TECHNICAL FIELD OF THE INVENTION
[001] The present invention relates to a thermoresponsive injectable
biomaterial.
[002] In particular, the invention discloses the thermoresponsive injectable
biomaterial with application as a scaffold or filling material for bone
tissue, intervertebral disc or dental implant.
[003] Further, the invention relates to thermoresponsive hydrogel.
[004] Further, the disclosed biomaterial is sterile, injectable and ready to
use.
[005] Further, the said biomaterial enhances the process of bone tissue
regeneration and provides support to the damaged tissues such as
intervertebral disc.
BACKGROUND
[006] Low back pain is prevalent in society and is the number one cause of
disability globally. 60–80% of adults experience varying degrees of
low back pain (Murray et al., 2012; Hartvigsen et al., 2018).
Although the etiology and pathology of low back pain are complex,
evidence suggests that low back pain is strongly associated with
intervertebral disk degeneration (IVDD). The IVD gradually
degenerates due to aging and tissue damage caused by multiple
stressors, resulting in vertebral instability, spinal canal stenosis, and
spinal segment deformity, causing low back pain and mobility
disability (Wang et al., 2016).
[007] The available treatments aim at relieving pain but do not work on the
regeneration of damaged tissue. Such regenerative approaches
involve renewal of the damaged matrix by the active growth of matrix
secreting cells. However, the current surgical interventions add to
the sufferings of the patients as the surgeries result in collateral
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damage to neighboring tissues and eventually elevate the pain. There
are various materials that are been studied for their regenerative
properties like PLGA-PEG-PLGA triblock copolymer, Sulfonatednanocellulose (pNC), etc. these materials are known to change under
pressure, temperature, pH or photopolymerization treatment (UV
light) from a solution to a semi-solid/gel state known as sol-gel
transition.
[008] There are commercially available biomaterials such as (a) DentGist
NanoCom Restorative; (b) DentGist NanoCom; and (c) Sdi Wave
Flowable Composite MV, Flowable Composite are known for their
application as dental fillers and are used as binding material to treat
fractures in long bones. But these materials are not reported to be
used for the regeneration of tissue in the intervertebral disc (IVD).
[009] Hence there is a need for a biomaterial which can be used for
regeneration of bone tissue and provide support to the damaged
tissues and generate conducive microenvironment for growth and
survival of indigenous cellS.
Description:OBJECTS OF THE INVENTION
[0010]
It is an object of the invention to provide a thermoresponsive injectable biomaterial.
[0011]
It is another object of the invention to provide a method of preparing a thermoresponsive biomaterial.
[0012]
It is yet another object of the invention to provide a biomaterial, which is used to prepare the scaffolds or filling material, which is further used for regeneration of bone and cartilage tissues in intervertebral disc injuries and providing support to the damaged tissues.
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SUMMARY OF THE DISCLOSURE
[0013]
The present disclosure relates to a thermoresponsive injectable biomaterial, comprising of collagen, hydroxyapatite and polymer of chitosan-beta glycerophosphate-sodium bicarbonate.
[0014]
The present disclosure relates to a method of preparing a thermoresponsive injectable biomaterial, comprising of: a) dissolving chitosan in 0.1% acetic acid to prepare a homogenous mixture; b) autoclaving the chitosan mixture obtained at step a) at 121?, 15 psi for 15 mins; c) storing the chitosan mixture at 4? for 12hr before further processing; d) autoclaving Hydroxyapatite nanoparticles at 121?, 15 psi for 15 mins; e) sterilizing collagen under UV for 2 hours; f) dissolving Glycerophosphate solution in PBS containing sodium bicarbonate filter sterilized and store at 4? at least 2hr before use; g) mixing the chitosan mixture, the hydroxyapatite, and the collagen under sterile conditions at 4? until formation of homogenous mixture; h) adding glycerophosphate dropwise and mixed for 1hr at 4? to form biomaterial which is stored at 4? until used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015]
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 a detailed description below form a part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in
5
accordance with the present disclosure wherein:
[0016]
Figure 1 depicts a pictorial representation of a thermoresponsive injectable biomaterial.
[0017]
Figure 2 depicts a process for preparing a thermoresponsive injectable biomaterial.
[0018]
Figure 3 depicts cyto-compatibility of the biomaterial using MTT assay.
[0019]
Figure 4 depicts SEM analysis of hydrogel.
[0020]
Figure 5 depicts FTIR analysis of thermo-responsive hydrogel.
[0021]
Figure 6 depicts rheological analysis of thermo-responsive hydrogel.
