Description:FIELD OF THE INVENTION:

[0001] The present disclosure generally relates to the field of pharmaceuticals. Specifically, the present disclosure relates to a three-dimensional (3D) extracellular matrix and uses thereof as bone graft substitute. More specifically, the present disclosure relates to 3D-extracellular matrix and pre-differentiated osteoblast derived exosomes. The present disclosure also relates to a method of preparing 3D extracellular matrix scaffold, method of preparing porous polymer scaffold, and pre-differentiated osteoblast derived exosomes.

BACKGROUND OF THE INVENTION

[0002] Bone grafts are used to fill large volumes of bone loss from fracture, traumatic injury or other physical disorder that affects bone architecture and function in the body. Such types of bone loss lead to formation of critical size defects (CSD), which without grafting or other intervention usually lead to non-union. Bone grafts must have adequate mechanical properties to Support new bone formation in the defect and be osteogenic and resorbable to maintain continuity of Support during bone regeneration. Bone regeneration is a very complex, but well-orchestrated continuous remodelling process of bone formation. The entire adult life witnesses, bone as a tissue which possesses a tremendous inherent capacity for regeneration through the coordinated activity of osteoclasts (OCs) and osteoblasts (OBs). However, in case of large defects, where the injury goes beyond a critical limit, the bone will not be able to self-heal without surgical and therapeutic intervention. For example, in the case of metastatic osteosarcoma, the tumour site is often surgically removed after a combination of chemo and radiotherapy, which creates a large gap in the bone. In case of a major bone fracture, a part of the bone is completely or partially broken causing pain and loss of functionality. Therefore, a variety of currently available bone grafts are being used to replace the missing bone.

[0003] Critical-size bone defects have been traditionally treated with autografts, allografts and xenografts. However, these materials can cause disease transmission, donor site morbidity, and acute immunological responses that can lead to bone resorption and graft rejection. Also, resources for allografts and autografts are limited. Moreover, due to some severe limitations of these grafts, advanced strategies have been developed through tissue engineering and regenerative medicine approaches. With the use of different bone tissue scaffolds like hydrogels, nanofibers, and porous 3D scaffolds, the delivery of important bioactive molecules is done successfully. Mesenchymal stem cell (MSCs) based bone tissue engineering is a promising strategy in this field. Studies have demonstrated that the transplanted MSCs could efficiently restore critical-size bone defects as well as accelerate bone regeneration. However, due to the notable limitations involved with the direct use of MSCs, the recent study has shifted its gear towards the use of cell-free therapy in bone regeneration. Studies also indicate that the therapeutic effects of MSCs are controlled by the paracrine factors secreted from these that stimulate the activity of the recipient cells, instead of the direct cell replacement.

[0004] Current approaches to bone repair have been described to involve the fabrication of a scaffold using a 3-D technique. These fabricated scaffold may then be implanted into a patient at a site in need of repair of a small to large bone fracture. Conventional methods that are being used for fabrication of 3D scaffolds are labor-intensive, time-consuming, and do not provide precise control over the scaffolds architecture.

[0005] Further, for localized therapeutics in bone, native exosomes are not suitable as implant materials.

[0006] Therefore, there is a vital need for new bone graft substitutes that can rapidly heal these defects to reduce patient discomfort, medical care costs as well as biodegradable, which is the key factor towards the initiation of bone regeneration. Hence, the present disclosure aims to provide a 3D extracellular matrix comprising osteoblast precursor cell-derived exosomes that have bone mineralization potential.

OBJECT OF THE INVENTION:

[0007] Some of the objects of the present disclosure, with at least one embodiment herein satisfy, are listed herein below:

[0008] It is one of the primary object of the present disclosure to provide a 3D extracellaular matrix and its use as a bone graft substitute.

[0009] It is another objective of the present disclosure to provide 3D-extracellular matrix scaffold with shelf-life of six months, which reduces patient discomfort, medical care costs as well as it is biodegradable.

[0010] It is another objective of the present disclosure to provide a method for obtaining exosomes by mineralizing pre-osteoblasts.

[0011] It is yet another objective of the present disclosure to provide a method of preparing a porous polymer scaffold.

[0012] It is yet another objective of the present disclosure to provide a method of preparing a 3D-extracellular matrix.

SUMMARY OF THE INVENTION:

[0013] The present disclosure relates to a 3D-extracellular matrix scaffold comprising: a porous polymer scaffold; pre-differentiated osteoblast derived exosomes, wherein a particle size of the pre-differentiated osteoblast derived exosomes is in the range of 50 nm-150 nm; and wherein the pre-differentiated osteoblast derived exosomes cross-linked with the porous polymer scaffold to form the 3D extracellular matrix scaffold.

[0014] The present disclosure further relates to a method of preparing pre-differentiated osteoblast derived exosomes comprising the steps of: preparing an exosome-depleted media in 20% fetal bovine serum (FBS) containing α-MEM (α-Minimum Essential Medium) by ultracentrifuging the said media at 1,50,000 xg for 18 hr at 4°C; mineralizing pre-osteoblast cell line MC3T3-E1 in 50 µg/mL ascorbic acid, 100nM dexamethasone, and 10 mM β- glycerophosphate; incubating the MC3T3-E1 cells with α-MEM containing 10% exosome depleted media at temperature of 37°C with 5% CO2 and 100% relative humidity for 48 hours; cell culturing in α-MEM supplementing with 10% FBS and 1% (v/v) penicillin/streptomycin to form MC3T3-E1; collecting and processing the cell conditioned media with differential centrifugation to remove dead cells, larger particles, and microvesicles; isolating exosomes from the processed conditioned media by ultracentrifuging at 1,50,000 xg for 90 min at 4°C to form pellets; and resuspending the pellets containing exosomes in 1X PBS.

[0015] The present disclosure also relates to a method of preparing extracellular matrix comprising the steps of incubating pre-differentiated osteoblast derived exosomes with a porous polymer scaffold at a temperature in the range of 23°C to 32°C.

BRIEF DESCRIPTION OF DRAWINGS:

[0016] The illustrated embodiments of the subject matter will be best understood by reference to the drawings. The following description is intended only by way of example, and simply illustrates certain selected embodiments of reagents and processes that are consistent with the subject matter as claimed herein, wherein:

[0017] Fig. 1 illustrates an image of a rat model with critical-sized calvarial defects and implanted the cross-linked polymeric scaffold construct at the target site, which was done in vivo.

