Method for self-assembling three-dimensional tissue and therapeutic agent delivery

Abstract

Methods of self-assembling three-dimensional tissues and therapeutic agent delivery are disclosed. Specifically disclosed is an injectable hydrogel comprising a carboxymethyl chitosan (CMCTS) / oxidized hyaluronic acid (oHA) hydrogel bound to silver sulfadiazine (AgSD), where the hydrogel has a uniform porous structure on the surface.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511168514.2 (22) Application Date 2025.08.20 (30) Priority Data 63 / 685,034 2024.08.20 US (71) Applicant: University of Hong Kong Address: Pok Fu Lam Road, Hong Kong, China (72) Inventor: Li Shangzhen (74) Patent Agency: Beijing Liu Shen Law Firm 11105 Patent Attorney: Mou Ke (51) Int.Cl. A61L 27 / 20 (2006.01) A61L 27 / 38 (2006.01) A61L 27 / 02 (2006.01) A61L 27 / 50 (2006.01) A61L 27 / 52 (2006.01) A61L 27 / 54 (2006.01) A61L 27 / 56 (2006.01) A61L 27 / 58 (2006.01) (54) Invention Title: Method for Self-Assembling Three-Dimensional Tissue and Delivery of Therapeutic Agents (57) Abstract: A method for self-assembling three-dimensional tissue and delivery of therapeutic agents is disclosed. Specifically, an injectable hydrogel is disclosed comprising a carboxymethyl chitosan (CMCTS) / oxidized hyaluronic acid (oHA) hydrogel bound to silver sulfadiazine (AgSD), wherein the hydrogel has a uniform porous structure on its surface. Claims 1 page, Description 19 pages, Drawings 20 pages, CN 121588279 A 2026.03.03 CN 1 21 58 82 79 A 1. An injectable hydrogel comprising a carboxymethyl chitosan (CMCTS) / oxidized hyaluronic acid (oHA) hydrogel bound to silver sulfadiazine (AgSD), wherein the hydrogel has a uniform porous structure on its surface. 2. The injectable hydrogel of claim 1, comprising about 0.1% AgSD. 3. The injectable hydrogel of claim 1, in the form of high-density dental pulp stem cells (DPSCs) encapsulated within a carboxymethyl chitosan (CMCTS) and oHA hydrogel system reinforced with antibacterial silver sulfadiazine (AgSD) material. 4. The injectable hydrogel of claim 1, in the form of 4% CMCTS / 3% oHA + 0.x% AgSD, wherein x is one of 0.1%, 0.25%, and 0.5%. 5. The injectable hydrogel of claim 4, wherein x equals 0.1% AgSD. 6. A method for preparing an injectable hydrogel, comprising the steps of: directly combining carboxymethyl chitosan (CMCTS), oxidized hyaluronic acid (oHA), human cells, and silver sulfadiazine (AgSD) to form a hydrogel construct at room temperature. 7. The method of claim 6, wherein AgSD is incorporated into a CMCTS macromonomer solution.Then, it is combined with oHA to produce a CMCTS / oHA / AgSD hydrogel. 8. The method of claim 6, wherein oHA is synthesized by oxidatively cleaving the cyclic structure of hyaluronic acid (HA) into an open chain using sodium periodate, thereby generating two aldehyde groups from the vicinal diol unit of the HA backbone. 9. The method of claim 6, wherein the oHA is prepared by the following steps: adding HA to deionized water and stirring at room temperature until completely dissolved; adding NaIO4 pre-dissolved in Milli-Q water to the HA solution and stirring vigorously; placing the solution in the dark overnight; then adding excess ethylene glycol to the solution to terminate the reaction by continuing at room temperature for 30 minutes; dialyzing the solution using a dialysis bag for 7 days, changing the deionized water every 12 hours; lyophilizing the purified solution for 1 week to obtain a clear oHA composite material, wherein the lyophilized composite material is stored at -20 °C until use. 10. The method of claim 6, wherein the CMCTS / oHA is produced by a tunnel mixer or pipette mixing method. 11. The method of claim 6, wherein the human cells are dental pulp stem cells (DPSCs). Claims 1 / 1 page 2 CN 121588279 A Method for Self-Assembling Three-Dimensional Tissue and Delivery of Therapeutic Agents Technical Field

[0001] The present invention relates to injectable hydrogels, and more particularly to the use of injectable hydrogels for three-dimensional tissue assembly. Background Art

[0002] Human tissues may be accidentally damaged due to severe trauma, injury, and tumor resection. Over the past decade, various novel tissue engineering approaches have aimed to restore damaged tissues by fully utilizing biomaterials, cells, and growth factors to stimulate repair and regeneration. Key strategies include scaffold-based tissue replacement, cell-based therapies, and the use of bioactive molecules to enhance the body's natural healing processes.

[0003] Among biomaterial approaches for restoring damaged tissues, injectable hydrogel systems have become a widely used scaffold approach. A key advantage is their ability to be delivered in a minimally invasive manner, allowing the hydrogel to conform to complex tissue geometries at the site of injury. In addition to stem cells, injectable hydrogels can also be engineered to incorporate bioactive factors, such as growth factors or therapeutic agents, to stimulate and guide tissue regeneration processes. Various types of injectable hydrogels have been studied, including supramolecular peptide hydrogels and thermoresponsive hydrogels. While these hydrogel systems have demonstrated excellent performance in terms of regenerative outcomes, they also face several challenges. For example, the preparation of peptide-based hydrogels can be expensive, limiting large-scale production. Thermoresponsive hydrogels form in response to temperature changes, but the process can take several minutes to solidify, which may cause discomfort to patients waiting on the operating table or treatment chair. A maintenance period is also necessary.Moreover, hydrogels are typically formed from aqueous solutions when body temperature is maintained at an appropriate level, meaning they cannot form hydrogels at room temperature. In the case of ultraviolet (UV)-based hydrogels, they begin as soluble hydrogels and require UV cross-linking after application to the target area, making them difficult to use in situ as an immediate injectable form. These limitations hinder the ease of rapid delivery of various molecules or transplanted stem cells loaded in hydrogels, severely restricting their clinical application in rapid treatments.

[0004] As a tool for regenerative medicine, stem cell therapy also plays a crucial role in inducing the regeneration of damaged tissues. In particular, the implantation of 3D tissue constructs (e.g., 3D spheres or aggregates) has become the most advanced system in the fields of tissue engineering and regenerative medicine. These 3D tissue-engineered grafts offer significant advantages over traditional cell-based therapies because they can more closely mimic the natural extracellular matrix and cell interactions in complex tissues. Three-dimensional (3D) tissue platforms play a transformative role in advancing regenerative strategies across a range of clinical applications. Many researchers have investigated the use of hydrogels to pursue 3D tissue remodeling by loading hydrogel systems with cell aggregates. For example, Zhang et al. developed an extracellular matrix hydrogel and manually embedded pre-fabricated 3D spheroids, which were then subcutaneously implanted into mice (Zhang J, Xu Y, Zhuo C, Shi R, Wang H, Hu Z, Chan HF, Kim H-W, Tao Y, Li M. Highly efficient fabrication of functional hepatocyte spheroids by a magnetic system for the rescue of acute liver failure. Biomaterials. 2023;294:122014). In another study, Kwon et al. fabricated a hydrogel loaded with spheroids.This demonstrates its potential for in vivo applications in tissue engineering and regenerative medicine (Kwon H, Lee S, Byun H, Huh SJ, Lee E, Kim E, Lee J, Shin H. Engineering prevascularized 3D tissue and rapid vascular integration with host blood vessels via cocultured spheroids-laden hydrogel. Biofabrication. Instructions for Use 1 / 19 pages 3 CN 121588279 A 2024;16:025029). Furthermore, it is well known that commercially available cell culture plates are not required for the generation of 3D spheroids (Buentello et al., “Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies,” Plos One, May 10, 2022 https: / / doi.org / 10.1371 / journal.pone.0262062).