[0022]
Figure 7 depicts the strength of the scaffold at room temperature at different strains.
[0023]
Figure 8 depicts results of immunofluorescence staining for in vitro osteogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) seeded on the hydrogel.
[0024]
Figure 9 depicts ectopic bone formation under in vivo environmental conditions.
[0025]
Figure 10 depicts regeneration of Intervertebral Disc (IVD) when hydrogel is used to treat IVD injury.
[0026]
Figure 11 depicts in vivo toxicity of the hydrogel when induced into the rat body.
DETAILED DESCRIPTION OF THE DRAWINGS
[0027]
The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as
6
to not unnecessarily obscure the embodiments herein. The examples
used herein 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 examples should not be construed as limiting the scope of the embodiments herein.
[0028]
As used herein, the term/phrase “Biomaterials” are materials that have been designed to interface with biological systems, for the treatment, augmentation, or replacement of biological functions. The biomaterial mentioned in the disclosure herein refers to be used for its application as a “scaffold” to provide shape and structure to the healing tissue and also generate conducive microenvironment for growth and survival of indigenous cells.
[0029]
The terms mentioned herein “the thermoresponsive injectable biomaterial” and the biomaterial are used interchangeably for the purpose of the draft.
[0030]
The term “Scaffolds” mentioned herein are three-dimensional (3D) porous, fibrous or permeable biomaterials intended to permit transport of body liquids and gases, promote cell interaction, viability and extracellular matrix (ECM) deposition with minimum inflammation and toxicity while biodegrading at a certain controlled rate.
[0031]
As mentioned, there is a need for a biomaterial which can be used for regeneration of bone tissue and providing support to the damaged tissues and generate conducive microenvironment for growth and survival of indigenous cells. The present invention discloses a thermoresponsive biomaterial with application as a scaffold or filling material for bone tissue or dental implant wherein the disclosed biomaterial is sterile, injectable, ready to use and thermoresponsive. The said scaffold can be easily administered to the IVD by using the
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currently
used medical devices and does not require development of any further medical invasive procedures or equipment.
[0032]
The present invention discloses a thermoresponsive injectable biomaterial, comprising of; a crosslinked chitosan; collagen; hydroxyapatite.
[0033]
In one embodiment, the biomaterial comprises: a crosslinked chitosan, wherein the concentration of cross-linked chitosan is 2% (w/v). The chitosan is cross-linked by electrostatic interaction by ß-glycerophosphate and sodium bicarbonate (0.1 M). ß- glycerophosphate initiates the crosslinking of chitosan to from a cohesive gel-based structure to the biomaterial. ß-glycerophosphate is used at a concentration of 7.5% to 10% (w/v). Collagen provides adhesive surface for cell attachment and is used at a concentration of 0.16 % (w/v) to 2 % (w/v). Hydroxyapatite nanoparticles are used at a concentration of 0.05 % to 1 % (w/v) and with an average size of 150-200 nm. The biomaterial is injectable, ready to use, a cohesive viscoelastic gel and thermoresponsive.
[0034]
In another embodiment, the biomaterial comprises: 2% (w/v) crosslinked chitosan, which is crosslinked using 7.5% (w/v) ß-glycerophosphate in 0.1 M sodium bicarbonate, collagen at a concentration of 2% (w/v) and hydroxyapatite at a concentration of 1% (w/v) with particle size of 150 - 200 nm.
[0035]
The following table shows the various concentrations of all ingredients used for preparing a thermoresponsive biomaterial. It further shows the optimal concentrations of chitosan, ß-glycerophosphate, collagen and hydroxyapatite, for which gel formation is stable and reversal of gel to sol form does not happen.
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Table 1
[0036]
In another embodiment, the biomaterial is liquid at a temperature of 4ºC but solidifies at 37ºC within 10-15 mins. In another embodiment the shelf life of the scaffold is found to be 3 months at 4ºC and did not show any bacterial or fungal growth with proper storage and appropriate temperature. The biomaterial is stable at a pH range of 7 to 7.6.
[0037]
It is seen in the experiments that reducing the percentage of acetic acid allows to keep the pH of the hydrogel less acidic and thus facilitating a higher pH and governing stability of the gel. It further allows to use more amount of chitosan and collagen in the final composition.