[0018] Fig. 2 illustrates the study of the osteogenic properties of MC3T3-E1 cells on (A) alkaline phosphatase (ALP) staining after i) 7 days and ii) 14 days of osteogenic media incubation. (B) ALP activity compared to the control. (C) Alizarin Red Solution (ARS) staining as compared to the i) control after ii) 7 days and iii) 14 days of treatment. (D) Expression of osteogenic genes ALP, Col-1 and RUNX-2 compared to the control. Growth media (GM), Differentiation media (DM), ***P<0.0005, **P<0.005, *P<0.05.

[0019] Fig. 3 illustrates the (A) Average size of isolated exosomes (180nm) as depicted from dynamic light scattering (DLS). (B) Negative surface charge (-20mV) detected by zeta sizer. (C) Morphology of exosomes observed by transmission electron microscopy (TEM). (D) Magnified TEM image of a single exosome particle. (E) Western blot analysis to show exosome markers CD81 and TSG-101.

[0020] Fig. 4 illustrates the uptake of exosomes by MC3T3-E1 cells. Representative confocal microscopy images of PKH67 tagged exosomes (green) uptake after incubation with MC3T3-E1 cells for 6h, 12h, and 24h. Cell cytoskeleton and nuclei were stained with rhodamine-B (red) and DAPI (blue) respectively. Scale bar 25µm.

[0021] Fig. 5 illustrates the effect of exosomes on proliferation, migration, and osteogenic mineralization of MC3T3-E1 cells. (A) WST-1 assay results showing cell proliferation. (B) i) Trans-well migration assay, ii) quantitative analysis of trans-well assay. (C) Relative expression of osteogenic genes ALP, Col-1, RUNX-2 and MGP after incubation with exosomes. (D) Elevated expression levels of osteogenic genes in MC3T3-E1 cells after incubation with DM as compared to GM. GM: Growth media, DM: Differentiation media, ****P<0.0001, ***P<0.0005, **P<0.005, *P<0.05.

[0022] Fig. 6 illustrates the characterization of CS-scaffolds (A) Covalent crosslinking of chitosan and collagen with methacrylation for hydrogel synthesis. (B) Proton NMR to ensure cross-linking. (C) scanning electron microscope (SEM) image showing porous structure. (D) Rheological property of scaffold. (E) Swelling property of scaffold. (F) Rate of scaffold degradation.

[0023] Fig. 7 illustrates the Exosome detection and release from scaffolds. (A) Representative fluorescence microscopy image of PKH67 tagged exosomes integrated on the scaffold. (B) In vitro release profile of exosomes from scaffolds.

[0024] Fig. 8 illustrates the characterization of Tripolyphosphate polyanion (TPP) cross-linked CS-scaffolds (A) Ionic crosslinking of chitosan to synthesize scaffold. (B) Proton Nuclear magnetic resonance (NMR) to ensure cross-linking. (C) SEM image showing highly porous structure. (D) Rheological property of scaffold. (E) Swelling property of scaffold. (F) Rate of scaffold degradation. (G) X-ray images of rat skulls showing the comparative bone growth between control and scaffold treated group.

[0025] Fig. 9 illustrates the Micro-CT analysis of bone regeneration at 8 weeks after surgery. (A) 3D reconstructed images of calvarial bone indicating new bone formation. Quantitative analysis of bone regeneration by (B) New bone volume, (C) Trabecular thickness, (D) Trabecular spacing, and (E) Trabecular number. ***P<0.0005, **P<0.005, *P<0.05.

[0026] Fig. 10 Shows the (A) fold change of the new bone formation in treated groups as compared to the control group (with no implant). (B) Tabular representation of the graph showing the values.

[0027] Fig. 11 illustrates the histological evaluation of bone formation after 8 weeks of implantation. (A) HE staining, (B) Masson’s trichrome staining. Mature collagen in the bone matrix was stained in blue. New bone (NB), Host bone (HB).

DETAILED DESCRIPTION OF INVENTION:

[0028] The detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such detail as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

[0029] It is also to be understood that various substitutions/arrangements/permutations or combinations may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects, and embodiments of the present disclosure, as well as specific examples, are intended to encompass equivalents thereof.

[0030] The terminology used herein is to describe particular embodiments only and is not intended to be limited to example embodiments. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms “comprise”, “comprising”, “includes” and/or “including” when used herein, specify the presence of stated process features, steps, ingredients, elements and/or components, but do not preclude the presence or addition of one or more other process features, steps, ingredients, elements, components and/or groups thereof.

[0031] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0032] The meaning of the term “3D-extracellular matrix scaffold” in the context of the present disclosure is a porous, biocompatible and biodegradable matrix that is used to accelerate bone regeneration. Hence, the 3D extracellular matrix is capable of biodegradation, which is the key factor towards the initiation of bone regeneration. Further, no additional components such as proteins, cells, inorganic minerals and drugs are required in the matrix for the bone formation.

[0033] The term “porous polymer scaffold” or “covalent polymer scaffold” is a covalently cross-linked with chitosan and collagen protein. These phrases can be interchangeably used in the present disclosure. Therefore, the present invention developed cross-linked polymeric scaffolds synthesized out of chitosan, which provide strength and a well-formed matrix for holding and releasing an appropriate dose of exosomes at the target site.

[0034] The phrase “pre-differentiated osteoblast derived exosomes” in the context of the present disclosure is defined as the isolation of exosomes after mineralization of the pre-osteoblast cells (MC3T3-E1)The phrases “pre-differentiated osteoblast derived exosomes,” “mineralized osteoblast derived exosomes” are interchangeably used in the specification.

[0035] The present disclosure relates to a 3D-extracellular matrix scaffold. The extracellular matrix of the present disclosure comprises a porous polymer scaffold, and pre-differentiated osteoblast derived exosomes.

[0036] In an embodiment of the present disclosure, the porous polymer scaffold used in the present disclosure is cross-linked chitosan and collagen protein. The polymer scaffold are not limited to the examples given here but also include those polymer having similar characteristics as those of the above-mentioned polymer scaffold.

[0037] In an embodiment of the present disclosure, the diameter of the porous polymer scaffold is in the range of 5 mm to 20 mm. Preferably, the diameter of the porous polymer scaffold is 9mm. The disclosure of the range of the diameter includes all the permutations and combinations that fall within the mentioned range of the diameter and also includes extreme values of the said range.