[0005] While the transplantation of 3D stem cell aggregates has shown promising regenerative potential, these systems still encounter some drawbacks. Notably, the fabrication of these constructs requires cumbersome cross-linking steps using complex equipment before implantation. Furthermore, residual hydrogel after transplantation can hinder the integration of the graft with the host tissue. Eliminating the long-term degradation of these persistent hydrogel materials requires the use of enzymes or other degrading agents, which necessitates additional inconvenient steps. There is room for further optimization and innovation regarding these drawbacks related to the preparation and post-implantation behavior of hydrogel-based stem cell constructs.

[0006] Various hydrogel systems have also been used to enhance in vivo tumor modeling. While these systems are designed to provide a supportive environment for tumor growth and vascularization, they face significant challenges. For example, Matrigel® is useful in cancer research, but it requires time to solidify and is not easily degraded when injected into the body. This can hinder tissue integration and its vascularized infiltration.

[0007] Therefore, developing more user-friendly and easily manageable hydrogel preparation methods that enable smoother integration with host tissues could help overcome these challenges and unlock the full regenerative potential of advanced 3D tissue engineering approaches. Unfortunately, a convenient injectable system capable of constructing tissue-like structures from cells and allowing for the natural biodegradation of hydrogels has not yet been clearly established.

[0008] In the inventors' previous reports,The successful preparation of a self-crosslinkable hydrogel was disclosed, in which water-soluble ethylene glycol chitosan (gC) and oxidized hyaluronic acid (oHA) were combined at room temperature (Lee SJ, Nah H, Heo DN, Kim K-H, Seok JM, Heo M, Moon H-J, Lee D, Lee JS, An SY. Induction of osteogenic differentiation in a rat calvarial bone defect model using an In situ forming graphene oxide incorporated glycol chitosan / oxidized hyaluronic acid injectable hydrogel. Carbon. 2020;168:264-277; Ko W-K, Lee SJ, Kim SJ, Han GH, Han I-b, Hong JB, Sheen SH, Sohn S. Direct injection of hydrogels embedding gold nanoparticles for local therapy after spinal cord injury. Biomacromolecules. 2021;22:2887-2901). This hydrogel system has shown promising results in vitro and in vivo when used for in situ therapeutic applications, as demonstrated by its implantation in rat models of skull defects and spinal cord injuries.

[0009] To address the problems of the prior art, the present invention proposes an in situ injectable hydrogel system without cross-linking agents and loaded with high-density human cells, wherein the hydrogel is formed by the self-assembly of a macromolecular monomer solution at room temperature, allowing cells to spontaneously undergo 3D assembly during hydrogel degradation.

[0010] According to the present invention, a carboxymethyl chitosan (CMCTS) / oxidized hyaluronic acid (oHA) hydrogel is combined with silver sulfadiazine (AgSD) for tissue engineering applications with inherent antimicrobial activity. Physicochemical analysis determined the optimal CMCTS / oHA hydrogel formulation, which exhibited excellent injectability for minimally invasive in situ delivery. In vitro testing determined 0.1% AgSD as the optimal concentration, which supports cell proliferation while exhibiting antimicrobial efficacy. In vivo studies showed complete hydrogel degradation and good biocompatibility without adverse tissue reactions. Notably, this hydrogel is capable of forming 3D cell aggregates and supporting tissue regeneration.This highlights its potential for future 3D tissue engineering applications. In our previous research specification, page 2 / 19, CN 121588279 A, ​​Lee et al. introduced self-healing hydrogels based on water-soluble CMCTS and oHA, which were validated in situ therapy in mouse models (Heo M, Lee SJ, Heo DN, Lee D, Lim H-N, Moon J-H, Kwon IK. Multilayered coelectrospun scaffold containing silver sulfadiazine as a prophylactic against osteomyelitis: Characterization and biological in vitro evaluations. Applied Surface Science. 2018;432:308-316). In this study, the injectable hydrogel was found to completely degrade in vivo. Based on its degradability, the loaded high-density stem cells were observed to spontaneously form three-dimensional (3D) tissues in vitro. Therefore, this injectable CMCTS / oHA / AgSD hydrogel holds significant promise for a wide range of biomedical applications, including antimicrobial substance delivery systems and 3D tissue engineering, and has the potential for future clinical translation.

[0011] In some embodiments, the human cells are dental pulp stem cells (DPSCs). In this case, the hydrogel of the present invention is in the form of high-density dental pulp stem cells (DPSCs) encapsulated in a carboxymethyl chitosan (CMCTS) and oHA hydrogel system reinforced with antimicrobial silver sulfadiazine (AgSD) material.

[0012] The present invention eliminates the need for unnecessary 3D tissue engineering tools and expensive hydrogel materials. Furthermore, since the hydrogel formulation is designed for in-situ self-degradation, it eliminates the inconvenience of having to remove excess hydrogel before implantation in vivo. Specifically, the injectable CMCTS / oHA hydrogel bound to AgSD can directly form functional 3D tissue constructs in vivo without the need for complex pre-fabrication or removal of excess material. This enhances the practicality and clinical translatability of the technology, as it enables minimally invasive, on-site tissue engineering applications.

[0013] Therefore, the system of the present invention relates to a self-degrading, immediately injectable hydrogel technology. By fully utilizing this degradation mechanism, the hydrogel is able to form three-dimensional tissue constructs when high-density cells are encapsulated within it. Furthermore, the hydrogel can be easily used for injectable applications via a simple delivery device.

[0014] The product utilizes commercially available components and handcrafted composite materials.It is then combined with a proprietary hydrogel. This results in the production of a self-assembling hydrogel formulation. By combining a commercially available product with the custom-designed components of this invention, the system provides a practical and readily available solution for in-situ tissue engineering. The ability to utilize readily available components, coupled with the self-assembling properties of the hydrogel, represents a significant advance in making this technology readily available and implementable. The injectable hydrogel system achieves versatility by strategically combining existing materials with proprietary hydrogels, highlighting the novelty and potential impact of this research.

[0015] The self-degradable properties of the hydrogel, coupled with its biocompatibility and ability to support 3D cell aggregation, highlight its multifunctional potential for a wide range of tissue repair and regeneration therapies as well as immunocellular therapies. This injectable hydrogel system represents a significant advance in the field of in-situ tissue engineering, providing a streamlined, clinically relevant, and easily deployable approach to tissue engineering compared to conventional 3D tissue engineering methods. Brief Description of the Drawings

[0016] This patent or application contains at least one drawing implemented in color. A copy of the color drawing of this patent or patent application publication will be provided by the Patent Office upon request and payment of the necessary fees.

[0017] The foregoing and other objects and advantages of the invention will become more apparent when considered in conjunction with the following detailed description and accompanying drawings, wherein like designations in the various views denote like elements, and wherein:

[0018] Figure 1A shows a schematic diagram of incorporating AgSD into a CMCTS macromonomer solution and then combining it with oHA to prepare a CMCTS / oHA / AgSD hydrogel; Figure 1B shows a schematic diagram of culturing a CMCTS / oHA / AgSD hydrogel loaded with high-density DPSCs for 3D cell aggregation and analyzing the CMCTS / oHA / AgSD hydrogel using inhibition region and colony-forming unit tests to evaluate antibacterial activity; and Figure 1C shows a schematic diagram of subcutaneously injecting an injectable CMCTS / oHA / AgSD hydrogel into mice to evaluate degradation and biocompatibility;

[0019] Figure 2A is a schematic diagram of the preparation of oHA by NaIO4 modification, and Figure 2B shows the 1H-NMR results of oHA, indicating that oHA Dialdehyde groups appeared in the macromonomer solution. Figure 2C shows the fabricated CMCTS / oHA and CMCTS / oHA+0.5% AgSD hydrogels. Figure 2D shows the well-formed hydrogels after lyophilization and SEM analysis. Figure 2E shows the FT-IR analysis of the original CMCTS powder, the synthesized oHA composite material, and the lyophilized CMCTS / oHA hydrogel.Figure 2E shows the rheological analysis of the 2% CMCTS / 3% oHA and 4% CMCTS / 3% oHA hydrogels prepared by tunnel mixer and pipetting;