[0038]
In yet another embodiment, the biocompatibility of the scaffold for Mesenchymal Stem Cells (MSCs) is determined by MTT assay. Fig. 3 shows cytocompatibility of the biomaterial using MTT assay. MTT assay reveals that the biomaterial hydrogel is not toxic to cells and is cytocompatible as displayed by higher cell survival and proliferation. This could be attributed to the presence of a larger surface area and availability of adhesion molecules in collagen. Mesenchymal Stem Cells (MSCs) used are gingival tissue derived mesenchymal stem cells. Statistical analysis is done by One-way
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A
NOVA. (*p<0.05).
[0039]
In yet another embodiment, fig. 4 depicts SEM analysis of biomaterial hydrogel at 37ºC. It displays an irregular morphology of the surface and an average pore-size of 100 to 250µm (Magnification 500x). This pore size is enough for cell-to-cell interactions and allows mobility of cells through the material. The porosity is confirmed using the Archimedes principle and density is calculated by measuring the dry and wet weight of the biomaterial (see below table).
Table 2
[0040]
In yet another embodiment, rheological analysis of thermoresponsive injectable biomaterial hydrogel is carried out to determine the fluid behavior of the biomaterial. The said method of rheological analysis helps to determine the exact temperature at which the hydrogel solidifies, and the time taken by the hydrogel to solidify at that temperature. The said method further aids to monitor reversal of gel to sol upon decrease of temperature, thereby determining the stability of the biomaterial for biological applications.
[0041]
In yet another embodiment, the biomaterial is used as a filling agent, as a support for delivery of various drug molecules and cells to promote and enhance regeneration of tissue. In yet another embodiment the components of the biomaterial are osteoconductive and chondroconductive and have the innate ability to facilitate bone or cartilage tissue regeneration. In yet another embodiment the
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thermoresponsive
biomaterial has its application for use as an injectable material for hard-to-get locations in the human body.
[0042]
In yet another embodiment the thermoresponsive injectable biomaterial is aimed to be used as a scaffold to provide shape and structure to the healing tissue and generate conducive microenvironment for growth and survival of indigenous cells and facilitating fast tissue regeneration. In yet another embodiment the thermoresponsive injectable biomaterial is aimed for use as a filling material for damaged bone tissues or as dental implants. The scaffolds mimic the composition of the extracellular matrix, MSCs impregnated in the scaffold may secrete ECM matrix, enabling better regeneration. The thermoresponsive hydrogels may include dispersed therein an active pharmaceutical ingredient (API), preferably an API falling in the Biopharmaceutic Classification System (BCS) class II. The dosage of gel depends on the size of injury or the site of administration.
[0043]
In one embodiment, a method of preparing a thermoresponsive hydrogel formulation comprising the steps of: a) dissolving 2% chitosan solution in 0.1% acetic acid to prepare a homogenous mixture; b) autoclaving the chitosan mixture obtained in step (a) at 121?, 15 psi for 15 mins; c) storing the above mixture at 4? for 12hr before further processing; d) autoclaving hydroxyapatite nanoparticles at 121?, 15 psi for 15 mins; e) sterilizing collagen under UV for 2 hours; f) dissolving glycerophosphate in distilled water containing 0.1 M sodium bicarbonate filter which is further sterilized and stored at 4? for minimum 2 hours before use; g) mixing the chitosan mixture, the hydroxyapatite, and the collagen under sterile conditions at 4? to form homogenous mixture; h) adding glycerophosphate solution dropwise and mixing for 1 hour at 4? to get thermoresponsive gel.
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[0044]
Examples
[0045]
Example 1: FTIR Analysis
FTIR analysis of thermo-responsive injectable biomaterial hydrogel was carried out. Fig. 5(A-G) displays the presence of specific functional groups representing presence of hydroxyapatite, collagen and chitosan in the scaffold. Collagen was processed for FTIR analysis in powder form and therefore lacks the -OH group. Hydroxyapatite was also processed for FTIR analysis in powder form and thus did not reveal the presence of -OH group. However, both ß-glycerophosphate and chitosan were analyzed in aqueous acidic medium and both revealed presence of -OH group during FTIR analysis. The formulation is a hydrogel and therefore its base is aqueous. Thus, the liquid formulation reveals presence of -OH group in FTIR analysis.
[0046]
Example 2: Rheological Analysis of biomaterial
As shown in fig. 6 (A & D), the biomaterial hydrogel was found to solidify at a temperature of 37°C. However, this process was found to be irreversible as the hydrogels did not liquify after they were brought back to 4°C. Fig. 6 (B, C) shows that as the time sweep at 30°C and 37°C, the freshly prepared hydrogels underwent solidification within 7 mins and 2 mins at a temp of 30°C and 37°C respectively. Whereas as shown in fig. 6 (E, F), when the hydrogel is subjected to solidification after storing it at 4°C for 1 month, the time required for solidification at 30°C and 37°C reduced to 4 mins and 1 min respectively, suggesting that storage at low temperature would decrease the solidification time.