[0038] In an embodiment of the present disclosure, the porous polymer scaffold has a pore size in the range of 50 to 200 nm. The disclosure of the pore size includes all the permutations and combinations that fall within the mentioned range of the pore size and also includes extreme values of the said range.

[0039] In another embodiment of the present disclosure, the pre-differentiated osteoblast derived exosomes used in the 3D extracellular matrix. The pre-differentiated osteoblast derived exosomes is used as a regulator for maintenance and repair of bone, involves both bone degradation, dismantling of old bone matrix, and its replacement with new bone matrix. The pre-differentiated osteoblast derived exosomes are not limited to the examples given here but also include those regulators having similar characteristics as those of the above-mentioned regulator.

[0040] One of the essential components of 3D extracellular matrix is pre-differentiated osteoblast derived exosomes. The said exosomes are an acceptable treatment approach for healing fractures. These exosomes help in treatment provided after the bone defect has occurred, it facilitates three times more new bone formation after eight weeks post implantation.

[0041] In an embodiment of the present disclosure, the exosomes isolated from mineralized osteoblasts have the potential to elevate the osteogenesis of MC3T3-E1 cells in vitro. However, for localized therapeutics in bone, native exosomes are not suitable as implant materials. Therefore, the present invention developed cross-linked polymeric scaffolds synthesized out of chitosan and observed that they provide strength and a well-formed matrix for holding and releasing an appropriate dose of exosomes at the target site.

[0042] In another embodiment of the present disclosure, the particle size of the pre-differentiated osteoblast derived exosomes is in the range of 50 nm-150 nm. The disclosure of the particle size includes all the permutations and combinations that fall within the mentioned range of the particle size and also includes extreme values of the said range.

[0043] In an embodiment of the present disclosure, the amount of the pre-differentiated osteoblast derived exosomes used in the extracellular matrix is in the range of 80 ug/ml to 100ug/ml. In particular, the amount of the pre-differentiated osteoblast derived exosomes used in the extracellular matrix is in the range of 80 ug/ml to 100 ug/ml per scaffold, wherein the pre-differentiated osteoblast derived exosomes have 9mm diameter and 1mm thickness. The disclosure of the amount includes all the permutations and combinations that fall within the mentioned range of the amount and also includes extreme values of the said range.

[0044] In an embodiment of the present disclosure, the ratio of the porous polymer scaffold with that of the pre-differentiated osteoblasts derived exosomes is in the range of 50:50in the 3D extracellular matrix.

[0045] The present disclosure also relates to the preparation of a porous polymer scaffold. The method of preparing the porous polymer scaffold comprises the step of cross-linking polysaccharide with protein via a methacrylation reaction using UV rays to form a polysaccharide-protein composite.

[0046] In an embodiment of the present disclosure, the polysaccharide is chitosan. The polysaccharide is not limited to chitosan but also includes polysaccharides having similar characteristics as of chitosan.

[0047] In an embodiment of the present disclosure, the protein is collagen. The protein is not limited to chitosan but also includes protein having similar characteristics as of collagen.

[0048] In an embodiment of the present disclosure, the amount of chitosan used to form the polysaccharide-protein composite is in the range of 2.5% to 4%.

[0049] In an embodiment of the present disclosure, the amount of collagen used to form the polysaccharide-protein composite is in the range of 1mg/ml to 3mg/ml.

[0050] In another embodiment of the present disclosure, crosslinking is carried out at temperature in the range between 23°C to 32°C. The disclosure of the temperature includes all the permutations and combinations that fall within the mentioned range and also includes extreme values of the said range.

[0051] In an embodiment of the present disclosure, the cross-linking is carried out at a pH in the range at pH ranging from 3.5 to 6. The disclosure of the pH includes all the permutations and combinations that fall within the mentioned range and also includes extreme values of the said range.

[0052] In an embodiment of the present disclosure, the method of preparing the porous polymer scaffold further comprises the step of freeze-drying the composite followed by lyophilizing to form a porous polymer scaffold.

[0053] In an embodiment of the present disclosure, the freeze drying is carried out at a temperature in the range between –20°C and –25°C. The disclosure of the temperature includes all the permutations and combinations that fall within the mentioned range and also includes extreme values of the said range.

[0054] The present disclosure also relates to the preparation of a pre-differentiated osteoblast derived exosomes. The method of preparing the pre-differentiated osteoblast derived exosomes comprises preparing an exosome-depleted media in fetal bovine serum (FBS) containing α-MEM (Minimum Essential Medium) by ultracentrifuging the said media, mineralizing pre-osteoblast cell line MC3T3-E1 in ascorbic acid, dexamethasone, and β- glycerophosphate; incubating the MC3T3-E1 cells with α-MEM containing 10% exosome depleted media with 5% CO2 and 100% relative humidity; cell culturing in α-minimal essential medium supplementing with 10% FBS and 1% (v/v) penicillin/streptomycin to form MC3T3-E1; collecting and processing the cell conditioned media with differential centrifugation to remove dead cells, larger particles, and microvesicles; isolating exosomes from the processed conditioned media by ultracentrifuging to form pellets; and resuspending the pellets containing exosomes in 1X PBS.

[0055] In an embodiment of the present disclosure, the method of preparing the pre-differentiated osteoblasts derived exosomes comprising the steps of: preparing an exosome-depleted media in 20% fetal bovine serum (FBS) containing α-MEM (Minimum Essential Medium) by ultracentrifuging the said media at 1,50,000 xg for 18 hr at 4°C; mineralizing pre-osteoblast cell line MC3T3-E1 in 50 µg/mL ascorbic acid, 100nM dexamethasone, and 10 mM β- glycerophosphate; incubating the MC3T3-E1 cells with α-MEM containing 10% exosome depleted media at temperature of 37°C with 5% CO2 and 100% relative humidity for 48 hours; cell culturing in α-minimal essential medium supplementing with 10% FBS in vitro and 1% (v/v) penicillin/streptomycin to form MC3T3-E1; collecting and processing the cell conditioned media with differential centrifugation to remove dead cells, larger particles, and microvesicles; isolating exosomes from the processed conditioned media by ultracentrifuging at 1,50,000 xg for 90 min at 4°C to form pellets; and resuspending the pellets containing exosomes in 1X PBS.

