Description:FIELD OF THE INVENTION:
The present disclosure relates to the field of regenerative dentistry and biomaterials. Specifically, the present invention discloses an injectable graft comprising exosomes and ECM (extracellular matrix) promoting polymer matrix, will aid in regenerating dental bone at the site of dental implantation. More specifically, present disclosure relates to the therapeutic use of human dental pulp stem cell (hDPSC)‑derived exosomes as cell‑free osteogenesis inducing agents; and injectable or implantable chitosan–collagen based polymer scaffolds that control local release of exosomes at the target sites particularly in alveolar and maxillofacial defects associated with tooth loss, periodontal disease, trauma or tumour resection.
BACKGROUND OF THE INVENTION
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, which arises from fractures due to accidents, osteoporosis, osteosarcoma, maxillofacial injuries where the injury goes beyond a critical limit, the bone will not be able to self-heal without surgical and therapeutic intervention.
Dental bone loss commonly arises from infections of the periodontal tissue, pulpal necrosis, traumatic injuries, or progressive bone degeneration. These conditions affect individuals across all age groups and, when left untreated, compromise tooth stability and dental bone health. Restoring dental bone health is crucial for maintaining a healthy life.
Although several grafting materials are clinically available, most do not provide sufficient biological signalling required for accelerated and functionally organized bone regeneration. Effective bone healing requires coordinated osteogenic, angiogenic, and immunomodulatory cues, which current grafts often fail to deliver. The existing grafting materials and procedures often lack adequate biological signalling to promote rapid and functional bone regeneration. Further, exosomes secreted by osteogenic cells have recently been emerged as potent biomolecular carriers capable of enhancing bone formation. However, native exosomes are not suitable for direct implantation due to poor structural stability, uncontrolled diffusion, and short retention at the defect site.
Prior research work in dental and orthopaedic tissue engineering has focused on combining biodegradable scaffold and biological component such as cells, growth factors, or extracellular vesicles. Among scaffold materials, chitosan and collagen have attracted interest due to their biocompatibility, extra cellular membrane (ECM) like nature and ease of chemical modification for controlled degradation and mechanical tuning. However, many chitosan– or collagen based dental scaffolds are simply used as passive carriers for bone substitutes or small molecules and do not themselves incorporate a well defined, cell secreted signalling system.
Therefore, there is a vital need for local, scaffold mediated delivery of exosomes in dental bone defects that provides mechanical stability, protects and retains exosomes, and releases them in a controlled fashion over the early healing period.
The present invention addresses these gaps by designing an hDPSC exosome loaded chitosan–collagen scaffold specifically for dental and maxillofacial bone regeneration, with defined structural, release and biological performance characteristics that are not disclosed in the prior art.
OBJECTS OF THE INVENTION
It is therefore an object of this invention to provide an implant material, using exosomes from the Donor’s pulp tissue which will augment bone regeneration at the site of implantation.
Another object of the present invention is to provide a tissue regenerative scaffold that can be placed in dental and maxillofacial bone defects, and that supports new bone formation in a defined manner.
Another object of the present invention is to provide a bioactive scaffold incorporating human dental pulp stem cell (hDPSC) derived exosomes that promote osteogenesis, mineralisation and bone formation at the defect site.
Still another objective of the present invention is to provide cross-linked polymeric scaffolds synthesized out of chitosan/collagen composite to provide strength and a well-formed matrix for holding and releasing an appropriate dose of exosomes at the target site.
Yet another object of the present invention is to design a chitosan–collagen based polymer scaffold that serves both as a three dimensional extracellular matrix like framework and as a depot for controlled release of exosomes.
Yet another object of the present invention is to obtain a scaffold that is biocompatible, has suitable mechanical strength, shows favourable swelling and degradation behaviour, and does not provoke an undesirable immune response in vitro.

Another objective of the present invention is to provide a method for preparing the exosome-loaded chitosan-collagen scaffold.
These and other objects and advantages of the present subject matter will be made apparent to a person skilled in the art from the following detailed description taken in conjunction with the accompanying drawings which illustrate preferred embodiments of the present invention.
SUMMARY OF THE INVENTION
In a first aspect, the invention relates to a bioactive scaffold composition incorporating human dental pulp stem cell (hDPSC) derived exosomes that promote osteogenesis, mineralisation and bone formation at the defect site wherein the scaffold composition is particularly suited for dental and maxillofacial bone regeneration, comprising:
a covalently cross-linked, porous polymer matrix formed from methacrylated chitosan and collagen with polyethylene glycol dimethacrylate (PEGDMA) as the crosslinker; and
exosomes derived from human dental pulp stem cells (hDPSCs) integrated within the porous structure of said matrix; wherein the scaffold exhibits:
interconnected porosity with pore sizes ranging from 50-200 nm as determined by scanning electron microscopy;
elastic mechanical behaviour characterised by storage modulus (G′) exceeding loss modulus (G″) under oscillatory rheological testing at physiological temperature;
controlled exosome release providing 70-80% cumulative release after 12 hours of incubation when immersed in solvent.