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[0047]
Example 3: UTM Analysis of biomaterial
The biomaterial is checked for compressive strength using Universal Testing Machine (UTM) (fig. 7). The hydrogel was allowed to solidify at a temperature of 37ºC and was subjected to compression at a rate of 0.01 mm/sec using a load cell of 200 N at room temperature. The UTM analysis depicts Young’s modulus of the hydrogel thus determining its elasticity and compressive strength. The graphs represent stress vs strain curve for -
Solidified hydrogel at room temperature, on applying 0 to 50 % and 10-15 % strain respectively - indicating Young’s modulus of 11,574.50 ± 535.50 Pascal (graph A & B respectively in fig. 7).
Solidified hydrogel stored in saline, on applying 10-15% of strain - Young’s modulus decreases to 4396.70 ± 1443.70 Pascal (graph C in fig. 7).
Reduction in Young’s modulus in presence of saline, indicating loss of elasticity (graph D in fig. 7).
[0048]
Example 4: Immunofluorescence Staining
Fig. 8 depicts in-vitro osteogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) seeded on the hydrogel and its determination by immunofluorescence staining. MSCs were seeded on the solidified hydrogel and were induced with osteogenic/ chondrogenic differentiation medium to determine the efficacy of biomaterial to support bone and cartilage regeneration respectively. After 15 days of induction, cell seeded hydrogels were stained for presence of osteocalcin (bone matrix marker; red stain) and collagen type II (cartilage marker, red stain) by immunofluorescent dyes and observed under confocal microscope (Magnification 20x, scale 50µm). Immunofluorescence staining displays differentiation of
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MSCs to (B) functional osteoblasts and (D) chondrocytes, upon induction. This indicates that the biomaterial hydrogel can support both bone regeneration as well as cartilage regeneration.
[0049]
Example 5: In-vivo Osteogenesis
Fig. 9 depicts results of ectopic bone formation when the thermoresponsive biomaterial hydrogel is used as a filling agent. In vivo experiments were carried out to determine whether the scaffold supports bone tissue formation under in vivo environmental conditions. The MSCs were implanted on the seeded biomaterial hydrogel in the sub-cutaneous pockets of immunocompromised mouse. After 6 weeks of in vivo implantation at an ectopic site, the retrieved implants seeded with MSCs displayed the presence of bone matrix protein, osteocalcin (green) (A, D and G in fig. 9); collagen type I (red) (B, E and H in fig. 9), by immunofluorescence staining (inset magnification 60x, scale 100 µm; Magnification 10x, scale 50µm) and further displayed enhancement in levels of calcium and phosphate (C, F in fig. 9), as observed by SEM (Magnification 1000x, scale 60 µm) and (I) EDAX analysis of retrieved implants.
[0050]
Example 6: Regeneration of bone tissues in Intervertebral Discs (IVD) using thermoresponsive biomaterial
IVD injury was created using needle puncture model in rat tails and the hydrogel loaded with MSCs was injected at the site of damage. After 6 weeks the IVDs were retrieved, sectioned and stained with H&E and safranine O to determine regeneration of cartilage in the damaged IVDs. The healthy IVD as shown in fig. 10, A and E pictures, has lamellar organization of the nucleus pulposus-annulus fibrosus boundary, defined boundary between nucleus pulposus-annulus fibrosus and presence of glycosaminoglycan in the inter-
14
lamellar matrix (red stained area). Whereas the diseased animals (fig. 10, B and F pictures) showed distorted boundary between nucleus pulposus-annulus fibrosus as well as disturbed lamellar arrangement and lowered content of glycosaminoglycan (red stained area). When the scaffold and MSCs loaded scaffold are administered, as shown in (fig. 10, C and G) and (fig. 10, D and H) respectively, there is a regeneration of lamellar organization at the nucleus pulposus-annulus fibrosus boundary, reappearance of boundary between nucleus pulposus-annulus fibrosus and increased content of glycosaminoglycan in the inter-lamellar matrix (Magnification 1x, scale 5mm).
