molecular Orbital (FMO)
The energy difference between the highest occupied molecular orbital (HOMO) energy (EHOMO) and lowest unoccupied molecular orbital (LUMO) energy (ELUMO) is known as the HOMO–LUMO energy gap (HLEG). In the frontier molecular orbital (FMO) theory, these HOMOs and LUMOs have been sometimes commonly termed the FMOs. The HLEG gap between such two FMOs can be deployed in predicting the strength and stability of the chemical species. Species approaching zero HLEG value absorb light efficiently. As the HLEG is directly connected to valence and conduction band, the electron in the HOMO would easily move into the LUMO if the value of the HLEG approaches zero (i.e. it needs little energy and electrons are free to move showing high electrical conductivity). HLEG also helps describe the chemical behavior of any molecular system with lower HLEGs usually being indicative of higher reactivity (i.e. lower stability) of the particular species since they frequently have some interesting optical features. Moreover, a larger HLEG implies that in order to excite the electron in the HOMO, light of higher energy is required when the excited electron relaxes back to the HOMO. The EHOMO and ELUMO are normally associated with the electron-donating and electron-accepting abilities of a molecule, respectively from which the global chemical hardness is calculated. Additionally, a system having a small HLEG value signifies its tendency towards easy photo-chemical excitation.

The HOMO-LUMO surface maps can be discerned from Figure 16. The HOMO-LUMO plots of the PAM-TiO2 species shows that the HOMOs (in plane) are primarily spread over almost all the six-membered rings of the CNT fragment whereas the LUMOs (out of plane) are located over only three six-membered ring of the CNT along with minor contribution on another six-membered ring of the same. Transition from π to π* appears to take place in the case of PAM-CNT dimer complex. A small contribution of the HOMOs (in plane) of the PAM-TiO2 complex seems to be close to two terminal Ti atoms while the LUMOs were seen close to all four Ti atoms (out of plane) and showing π to π* transition. Like the PAM-CNT dimer complex, the HOMOs (in plane) were positioned over almost all the six-membered rings of the CNT fragment and the LUMOs (out of plane) were found to be close to only three six-membered ring of the CNT, illustrating π to π* transition.

Some key electronic parameters are provided in Table 7.

Table 7: Binding Energy and Electronic Property Based Parameters of All the Species Optimized at B3LYP/6-31G Level of Theory
System BE (kcal/mol) EHOMO (eV) ELUMO (eV) EGap(eV) Hardness Dipole Moment (Debye)
PAM-TiO2 -46.5 -4.136 -2.737 1.399 0.7 9.1
PAM-CNT -31.9 -5.524 -3.766 1.758 0.879 6.7
PAM-TiO2-CNT -81.9 -5.755 -3.984 1.771 0.886 11.3

As can be seen from the above table, the HOMO–LUMO energy gap (HLEG) is the highest for the PAM-TiO2-CNT composite, therefore being depictive of higher stability as compared to PAM-TiO2 and PAM-CNT composites. The trimer complex which showcases the highest HLEG value was thus found to be the most stable and least chemically reactive species. Based on aforesaid HLEG values, the PAM-TiO2 dimer complex was found to have the lowest HLEG value and it is therefore the most chemically reactive (or least stable) species.

The electronic parameter of hardness value also clearly demonstrates that the stability order is PAM-TiO2-CNT (0.886) > PAM-CNT (0.879) > PAM-TiO2 (0.7).

Molecular Electrostatic Potential (MEP) Surface
The molecular electrostatic potential (MEP) simultaneously exhibits the molecular size, shape and charge distribution and it is connected to the parameter of electron density. MEP has broadly been employed in understanding relative polarity, NCIs, electrophilic and nucleophilic attack; providing for the recognition of molecular systems. The MEP maps of the monomer constituents, dimer, and trimer composites can be seen in Figure 17.