In a second aspect, the invention provides a method for preparing the aforementioned scaffold composition, comprising:
Extracting and -culturing hDPSCs from human dental pulp tissue to obtain conditioned medium;
isolating exosomes from said conditioned medium by ultracentrifugation and mineralization of the cells by Exos treatment
synthesising methacrylated chitosan by reaction with methacrylic anhydride followed by dialysis purification;
mixing the methacrylated polymers and crosslinker PEGDMA in 1:1 ratio with photoinitiator (Irgacure), and collagen subjecting to UV cross-linking to form hydrogel;
lyophilising the hydrogel to obtain 3D porous structure of the cross-linked polymer scaffold.
In a third aspect, the invention provides use of the scaffold composition of the first aspect in the manufacture of a bone regenerative implant for treating alveolar ridge defects, peri-implant bone loss, and/or maxillofacial bone defects in human subjects.
The hDPSC-derived exosomes incorporated in the scaffold are characterised by: average diameter of 80-150 nm; zeta potential of -10 to -25 mV; expression of EV markers CD81 and CD63; and demonstrated capacity to enhance osteogenic differentiation of recipient stem cells as evidenced by increased mineralisation (Alizarin Red S staining) and upregulated expression of COL1A1, OCN and VEGF genes.
The summary hereinabove is provided only to introduce selected aspects of the invention in a simplified form and is not intended to identify every feature or to define or limit the claimed scope in any manner. The invention is described in further detail in the following specification, and the scope of protection is to be determined solely from the appended claims, interpreted considering the full description and drawings.
BRIEF DESCRIPTION OF DRAWINGS
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:
Schematic-1: Representation of pulp tissue extraction, seeding and colony formation of hDPSCs (Sonoda S. et. al., STAR protocols, 2022, 101386).
Figure 1 shows the processing of dental pulp tissue and the formation of hDPSC colonies in culture. (A) i) Dental pulp tissues in washing media 1XPBS, ii) Washed pulp tissues from donor nos. 1 and 2, iii) Washed pulp tissues from donor no. 3. (B) Microscopic images of i) multiplied hDPSCs on cell culture flask, ii) immature adherent cell cluster after 1 week of seeding, iii) mature adherent cell cluster after 2 weeks of seeding. Scale bar: 100 mm.
Figure 2 shows the stemness profile of hDPSCs from 4 different donors (A,B,C,D), by identifying the surface markers of hDPSCs including expression of CD44 and CD90 measured by flow cytometry. i) unstained, ii) CD44-PE positive, iii) CD90-FITC positive and iv) CD44-CD90 double positive cells, which indicated >90% positive signal.

Figure 3 shows Tri-lineage differentiation potential of hDPSCs to adipocytes, osteocytes, and chondrocytes after incubation with the respective supplements for 21 days as evident from (A) Oil-red O staining, (B) Alizarin Red staining, and (C) Alcian Blue staining.
Figure 4 shows characterization of hDPSC derived exosomes by determination of average size, zeta potential, TEM imaging and Western blotting analyses. (A) DLS peak representing the nano size of the exosomes (approx. 90nm), (B) Zeta peak representing the negative zeta potential of exosomes (approx. -15mV). (C) Comparison of average size, (D) Zeta, and (E) Yield of exosomes isolated using different methods. (F) Yield of exosomes normalized to 10 million cells. (G) TEM micrographs representing the cup-shaped irregular structure and uniform morphology of the exosomes. (H) Western blots showing the presence of EV markers (CD81, CD63) in exosomes and the absence of calnexin (ER marker).
Figure 5 shows representative confocal microscopy images of PKH67 tagged exosomes (green) uptake after incubation with hDPSCs for 6h, 12h, 24h, and 48h. Cell cytoskeleton and nuclei were stained with rhodamine-B (red) and DAPI (blue), respectively.
Figure 6 shows cell proliferation and mineralization potential of stem cells hDPSCs and differentiation potential of hDPSC exosomes into osteogenic lineage. (A) MTT assay results showing cell proliferation (n=4). (B) RT-PCR analysis of osteogenic markers (ALP, COL1-A1, RUNX2, and OCN, and VEGF) showing increased osteogenesis with the hDPSC-Exos treatment (n=4). Control was cells without any treatment. (C) ARS staining (D) Crystal violet staining of control and Exos treated cells.
Figure 7 shows dimension of the synthesized scaffolds using different ratio of chitosan & PEGDMA (A) 1:1, (B) 1:0.5, (C) 0.75:1, with 0.1% (w/v) collagen. The 0.1% of collagen is with respect to the total volume of the formulation.
Figure 8 shows fluorescent images confirming uptake of fluorescent tagged (PKH67) exosomes by the scaffolds (Chi:PEGDMA= 1:1) under a fluorescent microscope.
Figure 9 shows the release profile of fluorophore-tagged exosomes from the polymer scaffolds (Chi:PEGDMA= 1:1). (A) Histogram at different time points as a representative of the gating used in flow cytometry. (B) Quantification with FlowJo software.