[0056] The present disclosure also relates to a method of preparing extracelluar matrix comprising the step of incubating an pre-differentiated osteoblast derived exosomes with a porous polymer scaffold at room temperature. The room temperature in the context of the present disclosure is defined as being in the range of 23°C to 32°C. The disclosed range of temperature is not limited to the mentioned range but also includes any kind of permutation and combination as known in the person skilled in the art that can achieve the desired objective of the present application and also includes the end of the ranges.

ADVANTAGES OF THE PRESENT INVENTION:

[0057] The 3D-extracellular matrix of the present disclosure has the following advantages:

• The shelf-life of 3D-extracellular matrix scaffold is six months with sustained osteogenic ability.
• The 3D scaffold is capable of biodegradation, which is the key factor towards the initiation of bone regeneration.
• No additional components such as proteins, cells, inorganic minerals and drugs are required in the scaffold composition for the bone formation to happen.
• The release rate of exosomes from the 3D polymer matrix is 80% in 48 hours.
• The bone formation at the critical size defect is upto 4 times the control in case of exosome embedded chitosan scaffold in 8 weeks.

[0058] The invention is further illustrated by the following example, which is provided to be exemplary of the invention and does not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

EXAMPLES

EXAMPLE 1:

MATERIALS & METHODS:

Cell culture:

Example 1 provides a mouse pre-osteoblast cell line MC3T3-E1 (ATCC®CRL-2593™) was purchased from American Type Cell Culture Collection (ATCC) and cultured in α-minimal essential medium (HIMEDIA, α-MEM, AL221A) supplemented with 10% fetal bovine serum (FBS) (In Vitro Technologies, New York) and 1% (v/v) penicillin/streptomycin (Gibco®, Life Technologies Pty Ltd., New York). Cells were incubated at 37°C and 5% CO2 with 100% relative humidity. In order to induce osteogenic differentiation, the cells were cultured in osteogenic medium containing 50 µg/mL ascorbic acid (Sigma), 100nM dexamethasone (Sigma) and 10 mM β-
glycerophosphate (Sigma).

EXAMPLE 2:

Isolation and characterization of exosomes:

Exosomes were isolated from the conditioned media of cell culture. 20% FBS containing α-MEM (Minimum Essential Medium) was ultracentrifuged at 1,50,000 xg for 18 hr at 4°C to get the exosome depleted FBS media. After cells reached sub-confluency the cell growth media was replaced with 10% exosomes depleted FBS containing media. The MC3T3-E1 conditioned media was collected after 48 hr of incubation and was centrifuged and filtered for removal of unwanted larger particles. The supernatants were centrifuged at 500xg for 10 min, 2000xg for 10 min and 10,000xg for 30 min to eliminate dead cells, cellular debris, and larger vesicles. The medium was then filterd through a 0.22 µm sterile filter followed by ultracentrifugation at 1,50,000 xg for 90 min at 4°C in Type Ti70 rotor using an Optima™XPN-100 ultracentrifuge (Beckman Coulter, United States) to isolate exosomes.

The dynamic light scattering and zeta potential determination for size and charge measurements were performed using a Zetasizer nanoseries instrument (Malvern, Nano ZS). 10µl of exosomes were diluted with 1000 µl of 1× PBS (1:100 dilution), at 25°C before taking measurement. The exosome size data refers to the scattering intensity distribution (z-average). All measurements were taken in 3 replicates.

Electron microscopic analysis was done to verify the presence of exosomes in the resuspended pellet.
For Transmission Electron Microscopy, 10µl of resuspended exosomes were mixed with equal volume of 4% PFA, overnight at 4°C for fixation. 5µl of the mixture was placed on to formavar-coated Cu TEM grids (Agar Scientific) for 20min in dry environment. After washing with PBS, the grids were stained with 1% uranyl acetate for 5min, followed by second wash. Grids were gently blotted on Whatmann filter paper and air-dried. The exosomes were imaged by TEM (JEOL, Japan) at 120 kV. For Scanning Electron Microscopy, 30µl exosomes were fixed in the similar way with equal volume of 3.7% glutaraldehyde. 30µl of sample was deposited over poly-L-lysine (Sigma) coated coverslips, left covered to dry in room temperature. After washing with PBS, dehydration was done with ascending concentrations of alcohol. After evaporation of ethanol, the coverslips were dried at room temperature and kept at 60°C for complete drying, and then analyzed by SEM (Zeiss) after gold-palladium sputtering.

Protein concentration of freshly isolated exosomes was analyzed using BCA Protein Assay kit (Pierce, Rockford, USA). Briefly, MC3T3-E1 cell derived exosomal pellets were resuspended with RIPA buffer (HIMEDIA) containing PMSF and 1% protease inhibitor and phosphatase inhibitor to collect the whole cell lysate, this was followed by water bath sonication and centrifugation at 12,000 g for 15 min at 4°C. Supernatant was collected for protein analysis or kept at -20°C until further use. To detect exosomal markers Western blotting was carried out with anti-CD81, anti-TSG101 and anti-β-actin antibodies. Equal amounts of proteins (20 µg) were resolved on an 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on the SDS-PAGE were transferred to a 0.45µm PVDF membrane and incubated with 5% Bovine serum albumin (Sigma) for 1h at room temperature for blocking. The blot was incubated with appropriate primary antibodies, (anti-CD81, anti-TSG101 and anti-β-actin) overnight at 4-8°C. Anti-GAPDH was taken as the normalization control. After washing with TBS-Tween 20 (0.1%), the blots were incubated with anti-mouse and anti-rabbit HRP conjugated secondary antibodies and washed thrice with TBS-Tween 20 (0.1%). Finally, protein expression was detected using chemiluminiscent reagent and imaged under ChemiDoc (BioRad).

To further quantify CD63 positive exosomes, flow cytometric analysis was done. Briefly, 0.1µl of CD63-PE (BD Biosciences) and its isotype were mixed with 20µl of resuspended exosomes separately and incubated at 4°C for 30min. To quench the dye binding reaction, 500µl of PBS with 1% BSA was added and mixed well by pipetting. The fluorescence intensity was analysed by CytoFlex (Beckman Coulter). Specific gate was applied to acquire and quantify the PE-positive particles. Flow cytometry results were analyzed with FlowJo software.