[0020] Figure 3A shows an image of the tunnel mixing system used to prepare the 4% CMCTS / 3% oHA and 4% CMCTS / 3% oHA+0.5% AgSD hydrogels, wherein gelation occurs at room temperature without a crosslinking agent, and Figure 3B shows an image of the hydrogel being applied using a 27-gauge syringe, wherein the injection proceeds smoothly while maintaining the desired viscosity;

[0021] Figure 4 shows SEM images of the hydrogels without (left) and with 0.5% AgSD (right) immediately after fixation and after staining procedures following binding, wherein discrete particulate matter consistent with AgSD is observed in the right image;

[0022] Figure 5A shows optical images of the freeze-dried residual hydrogel after degradation over time, Figure 5B shows a graph of degradation rate, Figure 5C shows a graph of residual mass decay, Figure 5D shows SEM images of the freeze-dried 4% CMCTS / 3% oHA hydrogel at 0, 1, 2, 3, 5 and 7 days, and Figure 5E shows the average pore size (e). The pore size gradually decreased over time;

[0023] Figure 6A shows images of CMCTS / oHA and CMCTS / oHA+0.5% AgSD hydrogels loaded with high-density DPSCs cultured in vitro for 7 days, Figure 6B shows bar graphs of cell proliferation rates of CMCTS / oHA and CMCTS / oHA+0.5% AgSD hydrogels at 7 days, Figure 6C shows the live / dead ratio at 7 days of culture, and Figure 6D shows histological analysis of the CMCTS / oHA group at 0, 1, and 7 days of culture using H&E and Safranin O with Fast Green staining;

[0024] Figure 7A shows images of CMCTS / oHA, CMCTS / oHA+0.1% AgSD, and CMCTS / oHA+0.25% AgSD hydrogels loaded with medium-density DPSCs cultured in vitro for 14 days, Figure 7B shows CMCTS / oHA, CMCTS / oHA+0.1% AgSD, and CMCTS / oHA+0.25% AgSD hydrogels. Bar graphs of cell proliferation rate on AgSD hydrogels at 7 days, Figure 7C shows the live / dead ratio at 7 days of culture, Figure 7D shows the live / dead ratio stained with F-actin at 7 days of culture, and Figure 7E shows histological analysis using H&E and Alcian blue staining with nuclear solid red to reveal strong cell aggregation in the CMCTS / oHA and CMCTS / oHA+0.1% AgSD groups.However, no strong cell aggregation was observed in the CMCTS / oHA+0.25% AgSD group;

[0025] Figure 8 is a series of SEM images revealing the cell aggregation patterns in the CMCTS / oHA, CMCTS / oHA+0.1% AgSD and CMCTS / oHA+0.25% AgSD groups;

[0026] Figure 9A shows the in vitro antibacterial test of CMCTS / oHA and CMCTS / oHA / AgSD hydrogels against Streptococcus mutans and Enterococcus faecalis, with antibacterial drugs as positive controls (1), empty cells as negative controls (2), CMCTS / oHA hydrogel (3), CMCTS / oHA+0.1% AgSD hydrogel (4), and CMCTS / oHA+0.25% AgSD hydrogel (5), and Figure 9B shows the results of Figure 9A. The results of the experiment are shown in bar charts, characterized by the inhibition area and measured by the diameter of the antibacterial area; Figures 9C and 9D show the same experimental group cultures for Streptococcus mutans and Enterococcus faecalis characterized by the CFU test without a positive control, and Figures 9E and 9F show bar charts of the quantitative results of Figures 9C and 9D;

[0027] Figure 10 shows mice immediately after the application of the hydrogel, in which the hydrogel was subcutaneously injected into the back of the mouse without any prior incision;

[0028] Figure 11 shows optical images of mice injected subcutaneously in situ with CMCTS / oHA, +0.1% AgSD, +0.25% AgSD and +0.5% AgSD hydrogels, with histological analysis 2 weeks after injection;

[0029] Figure 12 shows H&E staining of representative organs in the CMCTS / oHA+0.1% AgSD hydrogel group of Figure 11 2 weeks after injection;

[0030] Figure 13A shows 4% CMCTS / 3% AgSD hydrogels. A series of SEM images of oHA hydrogel at different ratios of 8:2, 5:5 and 2:8 (see page 4 / 19 of the manual, CN 121588279 A), and Figure 13B shows the gelation test results of the hydrogel;

[0031] Figure 14A shows the degradation test results of 4% CMCTS / 3% oHA in 1x PBS, 10% FBS and complete cell growth medium for 10 days, and Figure 14B shows the change in the volume of the remaining hydrogel after degradation over time;

[0032] Figure 15 shows the morphological analysis of CMCTS / oHA hydrogel before (A) and after (B) encapsulation of high-density HepG2 cells. (C) Tunnel mixing system. (D) Hydrogel encapsulating tumor cells is squeezed out through the needle using a syringe.(E) Morphology of the hydrogel immediately after extrusion in vitro. (F) Rheology of CMCTS / oHA hydrogels without HepG2 or loaded with HepG2. (G) Camera and (H) Microscopic images of the HepG2-loaded CMCTS / oHA hydrogel at 0D and 1D; (I) CMCTS / oHA hydrogel containing HepG2 in vitro for 7 days, and its (J) H&E and (K) F-actin staining with DAPI on day 3 of culture. (L) Live / dead staining after 7 days of culture;

[0033] Figure 16 Microscopic images of Rhodamine-labeled HepG2 in CMCTS / oHA hydrogel in vitro for 7 days. High-density HepG2 cells form aggregates (A) and exhibit strong cell-cell connections (B and C);

[0034] Figure 17 (A-B) Morphological changes of CMCTS / oHA hydrogels loaded with high-density Huh-7 cells from day 0 to day 1, showing a spontaneously degrading paste-like structure. (C) Microscopic images of the culture gradually showing aggregation over 7 days: cells are highly active, cohesive, and homogeneous. (D) H&E staining and (E) F-actin staining on day 3 of culture. On day 7 of culture, (F) live / dead staining shows that the cell aggregation structure is maintained and the cells are alive;

[0035] Figure 18 In vivo biocompatibility and biodegradability assessment of CMCTS / oHA hydrogels encapsulating HepG2. CMCTS / oHA hydrogel encapsulating high concentrations of HepG2 was subcutaneously injected into Balb / C mice, and histological analysis was performed on optical images at 1 and 2 weeks post-injection;

[0036] Figure 19: H&E, Alcian blue, and Safranin O staining at the injection site 2 weeks after subcutaneous injection of CMCTS / oHA hydrogel encapsulating high concentrations of HepG2;

[0037] Figure 20: Angiogenic tumor formation in an immunodeficient SCID mouse model. Subcutaneous injection of CMCTS / oHA hydrogel encapsulating high concentrations of HepG2 2 weeks later (A). GPC3 IF staining images of HepG2 and CD31 on the vascular system (B, C, and D);

[0038] Figure 21 Immunofluorescence staining analysis of CMCTS / oHA hydrogel encapsulating HepG2 before implantation;

[0039] Figure 22 Quantification of blood vessels after injection of high-density HepG2 and Huh-7 cells into mice, compared with Matrigel® and PBS controls without gel 2 weeks after injection. (A) Results of H&E, Masson's trichrome, and Safranin O staining with Fast Green on sections of tumor tissue. (B) Number of blood vessels in tumor tissue for each cell group. (C) Comparison of the area of ​​formed blood vessels. (D) CD31 IF staining for detecting newly formed blood vessels in tumor tissue;

[0040] Figure 23 shows the morphology of cells embedded in Matrigel or CMCTS / oHA hydrogel before implantation.Staining was performed using H&E, Masson's trichrome, and Safranin O with Fast Green. Detailed Description of the Invention

[0042] Building upon the inventors' previous discoveries, the present invention further proposes encapsulating high-density human cells in a carboxymethyl chitosan (CMCTS) and (oHA) hydrogel system reinforced with antimicrobial silver sulfadiazine (AgSD) material. AgSD is incorporated into the hydrogel crosslinking process to utilize the hydrogel's material delivery capabilities and enhance the antimicrobial properties of the construct.