Figure 10 shows characterization of cross-linked scaffolds. A) SEM image showing a porous structure (50−200 nm) of the ratio i) CS: PEGDMA=1:1, which is not prominent in the ii) 0.75:1 & iii) 1:0.5 ratios. (B) Rheological property of the scaffold in the i) 1:1 formulation ii) 0.75:1 & iii) 1:0.5. (C) Percentage of scaffold degradation in the i) 1:1 formulation ii) 0.75:1 & iii) 1:0.5. (D) Swelling ratio of the scaffold formulation in PBS showing their water uptake ability.

Figure 11 shows biocompatibility of the loaded scaffolds using A) PrestoBlue and B) MTT assay.
Figure 12 shows immune profile of the pro-inflammatory markers (IL-10, IL-6, and TGF-b) on hDPSCs seeded on the loaded scaffolds.
DETAILED DESCRIPTION OF THE INVENTION
The detailed description of certain illustrative embodiments of the present invention is set out below to enable a person skilled in the art to make and use the invention without undue experimentation. The description is intended to convey the core technical features and working of the invention; it is not intended to confine the invention to the specific examples or parameter ranges mentioned, and reasonable modifications or variations that a skilled person would consider in light of this disclosure are intended to fall within the scope of the claims.
It should be understood that different structural or compositional options for the scaffold, and different ways of isolating and characterising human dental pulp stem cell (hDPSC) exosomes, may be employed while still relying on the same inventive concept of a chitosan–collagen based, exosome‑loaded scaffold for dental and maxillofacial bone regeneration. Unless the context clearly requires otherwise, singular terms such as "a", "an" and "the" include the plural, and terms such as "comprise" or "include" indicate the presence of stated features without excluding additional features. Technical and scientific terms used in this description have the meanings that would generally be given to them by an ordinarily skilled person in the relevant field, having regard to standard usage in biomaterials and tissue engineering.
The term "hDPSC-derived exosomes" in the context of the present invention refers to extracellular vesicles isolated from conditioned medium of cultured human dental pulp stem cells, characterised by expression of CD63 and CD81 markers, negative zeta potential, and demonstrated capacity to enhance osteogenic differentiation of recipient stem cells.
The term "methacrylated chitosan-collagen scaffold" or "polymer scaffold" refers to a covalently cross-linked, porous three-dimensional (3D) matrix formed by UV photo-crosslinking of methacrylated chitosan and PEGDMA in defined ratios with collagen, exhibiting interconnected porosity (50-200 nm), elastic rheological behaviour (G′ > G″), and biodegradability in physiological conditions.
The phrase "controlled exosome release" refers to the scaffold's capacity to retain integrated exosomes and release 70-80% of the loaded cargo after 12 hours incubation in a solvent, as measured by flow cytometry of fluorescently labelled exosomes.
The present invention provides a bioactive scaffold composition for dental bone regeneration comprising a porous, covalently cross-linked chitosan-collagen polymer matrix integrated with exosomes derived from human dental pulp stem cells (hDPSCs). The scaffold serves dual functions: (i) mechanical support and space maintenance within alveolar or maxillofacial bone defects, and (ii) controlled local delivery of osteoinductive exosomes that promote stem cell proliferation, mineralisation and osteogenic gene expression.
In an embodiment of the present invention, the polymer scaffold is formed from methacrylated chitosan and PEGDMA present in a weight ratio of 1:1 with 0.1% of collagen. The scaffold is not limited to this specific ratio but includes other ratios (1:0.5, 0.75:1) that yield comparable porosity, mechanical properties and degradation profiles. The disclosure of weight ratios includes all permutations and combinations falling within these ranges and the extreme values thereof.
In another embodiment, the scaffold exhibits pore sizes in the range of 50-200 nm as observed by scanning electron microscopy. The disclosure of pore size includes all values and combinations within this range.
In a further embodiment, the hDPSC-derived exosomes have an average hydrodynamic diameter of 80-150 nm and zeta potential between -10 mV and -25 mV, as determined by dynamic light scattering.
ADVANTAGES OF THE INVENTION
Tissue Regenerative Polymer Scaffold utilizing Human Dental Pulp Stem Cells Exosomes of the present invention offers several technical and practical advantages:
It is a cell free system that avoids the regulatory complexities associated with implantation of live cells, while retaining the biological activity of hDPSC exosomes.
The methacrylated chitosan–collagen scaffold provides a predictable 3D structure and mechanical support with controlled bioactive signalling which makes it suitable for dental sockets, peri-implant and maxillofacial defects.
Exosomes are loaded and released in a controlled manner, with a substantial fraction delivered during the early phase of healing (12-24h), thereby amplifying osteogenic signalling precisely when needed.
Demonstrated dental stem cell osteogenesis and low immunogenicity.
The construct is prepared from clinically accessible dental pulp tissue and widely used biomaterials, supporting scalability and potential cost effectiveness for routine dental practice.
EXAMPLES OF THE INVENTION
EXAMPLE 1
Isolation and culture of human dental pulp stem cells (hDPSCs)
Example 1 provides that the human third molar was extracted from the donor/volunteer under general surgical conditions in the dental hospital (Institute of Dental Sciences, SOA) after signing the consent form. Immediately post-extraction, tooth was placed in normal saline. The crown enamel and dentin were removed using a sharp carbide bur to access the pulp chamber. Dental pulp tissue was carefully pulled out from the cavity using sterile needle and pointed forceps and transferred to ice-cold Alpha-MEM containing 10% FBS as transport medium and transported to the laboratory on ice.