EXAMPLE 3:

Exosome uptake analysis

The uptake of exosomes by host cells was monitored with PKH67 Green Fluorescent Cell Linker Midi Kit (Lot#MIDI67-1KT, Sigma-Aldrich, NSW, Australia) according to the manufacturer’s protocols. Briefly, 400µl of Diluent C was added to freshly isolated 100µl of exosomes to which 6µl of PKH67 ethanolic dye solution was added and mixed well by mild vortexing. For control group, 100µl of PBS was taken in place of exosomes and mixed well with 6µl of PKH67 dye solution. After 5min of incubation, the staining process was terminated with the addition of 1ml 1% FBS-containing DMEM (exosome-free) for 1 min. Serum-free DMEM was added to make the final volume 8ml and the mixture was centrifuged at 2,00,000 g for 2hour at 4°C in a SW41Ti rotor using an Optima™XPN-100 ultracentrifuge (Beckman Coulter, United States) to remove unbound excess dye. The pellet was resuspended with 1X PBS.

3x 105 cells were seeded on glass coverslips and left overnight at 37°C and 5% CO2 to get attached. The labelled exosomes were incubated with recipient cells for 0hour, 2hour, 6hour, 12hour, 18hour and 24hour at 37°C and 5% CO2. Samples were washed twice with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.1% Triton X-100 (Sigma), stained with RhodamineB-Phalloidin (Invitrogen, Thermo Fisher) and mounted on glass slides with ProLong® anti-fade reagents (Life Technologies Pty Ltd., Australia). All the sides of the coverslips were sealed with transparent nail polish and dried overnight at room temperature. Images were captured using a confocal laser scanning microscope (Leica) under 63 × objectives.

EXAMPLE 4:

Cell proliferation assay and cell migration assay

To investigate the effect of exosomes on MC3T3-E1 cells, various concentrations of exosomes (3, 10, 25, 50, and 100 µg/ml) were treated with cells for 1 day, 3 days, 5 days and 7 days. After 2 days of treatment, the cells were incubated with WST-1 reagent for 30 min and reading was taken at an absorbance of 440nm.

The migration of hBMSCs was evaluated by Transwell assay. Briefly, the optimum numbers of 1×105 cells were seeded on the upper compartment of a Transwell with 8 µm pore size. 500 µl of medium with 10, 25, and 50 µg/ml of exosomes or without exosomes were added to the lower compartment. After culturing for 24h, the chamber was gently washed by PBS. To remove the non-migrated cells, the upper side of the membrane was wiped with a cotton swab. Then the membranes were fixed with 4% paraformaldehyde and stained with DAPI for 5m. After washing with PBS, images were taken in a fluorescence microscope. For quantification with ImageJ software, the number of migrated cells was counted in 5 randomly selected microscopic fields.

EXAMPLE 5:

Studying the in vitro osteogenic properties of MC3T3-E1 cells

To compare the extent of mineralization of MC3T3-E1 cells treated with osteogenic medium for 7 days and 14 days, ALP assay, Alizarin assay and RT-PCR was performed. MC3T3-E1 cells were seeded on 12- well plates and treated with osteogenic media containing 50ug/ml L-ascorbic acid, 1mM β-glycero phosphate and 10nM dexamethasone along with alpha MEM for 7 days and 14 days. Respective control cells (untreated) were grown with the normal alpha MEM media with 10% FBS and 1% Penicillin-Streptomycin. After the given time points, RNAs were isolated using TRIZOL-Chloroform phase separation followed by the isopropanol precipitation method. 1ug of cDNA was synthesized from the respective RNAs and was set for RT-PCR to check the expression of three important osteogenic genes such as ALP, Collagen-1 and RUNX-2. For ALP and Alizarin assays, the cells were fixed and incubated with the respective staining agents for the required duration. For ALP quantification, the cells were scraped and collected after PBS washing. Then the cells were lysed with the lysis buffer provided in the ALP quantification kit (ab83369) and the ALP activity was quantified according to the manufacturer’s protocol. For Alizarin staining, cells were washed, fixed with 10% formaldehyde, and then incubated with 2% Alizarin Red S (Sigma) solution for 45min. After washing with miliQ water, the matrix calcium deposition was checked under a brightfield microscope.

EXAMPLE 6:

Synthesis & physical characterization of chitosan scaffolds

To synthesize cross-linked chitosan scaffolds, chitosan and collagen were methacrylated along with UV cross-linking to get a covalent bonding between the polysaccharide and protein. The synthesized chitosan hydrogel composites were freeze-dried to get porous 3D scaffolds. Freezing at temperatures between –20°C and –25°C and subsequent lyophilization led to an interconnected porous structure.

EXAMPLE 7:

Characterization of chitosan scaffolds

The degree of methacrylation between chitosan and proteins was checked using proton NMR which could indicate exactly how many protons were substituted by the proteins as an indirect measure of methacrylation. The morphology of the scaffolds was examined under SEM. The rheological properties were measured using rheometer (AntonoPar). The storage modulus (G’) and the loss modulus (G”) were analyzed which indicated their elastic properties. To check the swelling ability, the scaffolds were immersed in 1X PBS and miliQ water at 37ºC. The initial dry weights of the scaffolds after lyophilization were noted down. Then the scaffolds were removed from the solvents and weighed after soaking the excess at the required time points. The swelling ratio was calculated using the below formula:

Swelling ratio= {(Wt – W0)/ W0} X 100 %
Where, W0 is the initial dry weight of the scaffold
Wt is the weight of the scaffold after time t.

To check the degradation of the scaffolds, these were immersed in 1X PBS at 37ºC with gentle shaking (50 rpm) for the defined time points. Scaffolds were then freezed and lyophilized to get the final dry weight. The percentage rate of degradation was analysed using the same formula as given above.

EXAMPLE 8:

Integration of exosomes over the scaffold

To investigate the exosome encapsulation ability of chitosan scaffolds, fluorescence microscopic analysis was done after incubating PKH67 labelled exosomes with chitosan scaffolds for 24h at 37°C with 5% CO2. Scaffolds were pre-treated with serum-free media for 24h. To check the release profile of exosomes from the scaffolds, the PKH67 labelled exosomes treated scaffolds were put into PBS and the solvent was collected at each time point. The solvents containing the labelled exosomes were acquired in a flow cytometer to check the percent positive population for PKH67. The cumulative release of exosomes was calculated considering the initial amount of exosomes taken for incubation with the scaffolds.