The three-dimensional MEP surface plot helps in understanding the reactivity of the species. The electron poor region (i.e. reactive site) is highlighted in blue color scheme which show absolute positive charge distribution as a high probability of nucleophilic attack may be possible. The green color scheme illustrates about the neutral region. The red color scheme is the diagnostic of electrophilic attack which illustrates the strong negative region (absolutely negative charge distribution). Figure 17 shows that the negative regions are located over the CNT constituent while positive regions are positioned over the NH2 group of the PAM constituent in all cases and the H atoms of the COOH groups of the CNT alone as well as PAM-CNT dimer complex. As reported in the below section, natural population analysis (NPA) conducted on the hydrogels also confirmed that the CNT and TiO2 constituents in the dimer and trimer complexes contain negative charge while the PAM associated with the dimer and trimer shows positive charge.

Natural Population Analysis (NPA)
Natural population analysis (NPA) by performing QCC is a broadly used technique in analyzing the electron population of each atom of any molecular species to analyze charge distribution on the nuclei, bond order, and other related information. In all three cases (two dimer and one trimer complexes), PAM constituent individually contained positive charge while the TiO2 and CNT constituents individually had the negative charges.

The PAM and CNT had positive (0.119e) and negative charges (-0.119e), respectively, revealing that the charge transfer can take place from PAM to the CNT constituent in its dimer complex PAM-CNT. Similarly, in the case of PAM-TiO2 dimer complex, the PAM having positive natural charge of 0.353e and the TiO2having a negative natural charge of -0.353e appeared to show the charge transfer phenomenon from PAM to the TiO2 constituent.

Finally, analysis of the combination of all three constituents forming a composite of trimer complex showed that the PAM monomer unit had a positive natural charge as 0.437e whereas both the TiO2 and CNT monomer units had negative natural charges as -0.366 and -0.071e, respectively. The order of charges (positive and negative) analyzed in all three species was PAM-TiO2-CNT (0.437e, -0.437e) > PAM-TiO2 (0.353e, -0.353e) > PAM-CNT (0.119e, -0.119e).

This finding confirmed that the strongest stabilizing interaction occurs in the trimer complex as there is the highest charge difference between the positive (0.437e) and negative (-0.437) natural charges. Such outcomes demonstrate that the trimer complex (PAM-TiO2-CNT) is the most stable among all three hydrogel species - said observation being further supported by the IE values and NCI plots.

Thus, from the above discussion, it is clear that the addition of TiO2 and CNTs in PAM matrix can alter the hydrophilicity, mechanical properties, self-healing and bioactivity in an effective manner. Figure 18 is the schematic representation of the summary of the present work, which shows that four types or species of hydrogels were subjected to compression and needle insertion tests. The PAM hydrogel alone exhibited comparatively lower compressive stress and puncture resistance whereas PAM-TiO2-CNT displayed highest mechanical strength. The self-healing performance, bioactivity and cytocompatibility in vitro were also found improved in PAM-TiO2-CNT (Figure 18) due to the strong interfacial strengthening by TiO2 and CNT nanoparticles with PAM.

From the computational studies it was observed that functionalized CNT acted as a reactive site for PAM and TiO2 in PAM-TiO2-CNT composite hydrogel and furnished strong metal-nonmetal Ti-O-C, Ti⋯N and O-OH hydrogen bonding altogether. Thus, the PAM-TiO2-CNT nanocomposite hydrogel was found to be an efficient biomaterial to consider for applications such as but not limited to cartilage repair applications.

A summary of all of the above, drawing a comparison between the dimer and trimer-based hydrogels is provided in the below table (Table 8).