In the laboratory, pulp tissues were washed three times with 1x PBS by centrifugation at 2,500xg for 5min at 4°C to remove remaining tooth chips and debris. Tissues were minced to small fragments with a sterile scalpel (Fig. 1A). The tissue fragments were then digested in a tissue dissociation solution containing collagenase (3 mg/ml)-dispase (4 mg/ml) (MERCK, SIGMA) followed by mixing with mild vortexing and incubation at 37°C for 1h with intermittent mixing. The dissociated tissues were centrifuged at 2,500xg, for 5min at 4°C and the cell pellets were resuspended in complete growth media (Alpha-MEM + 10% FBS + 1% Pen-strp) and seeded into T-25 flasks for culturing of human dental pulp stem cells (hDPSCs). Cultures are maintained at 37°C, 5% CO₂ in a humidified atmosphere. Stem cell attachment, growth and colony formation were observed microscopically regularly within 72 hours to 2 weeks (Schematic-1).

EXAMPLE 2
Characterisation of hDPSC stemness and differentiation potential

To check the stemness of hDPSCs, the expression level of stem cell markers was checked by flow cytometry. hDPSCs of passage-3 to 5 were used. hDPSCs (0.3x106) were trypsinized, collected, and washed with 1xPBS. The cells were then labelled with stem cell markers; anti-CD90 (Cat. No. #11-0909-41, ThermoScientific), and anti-CD-44 (Cat. No. #12-0441-81, ThermoScientific) in dark for 30-45 minutes at room temperature. The cells were centrifuged and resuspended in 300µl of 1x PBS and analysed by flow cytometery (CytoFlex, Beckman coulter).

Tri-lineage differentiation was performed by seeding 1x105 hDPSCs per well in 12 well plates. To check the osteogenic differentiation ability of hDPSCs, the cells were cultured in 12-well plates with growth medium (Cat. No. AL221A, Alpha-MEM, HIMEDIA). After the cells reached 80% confluency, the media were changed.

Osteogenic differentiation: Control cells were cultured in growth media (GM), for osteogenic differentiation, the cells were cultured in differentiation media (DM) containing 50 µg/ml L-ascorbic acid (Sigma), 100nM dexamethasone (Sigma) and 10 mM β-glycerophosphate (Sigma). All media were replaced in every 3 days. After 21 days of incubation, to evaluate the cell mineralization and osteogenesis potential, the cells were washed with PBS, fixed with 10% formaldehyde, and incubated with 2% Alizarin Red S (Sigma) solution and ALP reagent for 45min. After washing with miliQ water, the matrix calcium deposition and ALP concentration were checked under a brightfield microscope.

Adipogenic Differentiation: After 21 days of incubation, the cells are fixed and stained at room temperature with Oil Red O.

Chondrogenic Differentiation: 1 million cells were pelleted down to form spheroids and incubated with normal growth media (Control) and chondrogenic differentiation media containing the chondrogenic supplement for 21 days. The media was changed every 3 days. After 21 days, the spheroids were fixed and stained with Alcian blue as per the user’s guidelines provided. Images were taken with a brightfield microscope.
EXAMPLE 3
Isolation and characterisation of hDPSC-derived exosomes
To isolate exosomes, hDPSCs were cultured till 60- 70% confluency, after which the media was changed from complete growth media to serum-free media containing alpha-MEM and Pen-strp and were cultured for 48h. The conditioned media were collected and centrifuged at 4°C: centrifuged at 500×g for 10 minutes, 5000×g for 15 minutes and 10,000×g for 30 minutes to remove dead cells, debris, and large molecules. The filtered media were then ultracentrifuged at 150,000×g for 90 minutes by ultracentrifugation (Optima™ XPN, Beckman Coulter, California, USA); the supernatant was discarded, and the pellet at the bottom of the tube was resuspended in PBS. The total concentration of exosomes was determined by a bicinchoninic acid (BCA) kit (Invitrogen). hDPSC-EXO was either stored at −80°C for follow-up experiments or used immediately.
Ultracentrifugation method:
• Centrifuged at 10,000g, 45 min, 4°C (to remove debris)
• Supernatant ultracentrifuged at 150,000g, 90 min, 4°C
• Exosome pellet resuspended in 1X PBS, stored at -20°C
Kit-based method:
• Centrifuged at 10,000g, 45 min, 4°C (to remove debris)
• Supernatant + precipitation reagent (2:1) + thorough mixing, overnight at 4°C
• Centrifuged at 10,000g, 1 hour, 4°C; pellet resuspended in 1X PBS, stored at -20°C
EXAMPLE 4
Characterization of hDPSCs-Exos
For size and charge measurements, hDPSC-EXO were mixed with 1x sterile PBS in a ratio of 1:9 and mixed thoroughly. The dynamic light scattering and zeta potential determination for size and charge measurements were performed using Nanosight (Malvern, Nano ZS). The exosome size data refers to the scattering intensity distribution (z-average). All measurements were taken in 3 replicates.