[0043] In some embodiments, the human cells are dental pulp stem cells (DPSCs). In this case, the hydrogel of the present invention is in the form of high-density dental pulp stem cells (DPSCs) encapsulated in a carboxymethyl chitosan (CMCTS) and oHA hydrogel system reinforced with antimicrobial silver sulfadiazine (AgSD) material. DPSCs can be selected as the cell source because they are commonly used in craniofacial stem cell research and have gained considerable attention in regenerative medicine due to their unique properties. These properties include their self-manifestation capacity, multilineage differentiation potential, and immunomodulatory capabilities. Furthermore, DPSCs exhibit strong cell-cell communication, which promotes their self-aggregation. Given these regenerative properties, DPSCs have been effectively integrated with various biomaterials to develop biomimetic constructs for cell therapy applications, and are derived from discarded wisdom teeth, enabling us to extract valuable cells from waste resources. This detailed strategy is shown in Figures 1A-1C. CMCTS, oHA, DPSCs, and AgSD bind directly at room temperature to form a hydrogel construct (Figure 1A). Next, the 3D cell formulation, cell viability, and antimicrobial activity were characterized (Figure 1B), and then the optimal CMCTS / oHA / AgSD hydrogel was directly injected subcutaneously into mice without incision to verify self-degradation and cell compatibility (Figure 1C).

[0044] In this setup, as shown in Figure 1B, a CMCTS / oHA / AgSD hydrogel loaded with high-density DPSCs was cultured for 3D cell aggregation. The CMCTS / oHA / AgSD hydrogel was analyzed using inhibition zone and colony-forming unit tests to assess antimicrobial activity. Figure 1C. Subcutaneous injection of the CMCTS / oHA / AgSD hydrogel into mice was for the purpose of assessing the degradation of the hydrogel and its biocompatibility.

[0045] Biomaterials such as CMCTS, oHA, and AgSD have been widely used in various biomedical engineering fields. These materials can be combined to form a single liquid "glue" (e.g., honey-like) through simple combinations. However, the concept of using hydrogels to contain AgSD and cells requires the formation of fixed gel-like hydrogels (e.g., jelly bear-like).Not just a honey-like glue. This is to prevent AgSD or cells from naturally spreading or spreading when they are contained in a hydrogel that acts as a containment material.

[0046] Using the materials developed in this invention, the method of assembling these materials into hydrogels is optimized through simple chemical modifications, adjustments to concentration and composition conditions. The stability of the system in 3D cell assembly and animal model implantation has been demonstrated.

[0047] The key advantages of this invention are: 1) no UV exposure required to form a gel from a liquid-sol, 2) no temperature control required, 3) self-degradation without additional induction, and 4) injectable form without surgical implantation. The most significant advantage is ease of handling and the ability to form gel-like injectable materials, rather than sol-like forms, through simple assembly.

[0048] The materials of the present invention may include (1) O-carboxymethylated chitosan (CMCTS, catalog number sc-358091) with a degree of deacetylation of ≥90% purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); (2) silver sulfadiazine (I) (AgSD, catalog number 481181), ethylene glycol (catalog number 102466); (3) deuterium oxide (D2O, catalog number 151882, isotopic purity of 99.9 atomic% D) purchased from Sigma-Aldrich (St. Louis, MO, USA) and (4) high molecular weight hyaluronic acid powder (HA, 1000 kDa, cosmetic and food grade) purchased from The Pharmers Market (Manchester, United Kingdom). Two Luer lock-tip 1.0-mL syringes (catalog number 309628) purchased from BD Biosciences (Becton Dickinson, USA) can be connected to a tunnel mixer (catalog number MTSEM01) purchased from NOBAMEDI Co., Ltd. (Yongin, Republic of Korea).

[0049] The preparation of CMCTS / oHA and CMCTS / oHA / AgSD hydrogels is as follows. First, according to the inventors' previous literature,oHA was prepared using sodium periodate (NaIO4) (Lee SJ, Nah H, Heo DN, Kim K-H, Seok JM, Heo M, Moon H-J, Lee D, Lee JS, An SY. Induction of osteogenic differentiation in a rat calvarial bone defect model using an In situ forming graphene oxide incorporated glycol chitosan / oxidized hyaluronic acid injectable hydrogel. Carbon. 2020;168:264-277; Ko W-K, Lee SJ, Kim SJ, Han GH, Han I-b, Hong JB, Sheen SH, Sohn S. Direct injection of hydrogels embedding gold nanoparticles for local therapy after spinal cord injury. Biomacromolecules. 2021;22:2887-2901). NaIO4 was used in the oxidation process to prepare oHA, with some degree of modification. In short, 3.8 g of HA was added to 360 mL of deionized water and stirred at room temperature until completely dissolved. Then, 1.068 g of NaIO4 (pre-dissolved in 40 mL of Milli-Q water, see page 6 / 19 of the specification, CN 121588279 A) was added to the HA solution and stirred vigorously overnight in the dark. Next, excess ethylene glycol (1 mL) was added to the solution at room temperature for 30 minutes to terminate the reaction. To remove any remaining compounds, the solution was dialyzed for 7 days using a dialysis bag (catalog number JHC0095, 8000-14000 MWCO, BioMomei, China), with the deionized water changed every 12 hours. Finally, the purified solution was lyophilized for 1 week to obtain a clear oHA composite material. The lyophilized composite material was kept at -20 °C until use.

[0050] The next major step is to prepare CMCTS / oHA and CMCTS / oHA / AgSD hydrogels. To optimize the hydrogel fabrication method, two different preparation methods (tunnel mixer or pipette mixing) and two different concentrations of CMCTS were used to fabricate CMCTS / oHA. In short,2% and 4% CMCTS composites were dissolved in PBS (weight / volume), and 3% oHA composites were dissolved in PBS (weight / volume). After thorough dissolution by vortexing, the 2% or 4% CMCTS and 3% oHA macromonomer solutions were uniformly mixed on clean culture dishes using a tunnel mixer or pipette. To determine the optimal hydrogel ratio, hydrogels with different CMCTS / oHA ratios of 20:80, 50:50, and 80:20 were prepared, and gelation time and formation probability were measured. First, 4 wt% CMCTS and 3 wt% oHA were prepared. Hydrogel components of different ratios were created and mixed using a manual mixing method, with a total volume of 200 μL (n=4) for each condition. Gelation time was measured by observing when the CMCTS / oHA hydrogel was fully formed and no residual macromonomer solution remained on the dish. After hydrogel formation, all samples were freeze-dried for 3 days. The cross-sectional morphology was characterized using scanning electron microscopy (SEM, SU1510, Hitachi, Japan). Based on these measurements, a tunnel mixer with a CMCTS / oHA ratio of 80:20 was selected for further in vitro and in vivo studies. To prepare AgSD-loaded CMCTS / oHA hydrogels, a series of AgSDs (0.1%, 0.25%, 0.5%, wt / v) were added to a CMCTS macromonomer solution, followed by the addition of oHA, to generate CMCTS / oHA+0.1% AgSD, CMCTS / oHA+0.25% AgSD, and CMCTS / oHA+0.5% AgSD hydrogels, respectively, at a tunnel mixer ratio of 80:20 (CMCTS / oHA).

[0051] To confirm the successful synthesis of oHA, the chemical structure of oHA was measured using a 1H-nuclear magnetic resonance (NMR) spectrometer (Bruker Avance 400, Bruker Corporation, Billerica, MA, USA) with D2O as the solvent. Furthermore, characteristic peaks in the infrared spectra of the CMCTS / oHA composite material and the lyophilized CMCTS / oHA composite material from the hydrogel sample were measured using a Fourier transform infrared (FT-IR) spectrometer (Spectrum Two™, PerkinElmer, Norwalk, CT, USA), with wavenumbers ranging from 500 to 4000 cm⁻¹.