Table 8: Comparison between dimer and trimer-based hydrogels
Parameter PAM hydrogel PAM-TiO2 PAM-CNT PAM-TiO2-CNT
Top surface morphology Smooth surface Dispersed TiO2 nanoparticles could be observed for PAM-TiO2 sample CNTs were embedded within the PAM matrix Combination of TiO2, as well as CNTs in the PAM matrix, was clearly visible in PAM-TiO2-CNT hydrogel, wherein both TiO2 and CNTs were encapsulated in the PAM matrix
Retention of phases in the hydrogels as confirmed by XRD analysis Pure polyacrylamide hydrogel (PAM) showed broad humps, indicating the amorphous nature of PAM Characteristic TiO2 peaks were visible CNTs peaks are not visible Characteristic TiO2 peaks were visible; CNTs peaks are not visible
FTIR analysis Strong vibration bond at ~3337 cm-1 was observed due to O-H bond while the characteristic N-H stretching vibration was observed at ~ 2953 cm-1 due to the presence of amide group in PAM hydrogel Shoulder peaks at ~3188 cm-1 in PAM-TiO2

Absorption peaks at ~1008 cm-1 and 1310 cm-1 Shoulder peaks at ~3186 cm-1 in PAM-CNT

A broader band from 2953 cm-1 to 2925 cm-1
Strong metal-nonmetal bonding of the PAM with the TiO2 (Ti-O-C) as well as H-bonding with functionalized CNTs (-O-OH).
Cytotoxicity (% cell viability after exposure) Cell viability of more than about 98%. Cell viability of more than about 98%. Cell viability of more than about 98%. Highest cell viability (100 %)

Hydrophilicity (contact angle) ~70º ~54º ~44º ~40º (most hydrophilic)
Swelling behaviour equilibrium swelling after about 10 hours of immersion equilibrium swelling after about 10 hours of immersion equilibrium swelling after about 10 hours of immersion stable volume of PAM-TiO2-CNT hydrogel even after about 25 hours, represented non-swellable behaviour of the material

Degradation behaviour (fraction of gel remaining after immersion in PBS) ~75% ~80% ~85% ~90%
(maximum fraction remaining)
Compressive strength (MPa) 0.15 >0.37 >0.31 >0.43 (highest compressive strength)
Maximum strain (%) 61 95 65 96 (highest applicable strain)
Insertion force (N) 0.16to 0.25 0.030 0.10 ~0.035
Self-healing property (recovery of depthafter creation of a 5mm hole- μm) 115 70 75 ~25
Elastic modulus at low and high strain(MPa) 0.011±0.01; 0.091±0.02
0.024±0.00; 1.599±0.1 0.013±0.01; 1.503±0.2 0.027±0.01; 2.340±0.4 (Highest)
Ti-O bond length ― 2.044 Å ― 2.035 Å
Intermolecular N-H⋯O hydrogen bond distances (HBD) [PAM-CNT] ― ― 1.85 Å 1.82 Å

Interaction energy (IE) (kcal/mol) NA (Monomer (i.e. PAM) can never have interaction energy until it makes a complex by interacting with other monomer(s)) -46.5 -31.9 -81.9 (highest stability)
HLEG value (eV) ― 1.399 1.758 1.771 (highest stability and lowest chemical reactivity)
Hardness value NA 0.7 0.879 0.886 (highest stability)
Natural Population Analysis – charge difference between positive and negative natural charges NA
(No charges since PAM is a neutral monomer component.) 0.353e, -0.353e 0.119e, -0.119e 0.437e, -0.437e (highest order of charges. Therefore, strongest stabilizing interaction)
*NA – not applicable
Taken together, the above shows the enhanced suitability of the trimer complex based hydrogel of the present disclosure as compared to pure hydrogel or dimer based hydrogel. The above reported improvements in the properties of the trimer complex based hydrogel as compared to the dimer complex based hydrogel, however, are unexpected and surprising, since the enhanced content of nanoparticles (TiO2 and CNT) in the trimer complex as compared to the dimer complex would otherwise be expected to be associated with increased tendency to agglomerate and yield unfavorable properties.

The foregoing description fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the general concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein, without departing from the principles of the disclosure.

Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.