[0051]
Example 7: Cytokine Profiling (fig. 11)
In order to determine the in vivo toxicity of the hydrogel in terms of inflammatory response induced by the hydrogel upon administration into the mouse body, the serum samples of small animals injected with the hydrogel were examined for the levels of inflammatory cytokines. It was observed that though few pro-inflammatory cytokines such as TNF-a, IFN-?, IL-13 showed some enhancement at day 2 of hydrogel administration, but their levels reduced till day 7, indicating that although there might be some inflammatory response, it is not sustained and thus is not significant to cause chronic toxicity in the animal body and eliminating the chances of graft versus host disease (GVHD).
Advantages
The thermoresponsive injectable biomaterial of the present invention is used to prepare the scaffolds or filling material, which is further used for regeneration of bone and cartilage tissues in intervertebral disc injuries and providing support to the damaged tissues thereby enhancing the process of bone tissue regeneration. The scaffold is
15
further used to provide shape and structure to the healing tissues and to generate conducive microenvironment for growth and survival of indigenous cells, thereby facilitating fast tissue regeneration. The thermoresponsive injectable biomaterial can be used as a filling material for damaged bone tissues, intervertebral disc or as dental implants. The biomaterial as a filling agent provides a support for delivery of various drug molecules and cells to promote and enhance regeneration of tissue. The components of the biomaterial are osteoconductive and chondroconductive and have the innate ability to facilitate bone or cartilage tissue regeneration. The said biomaterial can be used as an injectable material for hard-to-get locations in the human body. The said biomaterial has an application in an ink for three-dimensional (3D) printer including thermoresponsive hydrogels as well as in the treatment of skin wounds.
[0001] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has 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 application. , Claims:CLAIMS
I/ We claim,
1.
A thermoresponsive biomaterial comprising:
a)
a cross-linked chitosan, wherein, the chitosan is cross-linked by electrostatic interaction by ß-glycerophosphate and sodium bicarbonate;
b)
a collagen, which provides adhesive surface for the cell attachment;
c)
a hydroxyapatite, which provides mechanical strength to the hydrogel;
wherein, the ß-glycerophosphate is at a concentration of 7.5% to 10% (w/v); the sodium bicarbonate is 0.1 M; the collagen is at a concentration of 0.16 % (w/v) to 2 % (w/v); the hydroxyapatite nanoparticles are at a concentration of 0.05% to 1 % (w/v)
2.
A thermoresponsive biomaterial as claimed in claim 1, wherein, the ß-glycerophosphate is at 7.5% (w/v); the collagen is at 2 % (w/v); the hydroxyapatite nanoparticles are at 1 % (w/v) with particle size of 150 - 200 nm.
3.
A thermoresponsive biomaterial as claimed in claim 1, wherein, the thermoresponsive biomaterial may be colloidal gel, organogel or hydrogel; preferably be hydrogel.
4.
A thermoresponsive biomaterial as claimed in claim 1, wherein, the gel is liquid at a temperature of 4ºC and solidifies at 37ºC.
5.
A thermoresponsive biomaterial as claimed in claim 1, wherein, the gel is irreversible to sol form.
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6.
A thermoresponsive biomaterial as claimed in claim 1, wherein, the thermoresponsive biomaterial has pore-size of 100 to 250µm (Magnification 500x).
7.
A thermoresponsive biomaterial as claimed in claim 1, wherein, the components of the thermoresponsive biomaterial are osteoconductive and chondroconductive; the biomaterial has an innate ability to facilitate bone or cartilage tissue regeneration.
8.
A thermoresponsive biomaterial as claimed in claim 1, wherein, the thermoresponsive biomaterial is used to prepare a scaffold or a filling material which is further used for regeneration of cartilage tissues in intervertebral disc injuries and providing support to the damaged tissues, and in ectopic bone formation.
9.
A method of preparing a thermoresponsive biomaterial, comprising the steps of:
a)
dissolving 2% chitosan solution in 0.1% acetic acid to prepare a homogenous mixture;
b)
autoclaving the chitosan mixture obtained in step (a) at 121?, 15 psi for 15 mins;
c)
storing the above mixture at 4? for 12hr before further processing;
d)
autoclaving hydroxyapatite nanoparticles at 121?, 15 psi for 15 mins;
e)
sterilizing collagen under UV for 2 hours;
f)
dissolving glycerophosphate in distilled water containing 0.1 M sodium bicarbonate filter which is further sterilized and stored at 4? for minimum 2 hours before use;
g)
mixing the chitosan mixture, the hydroxyapatite, and the collagen under sterile conditions at 4? to form homogenous mixture;
h)
adding glycerophosphate solution dropwise and mixing for 1 hour at 4? to get thermoresponsive gel.
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10.
A thermoresponsive biomaterial may be administered through subcutaneous or injectable route, preferably through injectable route.