[0052] The hydrogel formation kinetics and mechanical properties of the CMCTS / oHA hydrogel were tested in oscillating mode at room temperature and 37 °C using an MCR 92 rheometer equipped with parallel plates (Anton Paar, GmbH, Germany). In short,Two different concentrations of CMCTS solutions (2% and 4%) were mixed with a 3% oHA solution using two different methods (tunnel mixer and pipette mixing). The hydrogels were then measured using a rotational rheometer with a plate-to-plate geometry of 8 mm diameter and 1 mm gap to investigate frequency-dependent viscoelastic behavior. Storage modulus (G') and loss modulus (G'') of each sample were measured under controlled room temperature and steady-state shear flow conditions. Frequency sweeps were performed at a constant strain of 1% in the range of 0.1–10 Hz.

[0053] 2% CMCTS / 3% oHA hydrogels and 4% CMCTS / 3% oHA hydrogels were prepared and weighed, and their initial weights (W0) were recorded after lyophilization for 48 hours. The lyophilized samples were placed in 12-well culture plates (catalog number CS016-0093, ExCell Bio, Shanghai, China) and immersed in 3 mL of phosphate-buffered saline (PBS) solution (pH=7.4). The culture plates were placed in a 5% CO2 incubator at 37°C, and the lyophilized hydrogels were incubated at 80 rpm in an orbital incubator (catalog number Stuart SI50, Rhys International Ltd., Bolton, China). The culture plate was shaken in the UK. The PBS in the culture plate was carefully changed daily. At predetermined time points (days 1, 2, 3, 5, 7 and 14, n=4 at each time point), hydrogel samples were collected, washed twice with deionized water, and then freeze-dried for 48 hours. The weight was measured and photographed to record the changes in mass and shape over time. The following formula was used to calculate the (1) degradation rate and (2) residual mass of the two types of hydrogels using the weight (Wt) after degradation at specific intervals as specified in the instruction manual 7 / 19, page 9, CN 121588279 A:

[0054]

[0055]

[0056] In addition, in order to determine the changes in the internal microstructure of the hydrogel during the degradation process, the hydrogel with better performance (4% CMCTS / 3%) was examined by scanning electron microscopy (SEM, catalog number SU-1510, Hitachi High-Technologies, Tokyo, Japan). The hydrogel (oHA) was examined. Specifically, hydrogels immersed in PBS at different time intervals were collected, pre-frozen in liquid nitrogen, lyophilized, and longitudinally cut to obtain cross-sections. Thin Au membranes were sputter-coated onto the cross-sections, and changes in the internal microstructure were observed by SEM characterization. Based on the SEM images, the average pore size of the hydrogel at different time points was calculated and analyzed using ImageJ software.

[0057] To evaluate the in vitro degradation of CMCTS / oHA hydrogels under different culture conditions, further degradation experiments were conducted under different types of conditions: 1) 1x PBS, 2) 10% FBS,3) Complete cell growth medium. Briefly, 4% CMCTS / 3% oHA hydrogel was placed directly into 12-well plates, and 3 ml of medium was added to completely submerge the hydrogel (n=3 per group). After this, the plates were placed in an incubator (TT-60-SI InQShake shaking incubator, Hercuvan laboratory system, UK) and shaken for 10 days at 5% CO2, 37 °C and 80 rpm. To maintain a constant concentration, the solution had to be changed and images taken daily. Finally, based on the hydrogel profile from the daily images, the volume of the hydrogel in different media was calculated using polygon selection and measurement functions in ImageJ software. The volume change of different groups over time was analyzed using Prism 10 software.

[0058] Based on the rheological and degradation results, 4% CMCTS / 3% oHA hydrogel was selected for subsequent experiments. To determine whether the addition of AgSD would affect the morphological characteristics of the hydrogels, CMCTS / oHA and CMCTS / oHA + 0.5% AgSD hydrogels were prepared under the same conditions, and images were taken to observe their morphology. The hydrogels were then frozen overnight at -80 °C and then lyophilized for 48 hours. The surface and interface characteristics of these hydrogels were evaluated by photographs and SEM.

[0059] DPSCs were purchased from Lonza (catalog number PT-5025, Basel, Switzerland) and cultured at 37 °C with 5% CO2 in α-modified minimum essential medium (α-MEM, catalog number 11900-024, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, catalog number 10270106, Thermo Fisher Scientific Inc.) and 1% penicillin-streptomycin (P / S, catalog number 15140163, Thermo Fisher Scientific Inc.). Cells were passaged 2 to 4 times in the experiments. After expansion in T-75 flasks, they were trypsinized using 0.25% trypsin-EDTA (catalog number 25200072, Thermo Fisher Scientific Inc.). The trypsinized DPSC pellets were directly encapsulated in CMCTS or CMCTS / AgSD macromonomer solutions at a density of 100 x 10⁶ cells / mL total hydrogel, and then bound to oHA. As part of the description of the preparation of CMCTS / oHA and CMCTS / oHA / AgSD hydrogels,The detailed hydrogel preparation process is as described above.

[0060] The antibacterial activity of the CMCTS / oHA / AgSD hydrogel was tested using Streptococcus mutans ATCC 700610 and Enterococcus faecalis ATCC 29212 strains, which are commonly found in dental caries and root canals. Samples of CMCTS / oHA / AgSD hydrogels with different concentrations of AgSD were irradiated with UV light for 30 minutes in a clean bench before use. For assays, 50 μL of each bacterial suspension was inoculated onto Mueller-Hinton agar (catalog number CM0337, Oxoid™, Basingstoke, Hampshire, UK) at a concentration of 1 × 10⁶ colony-forming units (CFU) / mL. Five uniform circular wells were created on each agar plate using a 200 μL pipette tip. Subsequently, 50 μL of each of the five different samples was added to the wells. Samples included: 0.5 mg / mL TAP (a triple antibiotic paste, a 1:1:1 mixture of ciprofloxacin (catalog number 17850, Sigma-Aldrich), minocycline (catalog number M9511, Sigma-Aldrich), and metronidazole (catalog number M1547, Sigma-Aldrich)) as a positive control; PBS as a negative control; CMCTS / oHA hydrogel (instructions page 8 / 19, CN 121588279 A); CMCTS / oHA + 0.1% AgSD hydrogel; and CMCTS / oHA + 0.25% AgSD hydrogel. The agar plates were then sealed and incubated overnight in an anaerobic culture chamber (Baker Ruskinn, Concept Ruskinn, Bridget, UK). The diameter of the bacterial inhibition zone was measured and photographed. The experiment was independently repeated three times to ensure reliability.

[0061] The antibacterial activity of the CMCTS / oHA / AgSD hydrogels was further evaluated using a CFU test. For this test, 150 μL of a 10⁶ CFU / mL suspension of *Streptococcus mutans* and *Enterococcus faecalis* was co-cultured with 100 μL of each sample for 24 hours. Samples included: 0.5 mg / mL TAP (a triple antibiotic paste, a 1:1:1 mixture of ciprofloxacin, minocycline, and metronidazole) as a positive control; PBS as a negative control; CMCTS / oHA hydrogel; CMCTS / oHA + 0.1% AgSD hydrogel; and CMCTS / oHA + 0.25% AgSD hydrogel. After co-culture,Bacterial suspensions were serially diluted 10,000-fold with Bacto Brain Heart Infusion (BHI, catalog number CM1135, Oxoid™) and inoculated onto solid agar plates (catalog number CM0337, Oxoid™). The agar plates were then sealed and incubated in an anaerobic chamber for 2 days to allow bacterial colonies to form. The number of bacterial colonies was then counted to assess the antimicrobial activity of different samples. The experiment was independently repeated three times to ensure reliability.

[0062] Animal experiments were conducted in accordance with the Animal Research Protocol (No. 22-268) approved by the Committee on the Use of Live Animals for Teaching and Research (CULATR) of the University of Hong Kong. The studies followed the Animal (Experimental Control) Regulations (Hong Kong) and the guidelines of the Centre for Comparative Medicine Research (CCMR) of the Li Ka Shing Faculty of Medicine, University of Hong Kong. Male BALB / c mice (6-8 weeks old) were used for the CMCTS / oHA hydrogel experiments. Mice were obtained from the University's Laboratory Animal Centre and had free access to food and water and were maintained under pathogen-free conditions. All procedures were performed under anesthesia using an intraperitoneal injection of a ketamine / xylazine mixture (100 mg / kg ketamine (catalog number 013004, AlfaMedic Ltd.) + 10 mg / kg xylazine (catalog number 013006, AlfaMedic Ltd.). Anesthesia was confirmed by checking body reflexes, and eye ointment (Duratears® eye ointment, catalog number 05686, Alcon, Fort Worth, TX, USA) was applied to prevent blindness. The subcutaneous injection sites of mice were decontaminated with 70% alcohol and povidone-iodine (Betadine®) alternately using sterile swabs. See Figure 10.