EXAMPLE 9:

Animal Experiments (Critical-sized calvarial defect model)

In the present embodiment, we have used twenty-four male Copenhagen rats (4-5 months old, 200-250 g body weight), an inbred strain as a model to study the effect of biomaterial construct on calvarial bone regeneration. All the surgical procedures and other animal experiments were approved by the Institutional Animal Ethics Committee (ILS/IAEC-202-AH/DEC20) of Institute of Life Sciences, Bhubaneswar, India.

The rats were randomly divided into 4 groups such as: (1) defects without implantation (control group), (2) defects implanted with scaffold only (scaffold only group), (3) defects treated with exosome integrated scaffolds (scaffold+Exo group), (4) defects treated with scaffolds seeded with exosome treated cells (scaffold+Cells+Exo group). 80µg/ml to 100µg/ml of exosomes were used for the treatment of scaffolds and cells before implantation. Rats were given inhalation anaesthesia with sevoflurane after which they were anaesthetized using ketamine (80 mg/kg body weight) and xylazine (8 mg/kg body weight) via intraperitoneal injection. Tramadol was also given intraperitoneally (10mg/kg) before surgery and hair were removed from the region between the eyes to the posterior end of the skull. The skin above the skull was retracted laterally and the calvarium was scored using a 9mm trephine bur. During the surgery, the skull site was continuously irrigated with normal saline to maintain temperature equilibrium and to remove the bone chips. With continuous slow speed and gentle pressure with the trephine bur, the critical size calvarial defect was created successfully. The combination of scaffold implantation materials was then implanted at the defect site and the skin incision was closed with non-absorbable nylon sutures. The rats were sacrificed by CO2 inhalation after 8 weeks of implantation. Skulls were retrieved and fixed with 10% formalin for further analysis.

EXAMPLE 10:

Micro-CT analysis
The rat skulls were scanned 360º with Micro-CT (Bruker) and three-dimensional (3D) reconstructions were applied to produce images of the skulls. Along with that to quantify the new bone formation, bone mineral density (BMD), new bone volume (BV/TV), trabecular number, trabecular thickness, and trabecular spacing were calculated using the software.

EXAMPLE 11:

Histological analysis

After Micro-CT scanning and analysis, rat calvarial bones were decalcified using 10% EDTA (pH 7.4) for 30 days. After bones became soft enough to cut, they were dehydrated and embedded in paraffin. 5 µm thin sections were prepared using microtome for histological evaluations. For Hematoxylin-eosin (H&E) staining and Masson’s Trichrome staining, the tissue slices were first deparaffinized followed by dehydration and staining according to the protocol. The slides were visualized under a bright-field microscope and for analysis, ImageJ software was used.

EXAMPLE 12:

Exosomal RNA isolation and small RNA library preparation for sequencing

Exosomal Ribonucleic acid (RNA) was isolated from the pellet by phase separation method using trizol-chloroform. The aqueous layer was then precipitated with isopropanol to get the total RNA. The purity and concentration were checked using NanoDrop (Thermo) and Qubit (Thermo) respectively. Total 200 ng of RNA was taken initially to prepare the small RNA library using NEBNext®Small RNA Library Prep kit. Qubit reading was taken again to ensure library preparation. The small RNA library was purified using magnetic beads provided in the MagSure small RNA purification kit. After this step, TapeStation was performed to check the small RNA enrichment. Apart from exosomal small RNA sequencing, the expression of some of the well-known miRNAs and mRNAs involved in the bone remodelling process can be checked using qRT-PCR.

Results

The present invention provides a bone graft substitute constituting of mineralized osteoblast-derived exosomes and further integrating them with synthesized polymeric scaffolds. The method of the present invention provides a successful integration of pre-differentiated osteoblast derived exosomes onto the polymer scaffold and their release at the target site of critical-size rat calvarial defects. It has been observed that after 8 weeks post implantation at the defect site, 3 times more new bone formation was observed as compared to the control. This invention indicates a remarkable potential of exosomes isolated from mineralized osteoblasts in bone regeneration.

Further, the present disclosure provides insight into the therapeutic use of exosome-based scaffold construct for future tissue and organ repair. The 3D extracellular matrix of the present disclosure is capable of biodegradation, which is the key factor towards the initiation of bone regeneration. Further, no additional components such as proteins, cells, inorganic minerals and drugs are required in the scaffold composition for the bone formation.

Studying the osteogenic properties of MC3T3-E1 cells on incubation with osteogenic media

Mouse pre-osteoblasts (MC3T3-E1 cells) were treated with the osteogenic media for 7 days and 14 days to check their ability to mineralize. From ALP staining and ALP activity assays we found that after 7 days of osteogenic media treatment, the cells are showing significantly higher ALP deposition (Fig. 2A-i, B) as compared to the control where cells were grown in growth media and after 14 days of treatment, the cells tend to show elevated ALP activity (Fig. 2A-ii, B). Similar results were obtained from the Alizarin Red staining assay, which showed an increased amount of calcium deposition with an increased time of incubation (Fig. 2C). Also, the mRNA expression of osteogenic markers such as ALP, Col-1 and RUNX-2 were highly enhanced at 7 days and 14 days as compared to the control (Fig. 2D). These results suggested that MC3T3-E1 cells can show mineralization activities even after 7 days of osteogenic media treatment.

Characterization of mineralized osteoblast-derived exosomes

Mouse pre-osteoblasts (MC3T3-E1) were treated with osteogenic media for 7 days and exosomes were isolated from the culture supernatants by using ultracentrifugation. Their size, surface charge, morphology, distribution and marker proteins were checked with the help of DLS, zeta, electron microscopy and western blotting respectively. From the data we found that the isolated vesicles were measured about 150-200 nm in diameter (Fig. 3A), having a negative surface charge (Fig. 3B) and uniform distribution with circular morphology (Fig. 3C, D). The presence of some EV marker proteins such as CD81 and Tsg-101 (Fig. 3E) further confirmed that the vesicles are exosomes.