The morphology of hDPSC-EXO was identified by transmission electron microscopy (TEM). For TEM, 10µl of re-suspended EVs were mixed with an equal volume of 4% PFA overnight at 4°C for fixation. 5µl of the mixture was placed on formavar-coated Cu TEM grids (Agar Scientific) for 20min in a dry environment. After washing with PBS, the grids were stained with 1% uranyl acetate for 5min, followed by a second wash. Grids were gently blotted on Whatman filter paper and air-dried. The EVs were imaged by TEM (TEM_2100plus, JEOL, Japan) at 120 kV.
Protein concentration of freshly isolated exosomes was analyzed using BCA Protein Assay kit (Pierce, Rockford, USA). Briefly, hDPSCs derived exosomal pellets were resuspended with lysis buffer (RIPA buffer+Protease Inhibitor+Phosphatase Inhibitor) in the ratio 2:1 to collect the whole cell lysate, this was followed by water bath sonication (4 cycles of 5 min each) 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-CD63, and anti-Calnexin antibodies. Equal amounts of proteins (20 µg) were resolved on 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on the gel were transferred to a 0.45µm PVDF membrane and incubated with 5% Bovine serum albumin (Sigma) for 1hr at room temperature for blocking. The blot was incubated with different primary antibodies, such as anti-CD81, anti-CD63, and anti-Calnexin overnight at 4-8°C. The blots were incubated with anti-mouse and anti-rabbit HRP conjugated secondary antibodies and protein expression was detected using chemiluminescent HRP substrate (ab5801) and imaged under ChemiDoc (BioRAD, Hercules, CA, USA).
EXAMPLE 5
Exosome uptake analysis and osteogenic effects
The uptake of exosomes by host cells was monitored with PKH67 Green Fluorescent Cell Linker Midi Kit (Sigma-Aldrich) according to the manufacturer’s protocols. PKH67-labelled exosomes are incubated with hDPSCs for 2hr, 6hr, 12hr, 24hr, and 48hr . Images were captured using a confocal laser scanning microscope.
To evaluate the proliferation of cells, 25µg/µl, 50µg/µl, 75µg/µl, and 100µg/µl of hDPSC-EXOs were treated to the cells for 24h and 48h. After treatment with MTT the absorbance were taken at 570 nm.
To evaluate the mineralization potential of hDPSC-EXOs on stem cells, 50µg/µl were treated to the cells for 14days. After which the cells were stained with alizarin red & crystal violet. The total RNA was extracted, and quantitative real-time-polymerase chain reaction (qRT‒PCR) was performed to detect the expression of osteogenesis-related genes such as ALP, COL1-A1, RUNX2, OCN, VEGF.
EXAMPLE 6
Mineralization of the cells by Exos treatment
For RNA isolation, cells were collected by centrifugation and supernatant was discarded. The pellet was mixed with TRIzol reagent and incubated at room temperature for 5 minutes. Chloroform was mixed and incubated at room temperature for 3 minutes. The sample was centrifuged in 12000g for 15 minutes at 4˚c for phase separation. The aqueous layer was collected and passed through the DNA column. Again, sample was centrifuged in 12000g for 1 minute at room temperature. Isopropanol was added and incubated for 10 minutes. The sample was centrifuged at 12000g for 10 minutes at room temperature. The supernatant was discarded and the pellet was resuspended with 75% ethanol. Again, sample was centrifuged in 14000g for 5 minutes at room temperature. The supernatant was discarded and the pellet was air-dried thoroughly. Then Nuclease-Free Water was added; concentration and purity of RNA was checked and further used for cDNA synthesis in PCR for 2h at 37˚C, 5min at 85˚C and in ice for 2min. After preparation of C-DNA sample, SYBR-Green was added to each sample and mixed accordingly. Upon adding the primer, sample was run in RT-PCR for 1h 30min and analysed accordingly.
EXAMPLE 7
Synthesis of methacrylated chitosan-collagen scaffolds
Methacrylated chitosan and collagen was synthesized by reacting chitosan with methacrylic anhydride. Briefly, 1.5% w/v chitosan solution and collagen (0.1% w/v of total volume) were prepared by dissolving chitosan in 4% v/v acetic acid at room temperature for 24h. Methacrylic anhydride was added slowly, and the mixture was mixed at room temperature, 60 rpm for 12h and protected from light. The mixture was redispersed in deionized water and purified by dialysis (MWCO: 12−14 kDa membrane) for 72h. The final products were freeze-dried in 48-well plates (9−10 mm diameter) for 72h and stored at 4°C for future use. The required scaffold was fabricated by taking chitosan−methacrylate and PEGDMA in a 1:1 w/v ratio with 0.1% of collagen. 0.6% w/v of Irgacure was added, and the combining solution was UV-cross-linked. Then, the resulting scaffolds were washed overnight and freeze-dried for 72h to obtain the final scaffold constructs having dimensions of 9 mm X 4 mm (diameter x thickness).