[0063] The CMCTS / oHA / AgSD hydrogel was then injected into the left and right backs of the mice, 500 μL at each site. The mice were placed in the intensive care unit (ICU) until they fully recovered, after which they were returned to their original feeding locations. Seven days after the injection, the mice were administered an overdose of pentobarbital (250 mg / kg) via intraperitoneal administration. (mg / kg) (Dorminal®, Catalogue No. 013003, AlfaMedic Ltd.) for euthanasia. Injection sites were collected and immediately fixed in 10% neutral buffered formalin (NBF) for 24 hours for histological analysis. Samples underwent a series of treatments with ethanol, xylene-ethanol, and xylene, 2 minutes each, followed by embedding in paraffin and sectioning to a thickness of 10 μm using a rotary microtome (Catalogue No. RM2155, Leica Microsystems, Wetzlar, Germany). Sections were dewaxed and rehydrated.The sample was stained with hematoxylin (Catalogue No. SH4777, Harris Hematoxylin, Cancer Diagnostic Inc., Durham, NC, USA) and eosin (Catalogue No. CS701, Dako, Glostrup, Denmark) (H&E), Alcian Blue (pH 2.5) with 0.1% nuclear solid red (Catalogue No. PH1082, Scientific Phygene®, Fuzhou, China), and Safranin O (Catalogue No. 02782-25, Polysciences Inc., Warrington, PA, USA) with solid green (Catalogue No. PH1852, Scientific Phygene®).

[0064] oHA was synthesized by oxidatively cleaving the cyclic structure of hyaluronic acid (HA) into an open chain using sodium periodate, which generates two aldehyde groups from the vicinal diol unit of the HA backbone (Figure 2A). The chemical structure of oHA was confirmed by 1H-NMR analysis. As shown in Figure 2B, the peaks at 4.9 ppm, 5.0 ppm, and 5.1 ppm represent the hemiacetal protons of oHA, indicating that the aldehyde group was successfully synthesized through an oxidation process. Therefore, the CMCTS / oHA hydrogel was formed by a Schiff base reaction between the amino group of CMCTS and the dialdehyde group of oHA (Figure 2C)

[0065] In this study, based on our analysis, the 8:2 ratio of CMCTS / oHA hydrogel was optimal (Figure 13). Specification 9 / 19 pages 11 CN 121588279 A We prepared mixtures of 4% CMCTS and 3% oHA in three different combinations: 2:8, 5:5, and 8:2 (Figure 13A). The gelation times of these mixtures are presented in Figure 13B. The results show that the 8:2 condition formed a complete hydrogel. Values ​​of p*** < 0.001 were considered statistically significant for 8:2 and 5:5. The 2:8 ratio of CMCTS / oHA required nearly 2 minutes to form a complete hydrogel. In contrast, the 5:5 and 8:2 ratios achieved gelation within 30 seconds, indicating that these two ratios are more suitable for hydrogel formation. Images of the hydrogel morphology show that the 8:2 ratio is optimal, as no residual water remains around the hydrogel after formation. In contrast, the other two groups showed significant residual liquid, indicating insufficient cross-linking of the polymer chains, which adversely affected their performance. Furthermore, cross-sectional SEM images showed that the internal pore morphology of the 8:2 ratio was more regular and had a more uniform pore structure than the other two ratios (Fig. 13A). Regular and uniform pores facilitate cell aggregation and 3D tissue formation in cell-loaded hydrogels. Based on these results,We determined that an 8:2 ratio maximizes stem cell encapsulation efficiency due to its rapid formulation and lack of residue. The slow hydrogel formation at a 2:8 ratio is attributed to the significantly lower concentration of amide groups from CMCTS compared to the dialdehyde groups from oHA, which may prevent hydrogel formation. For similar reasons, we used cross-linked hydrogels with high ethylene glycol chitosan content and low oHA content in our previous studies (Lee SJ, Nah H, Heo DN, Kim K-H, Seok JM, Heo M, et al. Induction of osteogenic differentiation in a rat calvarial bone defect model using an In situ forming graphene oxide incorporated glycol chitosan / oxidized hyaluronic acid injectable hydrogel. Carbon. 2020;168:264-77; Ko W-K, Lee SJ, Kim SJ, Han GH, Han I-b, Hong JB, et al. Direct injection of hydrogels embedding gold nanoparticles for local therapy after spinal cord injury. Biomacromolecules. 2021;22(7):2887-901).

[0066] The hydrogel formation process is described in Figures 3A and 3B, demonstrating the preparation of 4% CMCTS / 3% oHA and 4% CMCTS / 3% oHA+0.5% AgSD hydrogels. The tunneling mixing system gels without the need for a crosslinking agent and at room temperature. As shown in Figure 3A, both hydrogel formulations bind well. When the hydrogel is applied using a 27-gauge syringe, the injection proceeds smoothly while maintaining the desired viscosity (b). The inclusion of AgSD does not adversely affect the injectability of the hydrogel.

[0067] Morphological analysis of the hydrogels was performed using macroscopic photographs and SEM characterization. Furthermore, an attempt was made to add AgSD to 4% CMCTS / 3% oHA, which resulted in a significant color change due to the reaction. The color change is fundamentally caused by the reaction between the amino groups (-NH2) in CMCTS and the dialdehyde groups (-CHO) in oHA, which forms Schiff base bonds and thus promotes crosslinking. Silver (Ag) ions can coordinate with functional groups such as amino groups in hydrogels, establishing connections between polymer chains.This method increases the crosslinking density of the hydrogel. AgSD acts as a small molecule catalyst, accelerating the formation of imine bonds. This addresses the problems of poor structural stability and weak mechanical properties in hydrogels. Under conjugation, surface plasmon resonance occurs on the surface of silver nanoparticles (AgNPs), enabling these nanoparticles to absorb and scatter light, thus producing a specific color (Figs. 2C and 3A-B). As the concentration of Ag ions in the hydrogel increases, the number of AgNPs increases accordingly, leading to greater light absorption and scattering, and resulting in a deeper color of the hydrogel. Furthermore, the presence of AgSD is clearly observed in the stained section images (Figs. 4A and 4B). These images show safranin O staining of 4% CMCTS / 3% oHA and 4% CMCTS / 3% oHA+0.5% AgSD hydrogels on day 0. The hydrogels were fixed immediately after binding and subjected to the staining procedure. Cells were stained green by applying Fast Green, while the CMCTS / oHA matrix showed a bright red. In the 4% CMCTS / 3% oHA+0.5% AgSD hydrogel, discrete particulate matter consistent with AgSD was observed, which was well fixed in the hydrogel network. The encapsulated cells were also firmly embedded in the entire hydrogel structure.

[0068] After lyophilization, photographs and SEM images showed that the incorporation of AgSD had minimal impact on the surface and interface of the hydrogel as well as its original porous structure at both the macroscopic and microscopic levels. In addition, the hydrogel formulation was characterized by FT-IR spectroscopy (Figure 2D). Compared with oHA, the characteristic absorption peak of the aldehyde group at 1720 cm⁻¹ from the CMCTS / oHA hydrogel disappeared, while the imine stretching vibration absorption peak appeared at 1583 cm⁻¹, indicating that dynamic imine bonds were formed between CMCTS and oHA.