Uptake of exosomes by MC3T3-E1 cells

The uptake of fluorescence-labelled exosomes by the host cells (MC3T3-E1) was visualized with the help of confocal microscopy. The results showed that the PKH67 tagged exosomes were internalized by the host cells in a time dependent manner, with the highest uptake at 24 h of incubation as evident from the fluorescent particles surrounding the nucleus (Fig. 4).

Effects of exosomes on proliferation, migration and osteogenic differentiation of MC3T3-E1 cells

To get an appropriate concentration of exosome for further experiments, cell proliferation and migration assays were performed. For cell proliferation assay WST-1 was used by taking different concentrations of exosomes for variable time points. The OD value results showed that at day 3, 5, and 7, the exosomes can significantly promote cell proliferation with increasing concentration (Fig. 5A). The cell migration was assessed by trans-well assay. The results revealed that exosomes can also effectively promote cell migration through the membrane in a concentration dependent manner (Fig. 5B-i, ii). These studies revealed that exosomes are indeed important regulatory factors that enhance cell proliferation as well as migration. Then to investigate the in vitro mineralization ability, the appropriate concentration of exosomes (50 µg/ml) was incubated with MC3T3-E1 cells for 72h and we found a significant elevation in the gene expression level of some of the important osteogenic markers like ALP, Collagen and RUNX-2 (Fig. 5C). We also found that cells tend to undergo higher amount of mineralization when treated with differentiation media as compared to the cells treated with growth media (Fig. 5D).

Synthesis and characterization of cross-linked chitosan scaffolds

For achieving the therapeutic efficacy of naturally occurring exosomes, a matrix is needed for holding these at the target site and regulating their release in a sustained manner so as to achieve the required dosage at the defect site. A cross-linked polymeric scaffold was synthesized by using polysaccharides i.e., chitosan and collagen protein. The schematic diagram in Fig. 6A describes the mechanism of covalent cross-linking between the amino group and methyl group. The hydrogel was UV crosslinked and freeze-dried to get a 3D porous structure of chitosan scaffold (CS-scaffold). Their biocompatibility, osteoconductivity, mechanical strength, biodegradability and exosome release profile were checked. Proton NMR confirmed the cross-linking between the polysaccharide and protein (Fig. 6B). The morphology of the scaffolds was observed by SEM, which showed the porous structure of the scaffolds having a pore size ranging from 50 to 200nm (Fig. 6C). The rheological properties of the scaffolds are shown in Fig. 6D, which exhibited a higher G’ value than G’’ indicating that the elastic modulus of the scaffolds is increasing making it solid. The swelling properties of the scaffolds represent that the swelling ratio reached equilibrium at a faster rate at approximately after 5min of incubation in the solvent indicating their faster solvent retention capacity (Fig. 6E). The rate of degradation also shows faster loss of weight of scaffolds, with approximately 80% degradation within 3 days (Fig. 6F).

Exosome embedding and release from chitosan scaffold

To check the integration of exosomes on chitosan scaffolds, PKH67-tagged exosomes were incubated with the scaffolds overnight and imaged under fluorescence microscopy. From the images, we could find that the tagged exosomes are retained over the chitosan scaffolds (Fig. 7A). To know the release profile of exosomes from the scaffolds, the released exosomes were acquired in flow cytometry. Data suggested a fast exosome release profile related to the higher swelling ratio and degradation rate of the scaffolds (Fig. 7B).

Scaffold biodegradation as key factor for initiation of bone regeneration

Two types of 3D scaffolds using two different chemistries were prepared to compare the difference between the biodegradability. One of the porous 3D chitosan-base scaffolds were synthesized by ionic cross-linking and freeze-drying processes. Freezing at temperatures between –20°C and –25°C and subsequent lyophilization led to an interconnected porous system. It is known that non cross-linked chitosan membranes have poor chemical stability, will dissolve in an acid environment and therefore cross-linking is needed. Chitosan groups were cross-linked using sodium tripolyphosphate (TPP) (Fig. 8A). To confirm the level of cross-linking NMR analysis was done (Fig. 8B). SEM indicated a homogenous microporous structure with average sizes of the pores found to be 50-100 μm (Fig. 8C). The rheological properties of the scaffold showed higher G” compared to G’ indicating their good mechanical strength (Fig. 8D). The swelling degree profiles represented the mass increase of scaffolds after solvent absorption. It was found that at 24h, solvent uptake capacity reaches the maximum and remains constant thereafter (Fig. 8E). The rate of degradation of the scaffold in PBS along with lysozyme was found to be very slow till day 7 (Fig. 8F). In the in vivo model, no formation of new bone at the defect site is indicative of their very slow degradation rate, in comparison to the control, where with time the visible amount of new bone formation has occurred (Fig. 8G). The other type of scaffold was prepared by UV crosslinking method whose results are presented in Fig 6.

MicroCT analysis of bone regeneration

For in vivo assessment of bone regeneration, rat model of critical size calvarial defect (9 mm diameter) was created with the help of a trephine bur. The CS-scaffolds alone and in combination with exosomes were implanted at the defect site. After 8 weeks of implantation, the amount of new bone formation was compared to the control (with no implantation) and was evaluated by micro-CT scanning and analysis. From reconstructed micro-CT 3D images, it can be observed that very less amount of new bone was formed in the control group, while a significantly higher amount of new bone formation was found in the case of scaffold and scaffold exosome groups. Specifically, exosome-laden and exosome-treated cell-seeded scaffold groups showed the highest amount of new bone formation (Fig. 9A). Quantification of the microCT images in terms of new bone volume, trabecular thickness, trabecular spacing, and trabecular number provided further evidence that compared to other groups, significantly improved new bone formation was observed in the scaffold-exosome group (Fig. 9B-E). The comparative analysis of the average amount of new bone formation between the treated and the control group has been represented in graphical form (Fig. 10A). A tabular representation of the values showing three times new bone formation in treated groups than the control has been given below and also in Fig. 10B.
Control SC-only SC+Exo SC+Cells+Exo
Average fold change of new bone volume 1 1.88 3.2 3.0

Histological analysis of bone regeneration

After 4 weeks of decalcification, the skull bones were sectioned and stained with H&E, and Masson’s trichrome. HE staining of representative sections indicated the formation of fibrous connective tissues in the control group, while in the other three groups, solid new bone tissue formation was observed along the borders and in the centre of the defects (Fig. 11A). Masson’s staining showed more mature collagen formation in the scaffold-exosome group as compared to the control group with no implantation (Fig. 11B).