EXAMPLE 8
Characterization of the polymer scaffolds
a. SEM Analysis: The morphology of the scaffolds was examined using SEM (JEOL, Japan). For dry scaffolds, a small portion was directly taken for imaging. For cell-seeded scaffolds, after the required time point, the cell−scaffold construct was fixed with formalin and dried with an increased concentration of ethanol, followed by overnight drying inside the fume hood.
b. Mechanical Strength: The rheological properties were measured using a modular compact rheometer (MCR 102e MultiDrive, Anton Paar). The storage modulus (G′) and the loss modulus (G″) were analyzed, which indicated their elastic properties.
c. Swelling Ratio: To check the swelling ability, the scaffolds were immersed in 1× PBS and MilliQ water at 37°C. The initial dry weights of the scaffolds after lyophilization were noted down. Then, the scaffolds were removed from the solvents and weighed after the excess was soaked at the required time points. The swelling ratio was calculated using the below formula-
Swelling ratio = [(Wt -W0)/W0] × 100%
where W0 is the initial dry weight of the scaffold and
Wt is the weight of the scaffold after time t.
d. Degradation Profile: The degradation of scaffolds (n = 3) was performed with phosphate-buffered saline solution (1× PBS, pH= 7.4) containing 20 mg/L of lysozymes at 37°C on an orbital shaker at 50 rpm. Enzymatic degradation was monitored for 7 days, while the lysozyme solution was refreshed on the third day. At defined time points, samples were removed from the medium, rinsed with distilled water, and dried in an oven at 50 °C until constant mass. The samples were weighed before (m1) and after in vitro degradation (m2). The degradation degree (Δm) was determined as the weight loss with respect to the initial weight of the sample
Δm= (m1-m2/m1)*100
Scaffolds were then frozen and lyophilized to obtain the final dry weight. The percentage rate of degradation was analyzed using the same formula as above.
EXAMPLE 9
Integration of EVs over the Scaffold and Their Release
To investigate the integration of EVs on scaffolds, fluorescence microscopic analysis was done after incubating PKH67-labeled EVs with scaffolds for 24 h at 37°C with 5% CO2. Scaffolds were pretreated with serum-free media for 24 h. To check the release profile of EVs from the scaffolds, a total of 250 μg of EVs was taken at the beginning for PKH67 labeling. The labeled EVs were seeded over the scaffolds and incubated for 4 h at 37°C with 5% CO2 for complete integration. The Scaffold + EVs constructs were put into 1× PBS, and the solvent was collected at different time points, starting from 5 min to 48 h. The solvents containing the labeled EVs were acquired in a flow cytometer (CytoFLEX, Beckman Coulter) to check the percent positive population for PKH67. The labeled EVs were gated from the live cell population by taking the FITC green fluorescence channel with 488 nm excitation. Unlabeled EVs were taken as the control to set the gating limits. The cumulative release of EVs was calculated considering the initial amount of EVs taken for incubation with the scaffolds.
EXAMPLE 10
Immunogenicity profiling of EV loaded scaffolds
To determine the immune profile of proinflammatory and anti-inflammatory markers, like TGF-beta, IL-10, and IL-6 were procured from ELabscience. Cells were seeded on the scaffold-EV construct, keeping the controls (only scaffold), and only cells (hDPSCs). Approximately 1 million cells were seeded onto the scaffold and scaffold-EV; incubated for 48h. Cells were trypsinized, washed and pelleted in microcentrifuge tubes. To the 50ul of cell suspension, antibodies were added as per the recommended concentrations and incubated for 30min in dark. A tube of unstained cells was kept for initial gating. The labelled samples were then resuspended in 1XPBS and acquired in flow cytometer (LSR Fortessa) to check the FITC positive population.
RESULTS
The present disclosure provides a tissue regenerative construct that combines a methacrylated chitosan–collagen scaffold with exosomes derived from human dental pulp stem cells (hDPSCs). In summary, the disclosure demonstrates: (i) successful isolation and characterisation of hDPSC exosomes; (ii) their ability to promote proliferation and osteogenic differentiation of stem cells in vitro; and (iii) effective integration and controlled release of these exosomes from a porous polymer scaffold that is itself biocompatible and biodegradable. Together, these findings support the use of the construct as a cell free bone regenerative material for dental and maxillofacial applications.
Dental pulp tissue processing and hDPSC isolation
Human dental pulp tissue was successfully retrieved from extracted third molars. Dental pulp tissues were minced to small pieces with a sterile scalpel (Fig. 1A). The growth and colony formation of the cells were observed. Fig. 1B represents the attachment of hDPSCs to the culture plate (Fig. 1B-i) after 72h of seeding, and the adherent cell colony formation (Fig. 1B-ii, iii) after 1 and 2 weeks indicating robust and reproducible isolation of hDPSCs from samples. These observations confirm that dental pulp provides a practical stem cell source for generating exosome producing cultures.
Characterization of the stemness of hDPSCs
To check the stemness of the hDPSCs, the cells were tagged with stem cell markers and acquired in flowcytometer. The plots suggested that hDPSCs exhibited intense expression of stem cell markers (CD 90- 99.31%), (CD 44- 99.67%) (Fig. 2) as compared to the unstained control cells. The results indicated that the stemness characteristics of the extracted hDPSCs are in line with the characteristics of mesenchymal stem cells (MSCs).