[0069] The sol-to-gel transition of the hydrogel is demonstrated by the relative values ​​between G' and G". Figure 2E shows that at moderate frequencies (less than 6 Hz), both conditions exhibit semi-solid hydrogels because the energy storage modulus G' is greater than the loss modulus G"." At this point, the hydrogel exhibits the main elastic properties of a gel, enabling it to partially withstand external forces. However, as the frequency increases to 6 Hz, G" gradually surpasses G' and approaches a solution state under the 2% CMCTS / 3% oHA condition, while the elastic behavior under the 4% CMCTS / 3% oHA condition remains relatively stable until the frequency approaches 10 Hz. Furthermore, the 4% CMCTS / 3% oHA hydrogel has a much larger elastic modulus and loss modulus compared to the 2% CMCTS / 3% oHA condition, which is related to the number of potential crosslinking sites per chain and the increased internal crosslinking density accompanied by a higher carboxymethyl chitosan concentration. Under each condition,The two different preparation methods (tunneling and pipette mixing) had minimal impact on the rheological properties of the hydrogels. The G' values ​​for each type of hydrogel were no more than two orders of magnitude greater than G'', indicating a weak hydrogelation phenomenon. This offers great potential for producing injectable hydrogels rather than viscoelastic solid hydrogels from these two systems.

[0070] In summary, Figure 2A is a schematic diagram of the preparation of oHA via NaIO4 modification, and the CMCTS / oHA and CMCTS / oHA / AgSD hydrogels were characterized. Figure 2B shows the 1H-NMR results of oHA, indicating the presence of dialdehyde groups in the oHA macromonomer solution. Figure 2C shows the fabricated CMCTS / oHA and CMCTS / oHA+0.5% AgSD hydrogels. In Figure 2C, the hydrogels were well-formed and lyophilized for SEM analysis, exhibiting a uniform porous structure on the surface. Figure 2D shows the FT-IR analysis of the original CMCTS powder, the synthesized oHA composite, and the lyophilized CMCTS / oHA hydrogel, and Figure 2E shows the rheological analysis of the 2% CMCTS / 3% oHA and 4% CMCTS / 3% oHA hydrogels prepared by tunnel mixer and pipetting. Neither method affected the hydrogel properties and no difference was observed.

[0071] The in vitro degradation performance of the lyophilized 2% CMCTS / 3% oHA and 4% CMCTS / 3% oHA composites was characterized by macroscopic photographs, residual mass, degradation rate, and microstructural transformation. In Figures 5A and 5C, the volume and shape of the 2% CMCTS / 3% oHA hydrogel decreased significantly on day 1 compared to day 0, while the 4% CMCTS / 3% oHA hydrogel did not show noteworthy changes until day 3. Figure 5B shows the degradation kinetics, indicating that the 2% CMCTS / 3% oHA conditions exhibited a rapid degradation rate and almost complete degradation on day 3. In contrast, the 4% CMCTS / 3% oHA hydrogel exhibited moderate degradation by day 14. Therefore, it was determined that increased cross-linking strength could slow the degradation rate. Based on these results, the 4% CMCTS / 3% oHA hydrogel was selected for in vitro and in vivo experiments. As shown in Figures 5D and 5E, the internal microstructure of the 4% CMCTS / 3% oHA hydrogel was confirmed throughout the degradation process. After day 1 of immersion in PBS, the hydrogel swelled significantly, and the average pore size (93.32 μm) was much larger than that of the hydrogel prepared on day 0 (60.92 μm), providing a superior three-dimensional extracellular matrix for loading AgSD and DPSCs. After day 2, although the hydrogel maintained its porous structure, the average pore size continued to decrease until the degradation test was completed. Furthermore,In vitro degradation experiments of the CMCTS / oHA hydrogel were also conducted in different media (Figures 14A and 14B). The degradation of the CMCTS / oHA hydrogel varied depending on the environment, and the point of complete degradation was indicated by a yellow asterisk. The results showed that the CMCTS / oHA hydrogel could be completely degraded within 10 days, and its degradation trend was similar to that of the composite material. Notably, the initial swelling trend of the hydrogel in the FBS and culture medium groups was significantly lower than that in the PBS group. Among the groups, the 10% FBS group showed the fastest degradation behavior (Figure 15B). It is well known that hydrogel degradation can be affected by various in vitro culture medium conditions (including the presence of different enzymes and ionic strength). Specifically, this can be attributed to the fact that the addition of FBS alters the internal structure of the hydrogel. To compare the degradation rate between the CMCTS / oHA composite material and the hydrogel, the CMCTS / oHA hydrogel showed a slightly faster degradation rate. Based on these results, the 4% CMCTS / 3% oHA hydrogel was selected for in vitro and in vivo experiments.

[0072] Therefore, Figures 5A-5E show the degradation kinetics of the 2% CMCTS / 3% oHA and 4% CMCTS / 3% oHA hydrogels. Figure 5A shows optical images of the freeze-dried residual hydrogel after degradation over time, Figure 5B shows the degradation rate measurements as per specification page 11 / 19, CN 121588279 A, ​​and Figure 5C shows the residual mass. As these figures show, the degradation rate of the 2% CMCTS / 3% oHA hydrogel is faster than that of the 4% CMCTS / 3% oHA hydrogel. Figure 5E shows SEM images and average pore size of the freeze-dried 4% CMCTS / 3% oHA hydrogel at 0, 1, 2, 3, 5, and 7 days (d). The pore size gradually decreases over time. Values ​​of p*<0.05, p**<0.01, p***<0.001, and p****<0.0001 are considered statistically significant.

[0073] To verify biocompatibility, CMCTS / oHA and CMCTS / oHA+0.5% AgSD hydrogels loaded with high-density DPSCs were cultured for 7 days to confirm biocompatibility and intracellular behavior. In Figure 6A, the CMCTS / oHA group was placed in a cell culture plate with culture medium, and single cells were encapsulated in the hydrogel matrix at OD. Interestingly, the hydrogel gradually disappeared over time, and the high-density cells aggregated together to form 3D tissues similar to 3D spheroids. In contrast, the CMCTS / oHA+0.5% AgSD group did not show a similar tendency to form 3D tissues, and the cells were dispersed on the cell culture plate. This result indicates that the self-degradation of the CMCTS / oHA hydrogel and cell-to-cell adhesion in the CMCTS / oHA group promote 3D cell aggregation.The CMCTS / oHA+0.5% AgSD group failed to support 3D cell aggregation, possibly due to excessive AgSD leading to cell death. Indeed, silver-based materials exhibit cytotoxicity, requiring a proper balance in the biomaterial.

[0074] Next, cell viability was confirmed by CCK-8 assay (Fig. 6B). During 7 days of culture, DPSCs increased dramatically from day 3 to day 7, while DPSCs continuously decreased in the CMCTS / oHA+0.5% AgSD group. This result indicates that 0.5% AgSD exhibits cytotoxicity to DPSCs during culture. On day 7, both live and dead DPSCs were visualized (Fig. 6C). After live / dead staining, the stained cell aggregates were examined under a phase-contrast microscope (Eclipse Ti-S, Nikon, Tokyo, Japan) equipped with an LED lamp (D-LEDI, Nikon, Tokyo, Japan). 3D DPSC aggregates from the CMCTS / oHA group showed strong green (live) and no red (dead). However, most DPSCs in the CMCTS / oHA + 0.5% AgSD group appeared red (dead) instead of green (live), indicating significant cell death. This result was consistent with microscopic visualization (Fig. 6A) and CCK-9 assay (Fig. 6B). It was caused by the release of Ag ions from AgSD within the hydrogel, which exhibit cytotoxic properties. High concentrations of Ag ions disrupt cell membranes, leading to apoptosis or necrosis. Furthermore, Ag ions can induce the production of reactive oxygen species in cells, triggering oxidative stress responses that damage intracellular proteins, lipids, and DNA. Therefore, high concentrations of Ag ions reduce cell viability, thereby affecting cell proliferation and aggregation. 3D cell aggregates were confirmed to have been generated by the CMCTS / oHA hydrogel. Histological analysis was performed to examine the matrix of the 3D aggregates (Fig. 6D). As expected, at day 0, individual cells were encapsulated in CMCTS / oHA. From day 1, DPSCs aggregated, eventually forming 3D spherical structures by day 7, clearly demonstrating strong cell-to-cell aggregation. As mentioned above, this is facilitated by the self-degradation of CMCTS / oHA, which enables DPSCs to interact. In this study, high-density DPSCs were observed encapsulated in CMCTS / oHA hydrogel and spontaneously formed 3D tissues. This phenomenon can be explained by the spontaneous and rapid degradation properties of the hydrogel. In fact, various enzymes are commonly used to accelerate hydrogel degradation and promote tissue formation. This approach promotes faster intercellular binding (e.g., f-actin adhesion) and enhances cell-cell interactions. When cells come into contact, they can form junctions and generate cell clusters. As many cells establish junctions, they collectively form structured 3D cell aggregates.This is commonly referred to as 3D self-assembly. This was confirmed by Safranin O staining with a fast green. Compared to OD, the decellularized hydrogel matrix showed no red staining from day 1. Instead, a strong green was observed, indicating a cellular matrix. This result suggests that the CMCTS / oHA hydrogel self-degrades during culture. Importantly, enzymes such as sodium citrate or hyaluronidase were not used to break down the hydrogel matrix. Typically, after 3D cell assembly within the hydrogel matrix, the hydrogel is manually removed using chemical agents to obtain only cell aggregation. For example, Flores-Torres et al. highlighted the use of sodium citrate to degrade their alginate-based bioprinted constructs, thereby releasing 3D spheroids. The alginate construct is destabilized by removing calcium ions from the cross-linked chain via citrate cations (Flores-Torres S, Peza-Chavez O, Kuasne H, Munguia-Lopez JG, Kort-Mascort J, Ferri L, Jiang T, Rajadurai CV, Park M, Sangwan V. Alginate–gelatin–Matrigel hydrogels, manual 12 / 19 pages, 14 CN 121588279 A enable the development and multigenerational passaging of patient-derived 3D bioprinted cancer spheroid models. Biofabrication. 2021;13:025001).