SPECIFIC EMBODIMENTS OF THE PRESENT DISCLOSURE:

The present disclosure relates to a 3D-extracellular matrix scaffold comprising: a porous polymer scaffold; pre-differentiated osteoblast derived exosomes, wherein a particle size of the pre-differentiated osteoblast derived exosomes is in the range of 50 nm-150 nm; and wherein the pre-differentiated osteoblast derived exosomes cross-linked with the porous polymer scaffold to form the 3D extracellular matrix scaffold.

Such extracellular matrix is disclosed, wherein the porous polymer scaffold is cross-linked chitosan and collagen protein.

Such extracellular matrix is disclosed, a diameter of the porous polymer scaffold is in the range of 5 mm to 20mm, and wherein the porous polymer scaffold and the pre-differentiated osteoblast derived exosomes is present in the matrix having a ratio in the range of 50:50.

Such extracellular matrix is disclosed, wherein the pre-differentiated osteoblast derived exosomes is in an amount having a range 80 ug/ml to 100ug/ml.

Such extracellular matrix is disclosed, wherein the porous polymer scaffold has a pore size in the range of 50 to 200 nm.

The present disclosure relates to a method of preparing a porous polymer scaffold, comprising the steps of:
a) covalently cross-linking chitosan and collagen protein via a methacrylation reaction using UV rays to form a polysaccharide-protein composite;
b) freeze-drying the composite;
c) followed by lyophilizing to form a porous polymer scaffold.

Such method is disclosed, wherein the freeze drying is carried out at a temperature in the range between –20°C and –25°C.

Such method is disclosed, wherein the crosslinking is carried out at temperature in the range between 23°C to 32°C at pH ranging from 3.5 to 6.

The present disclosure also relates to a method of preparing pre-differentiated osteoblast derived exosomes comprising the steps of:
preparing an exosome-depleted media in 20% fetal bovine serum (FBS) containing α-MEM (Minimum Essential Medium) by ultracentrifuging the said media at 1,50,000 xg for 18 hr at 4°C;
mineralizing pre-osteoblast cell line MC3T3-E1 in 50 µg/mL ascorbic acid, 100 nM dexamethasone, and 10 mM β- glycerophosphate;
incubating the MC3T3-E1 cells with α-MEM containing 10% exosome depleted media at temperature of 37°C with 5% CO2 and 100% relative humidity for 48 hours;
cell culturing in α-minimal essential medium supplementing with 10% FBS in vitro and 1% (v/v) penicillin/streptomycin to form MC3T3-E1;
collecting and processing the cell conditioned media with differential centrifugation to remove dead cells, larger particles, and microvesicles;
isolating exosomes from the processed conditioned media by ultracentrifuging at 1,50,000 xg for 90 min at 4°C to form pellets; and
resuspending the pellets containing exosomes in 1X PBS.

The present disclosure further relates to a method of preparing extracellular matrix comprising the steps of incubating an pre-differentiated osteoblast derived exosomes with a porous polymer scaffold at a temperature in the range of 23°C to 32°C.

INDUSTRIAL APPLICATION:

[0059] The present disclosure relates to a 3D-extracellular matrix scaffold with shelf-life of six months, which reduces patient discomfort, medical care costs as well as biodegradable and which is the key factor towards the initiation of bone regeneration.
, Claims:WE CLAIM:
1. A 3D-extracellular matrix scaffold comprising:
a porous polymer scaffold;
pre-differentiated osteoblast derived exosomes, wherein a particle size of the pre-differentiated osteoblast derived exosomes is in the range of 50 nm-150 nm; and
wherein the pre-differentiated osteoblast derived exosomes cross-linked with the porous polymer scaffold to form the 3D extracellular matrix scaffold.

2. The extracellular matrix as claimed in claim 1, wherein the porous polymer scaffold is cross-linked chitosan and collagen protein.

3. The extracellular matrix as claimed in claim 1, wherein a diameter of the porous polymer scaffold is in the range of 5 mm to 20mm, and wherein the porous polymer scaffold and the pre-differentiated osteoblast derived exosomes is present in the matrix having a ratio in the range of 50 to 50

4. The extracellular matrix as claimed in claim 1, wherein the pre-differentiated osteoblast derived exosomes is in an amount having a range 80 ug/ml to 100ug/ml.

5. The extracellular matrix as claimed in claim 1, wherein the porous polymer scaffold has a pore size in the range of 50 to 200 nm.

6. A method of preparing a porous polymer scaffold as claimed in claim 2 comprising the steps of:
d) covalently cross-linking polysaccharide and protein via a methacrylation reaction using UV rays to form a polysaccharide-protein composite, wherein the polysaccharide is chitosan and the protein is collagen;
e) freeze-drying the composite;
f) followed by lyophilizing to form a porous polymer scaffold.

7. The method as claimed in claim 6, wherein the freeze drying is carried out at a temperature in the range between –20°C and –25°C.

8. The method as claimed in claim 6, wherein the crosslinking is carried out at temperature in the range between 23°C to 32°C at pH ranging from 3.5 to 6.

9. A method of preparing pre-differentiated osteoblast derived exosomes comprising the steps of:
preparing an exosome-depleted media in 20% fetal bovine serum (FBS) containing α-MEM (Minimum Essential Medium) by ultracentrifuging the said media at 1,50,000 xg for 18 hr at 4°C;
mineralizing pre-osteoblast cell line MC3T3-E1 in 50 µg/mL ascorbic acid, 100 nM dexamethasone, and 10 mM β- glycerophosphate;
incubating the MC3T3-E1 cells with α-MEM containing 10% exosome depleted media at temperature of 37°C with 5% CO2 and 100% relative humidity for 48 hours;
cell culturing in α-minimal essential medium supplementing with 10% FBS in vitro and 1% (v/v) penicillin/streptomycin to form MC3T3-E1;
collecting and processing the cell conditioned media with differential centrifugation to remove dead cells, larger particles, and microvesicles;
isolating exosomes from the processed conditioned media by ultracentrifuging at 1,50,000 xg for 90 min at 4°C to form pellets; and
resuspending the pellets containing exosomes in 1X PBS.

10. A method of preparing extracellular matrix comprising the step of incubating an pre-differentiated osteoblast derived exosomes with a porous polymer scaffold at a temperature in the range of 23°C to 32°C.