Lineage specific differentiation of hDPSCs
To check the osteogenic differentiation ability of hDPSCs, cells were cultured under adipogenic, osteogenic and chondrogenic differentiation conditions. The cells developed characteristic lineage specific morphological and staining patterns. Oil Red O positive lipid droplets for adipocytes (Fig. 3A), Alizarin Red S positive mineral deposits for osteogenic cultures (Fig. 3B), and Alcian Blue positive cartilaginous matrix in chondrogenic spheroids. (Fig. 3C) These results demonstrate that the isolated hDPSCs retain tri lineage differentiation potential, indicating suitability as an exosome source for bone tissue engineering applications.
Isolation and characterization of the hDPSC-Exosomes
Exosomes were successfully isolated from conditioned media of hDPSC cultures using both ultracentrifugation and kit based precipitation methods.
The DLS peaks indicated that the isolated vesicles are of 90 nm in size (Fig. 4A) with a negative surface potential of around -15 mV (Fig. 4B). TEM images showed that hDPSC-EXOs exhibited a bilayer membrane and cup-shaped morphology with a diameter of approximately 100–150 nm (Fig. 4F). Western blot images indicated the presence of EV markers (CD81, CD63) in exosomes (Fig. 4G). All these results proved that the nano-vesicles, exosomes were successfully isolated from the conditioned media of hDPSCs by using ultracentrifugation, Exsure kit and Invitrogen kit.
5. Uptake of hDPSC-Exosomes by stem cells
To verify uptake of hDPSC-EXOs by the host cells (hDPSCs), PKH67 labelled hDPSC EXOs were incubated with hDPSCs and visualised by confocal microscopy at various time points (2h, 6h, 12h, 24h, and 48h). Results showed that the PKH67-tagged hDPSC-EXOs were internalized by the host cells in a time-dependent manner as evident from the fluorescent particles surrounding the nucleus. (Fig. 5).
Mineralization potential of hDPSC-Exosomes
To evaluate the proliferation of cells, 25µg/µl, 50µg/µl, 75µg/µl, and 100µg/µl of hDPSC-EXOs were treated to the cells for 24h and 48h. After treatment with MTT the absorbance were taken at 570 nm which indicated proliferation of the stem cells hDPSCs with higher concentration of exosome treatment (Fig. 6A). To evaluate the mineralization potential of hDPSC-Exos on stem cells, 50µg/µl exosomes were treated to the cells for 14days. After which the cells were stained with alizarin red & crystal violet. The total RNA was extracted, and quantitative real-time-polymerase chain reaction (qRT‒PCR) was performed to detect the expression of osteogenesis-related genes such as ALP, COL1-A1, RUNX2, OCN, VEGF.
Results indicated that after 14 days of incubation with Exos, hDPSCs could differentiate into osteogenic lineage as evidenced by the higher amount of calcium deposition (Fig. 6C), and adhered cell matrix formation (Fig. 6D) as compared to the control cells. In line with the staining results, qRT-PCR results also indicated significantly higher expression of COL1-A1, OCN, and VEGF in the 14 day exosomes treated hDPSCs compared to the control cells (Fig. 6C). The results overall indicated that the hDPSC-Exos have osteogenic differentiation potential.
Synthesis of the polymer scaffolds
To control the release of the exosomes it was integrated within a cross-linked polymeric scaffold. The scaffold was synthesized by using polysaccharides, chitosan, and collagen protein mixed with different weight ratios of PEGDMA (Chitosan: PEGDMA= 1:1, 1:0.5, 0.75:1). The mixture formed a hydrogel which was UV cross-linked and freeze-dried to get a 3D porous structure of the cross-linked polymer scaffold. The pictures of the scaffolds showing the dimensions is given in fig. 7.
Integration of exosomes within the polymer scaffolds
PKH67-tagged EVs were incubated with cross-linked polymer scaffolds overnight and imaged under a fluorescence microscope to confirm their integration. The images showed that the tagged EVs were retained over the cross-linked polymer scaffolds (Fig. 8).
Release of the integrated exosomes from the crosslinked polymer scaffolds
To determine the release profile of exosomes from the scaffolds, the released exosomes were acquired using flow cytometry. Figure 9 shows the gating strategy used during the sample acquisition. The data suggested a faster EV release profile, with approximately 70− 80% release after 12 h of incubation in the solvent.
Characterization & optimization of the ratio of the polymer scaffolds
A cross-linked polymer scaffold was synthesized by using polysaccharides, chitosan, and collagen protein with the mechanism of covalent cross-linking between the amino group and the vinyl groups. The hydrogel formed was UV cross-linked and freeze-dried to get a 3D porous structure of the cross-linked polymer scaffold. The morphology of the scaffold was observed by SEM, which showed the porous structure of the scaffold having a pore size ranging from 50 to 200 nm (Figure 10A). The rheological properties of the scaffold are shown in Figure 10B, which exhibited a higher G′value than G, indicating that the elastic modulus of the scaffold is increasing. The rate of degradation also shows a faster loss of weight of the scaffold, with approximately 80% degradation within 3 days (Figure 10C).
Characterization of the loaded scaffolds
To check the biocompatibility of the loaded scaffolds, approx. 1 million cells were seeded for 48h. Cell viability in terms of fluorescence intensity was observed and plotted by using Prestoblue assay. Cell viability in terms of spectrophotometric reading was observed using MTT assay. Both these assays showed higher number of cells indicating the in vitro biocompatibility of the loaded scaffold system (Figure 11).
Immune profiling of the loaded scaffolds
To check the immunogenic activity of the loaded scaffolds in vitro, hDPSCs were seeded on to the scaffolds. After 48hrs, the cells were harvested and washed with PBS. As control, only cells without interaction with the loaded scaffolds were kept. For the assessment, pro-inflammatory markers IL-10, IL-6, and TGF-B were taken to check their expression level on the cells. All the fluorophore tagged markers were incubated with the samples for 30min, after which acquired with LSR Fortessa. Unstained cells were acquired to set the gate for live cells parent population, followed by the samples tagged with markers. Fig. 12 indicated that IL-10 and IL-6 were at a base level similar to the control showing no expression of the two markers. Whereas, TGF-b showed slight expression indicating minimal/ temporary immunogenicity by the cells.
Below are the remaining standard heads you would typically include, in a style consistent with the rest of your draft. You can tweak numbering/formatting to match your Form 2 house style.
SPECIFIC EMBODIMENTS OF THE PRESENT DISCLOSURE:
The present disclosure relates to a bioactive scaffold composition comprising: a covalently cross-linked, porous polymer matrix formed from methacrylated chitosan and collagen; and exosomes derived from human dental pulp stem cells (hDPSCs) integrated within the porous structure of said matrix; wherein a particle size of the pre-differentiated osteoblast derived exosomes is in the range of 50 nm-200 nm which cross-link with the porous polymer scaffold to form the 3D extracellular matrix scaffold.
The present disclosure also relates to a method for preparing the aforementioned scaffold composition, comprising:
a) isolating and culturing hDPSCs from human dental pulp tissue to obtain conditioned medium;
b) isolating exosomes from said conditioned medium by ultracentrifugation and mineralization of the cells by Exos treatment.
c) synthesising methacrylated chitosan by reaction with methacrylic anhydride followed by dialysis purification;
d) mixing the methacrylated polymers chitosan & PEGDMA in 1:1 ratio (w/v) with ).1% collagen w/v and photoinitiator (Irgacure) and subjecting to UV cross-linking to form hydrogel;
e) lyophilising/ freeze drying the hydrogel to obtain 3D porous structure of the cross-linked polymer scaffold; and
f) incubating the scaffold with isolated exosomes to achieve matrix integration.
Such method is disclosed, wherein the freeze drying is carried out at a temperature in the range between –50 °C to –80 °C for 72 hours.
Such method is disclosed, wherein the crosslinking is carried out at temperature in the range between 24°C to 27°C.
INDUSTRIAL APPLICATION
The present disclosure relates to a bioactive scaffold composition for use in dental and maxillofacial bone defects. The scaffold composition has industrial applicability in the fields of dental and cranio maxillofacial surgery, implantology and regenerative medicine. The exosome loaded scaffold can be manufactured as a sterile, ready to use product with defined shelf life. As it is prepared from clinically accessible dental pulp tissue and widely used biomaterials, it supports scalability and potential cost effectiveness for routine dental practice.
, Claims:WE CLAIM:
1. A tissue regenerative 3D porous cross-linked polymer scaffold composition comprising:
a porous, covalently cross linked polymer scaffold; and
exosomes derived from human dental pulp stem cells (hDPSCs), having a particle size in the range of 50 nm to 150 nm;
wherein the hDPSC derived exosomes are integrated within the porous polymer scaffold to form an exosome loaded scaffold suitable for bone regeneration in dental or maxillofacial defects.
2. The scaffold composition as claimed in claim 1, wherein the porous polymer scaffold comprises polysaccharides, chitosan, a hydrophilic biocompatible crosslinking monomer PEGDMA and protein collagen.
3. The scaffold composition as claimed in claim 2, wherein ratio of chitosan, and PEGDMA is 1:1, 1:0.5, 0.75:1.
4. The scaffold composition as claimed in claim 1, wherein a diameter of the porous polymer scaffold is in the range of 50 to 200 nm, and wherein the porous polymer scaffold and the hDPSC-derived exosomes are present in a 1:1 ratio.
5. A method of preparing a porous polymer scaffold as claimed in claim 1, comprising the steps of:
a) isolating and culturing hDPSCs from human dental pulp tissue to obtain conditioned medium;
b) isolating exosomes from said conditioned medium by ultracentrifugation and mineralization of the cells by exosome treatment.
c) Synthesising methacrylated chitosan by reaction with methacrylic anhydride followed by dialysis purification;
d) mixing the methacrylated polymers in chitosan & PEGDMA in 1:1 ratio with 0.1% collagen (w/v) with 0.6% w/v Irgacure and subjecting to UV cross-linking to form hydrogel;
e) freeze drying the hydrogel to obtain 3D porous structure of the cross-linked polymer scaffold.
6. The method as claimed in claim 6, wherein the freeze drying is carried out at a temperature in the range of –50 °C to –80 °C.
7. The method as claimed in claim 6, wherein the cross linking is carried out at a temperature in the range of 24°C to 27°C.