[0075] In another study, Wei et al. fabricated injectable HA hydrogels using seeded bone marrow mesenchymal stem cell spheroids. Similarly, the hydrogel was digested with hyaluronic acid to release 3D cell spheroids after chondrogenic differentiation (Wei J, Ran P, Li Q, Lu J, Zhao L, Liu Y, Li X. Hierarchically structured injectable hydrogels with loaded cell spheroids for cartilage repairing and osteoarthritis treatment. Chemical Engineering Journal. 2022;430:132211). Compared with the previous examples above, the CMCTS / oHA hydrogel system of the present invention has the significant advantage of forming 3D tissues through self-degradation. As mentioned above,The self-degradable CMCTS / oHA hydrogel effectively facilitates stem cell delivery and can encapsulate nanoparticles / microparticles. Therefore, we propose various applications for 3D tissue engineering and regenerative medicine using the CMCTS / oHA hydrogel platform. First, our method can induce simple co-cultured 3D tissue aggregation systems by combining multiple cellular resources. More specifically, we are able to inject multiple cellular resources in vivo to promote tissue formation. Furthermore, our hydrogel can encapsulate nanoparticles / microparticles and deliver them to target disease sites, making it suitable for minimally invasive treatments of complex conditions where the scope of manipulation in dentistry is limited, such as pulpitis or periodontitis. Additionally, it can be used clinically to promote stem cell injections for several types of tissue formation, such as craniofacial bone fissures. In summary, because it is in the form of an injectable formulation, it can be used as a medical device that does not create surgical scars due to incisions. Therefore, this method has proven to be very convenient and enables the easy production of 3D cell constructs.

[0076] In summary, Figure 6A shows 7 days of in vitro culture of CMCTS / oHA and CMCTS / oHA+0.5% AgSD hydrogels loaded with high-density DPSCs. The CMCTS / oHA hydrogel promoted 3D tissue aggregation, while the CMCTS / oHA+0.5% AgSD hydrogel did not. 3D tissue aggregates began to form in the CMCTS / oHA group on day 1 of culture. Figure 6B shows the cell proliferation rate of CMCTS / oHA and CMCTS / oHA+0.5% AgSD hydrogels at 7 days, and Figure 6C shows the live / dead cell assay at 7 days of culture. Figure 6D shows the histological analysis of the CMCTS / oHA group at 0, 1, and 7 days of culture using H&E and Safranin O staining with Fast Green. 3D tissue aggregation was observed on day 1, and strong cell-to-cell communication was confirmed on day 7. Safranin O staining was negative from day 1, indicating no residual CMCTS / oHA hydrogel due to self-degradation. Values ​​p* < 0.05 and p*** < 0.001 were considered statistically significant.

[0077] All values ​​are plotted as mean ± standard deviation. Statistical analysis of cell proliferation (Fig. 6B and Fig. 7B) and antimicrobial inhibition zone assay (Fig. 9B) was performed using two-way ANOVA (Bonferroni post-hoc test) in GraphPad® Prism 5.0 (GraphPad Software, San Diego, CA). Statistical analysis of antimicrobial CFU assay (Fig. 9E and 9F) was performed using one-way ANOVA (Bonferroni post-hoc test) in GraphPad® Prism 5.0.

[0078] Further in vitro studies were conducted to evaluate the cytotoxicity of AgSD at concentrations below 0.5%. In Fig. 7A,No 3D cell aggregates formed in the CMCTS / oHA + 0.25% AgSD group after 14 days of culture. Instead, cells spread out into a monolayer on the culture plate (Fig. 8), indicating better cell proliferation compared to the 0.5% AgSD group.

[0079] In Fig. 8, SEM imaging revealed the cell aggregation pattern. Robust cell aggregation was observed in both the CMCTS / oHA and CMCTS / oHA + 0.1% AgSD groups. In contrast, the 0.25% AgSD group showed a limited number of sparsely distributed cells adhering to the culture plate surface. The cell proliferation rate of the CMCTS / oHA + 0.1% AgSD group was lower than that of the CMCTS / oHA group. However, after 14 days of culture, 3D cell aggregates similar to those in the CMCTS / oHA group were observed (Fig. 8). This result indicates that 0.1% AgSD exhibits less cytotoxicity compared to the 0.25% and 0.5% AgSD groups. CCK-8 assay results showed that cell proliferation gradually increased in both the CMCTS / oHA group and the CMCTS / oHA+0.1% AgSD group over 7 days. Conversely, the CMCTS / oHA+0.25% AgSD group exhibited poor cell proliferation capacity, indicating that 0.25% AgSD had similar cytotoxic effects to 0.5% AgSD (Figure 7B).

[0080] Live / dead assays revealed extensive cell extension and a large number of live cells in both the CMCTS / oHA and CMCTS / oHA+0.1% AgSD groups, with only a small number of dead cells (Figure 7C). The CMCTS / oHA+0.25% AgSD group showed the fewest number of live cells. F-actin staining also showed strong cell extension and adhesion in the CMCTS / oHA and CMCTS / oHA+0.1% AgSD groups, but little cell extension and adhesion in the CMCTS / oHA+0.25% AgSD group (Fig. 7D). Histological analysis of 3D aggregates in the CMCTS / oHA and CMCTS / oHA+0.1% AgSD groups showed similar 3D cell matrix and strong cell-cell connections. In contrast, only a small number of cells were observed on the culture plate in the CMCTS / oHA+0.25% AgSD group (Fig. 7E). Based on these results, the optimal AgSD concentration in the CMCTS / oHA hydrogel was determined to be 0.1%.

[0081] In the antibacterial test, Streptococcus mutans and Enterococcus faecalis were used because they are prevalent in dental caries and infected root canals, respectively. Figure 9A shows the inhibition areas of Streptococcus mutans and Enterococcus faecalis in different samples, and Figure 9B shows the corresponding statistical measurements. Negative control (2) and CMCTS / oHA hydrogel (3) showed almost no inhibition area.This indicates limited antimicrobial activity against Streptococcus mutans and Enterococcus faecalis. In other samples, the diameter of the antimicrobial region increased in the following order: CMCTS / oHA+0.1% AgSD hydrogel (4), CMCTS / oHA+0.25% AgSD hydrogel (5), and TAP (1). For all three samples, the inhibition region against Streptococcus mutans was larger than that against Enterococcus faecalis. These results indicate that the CMCTS / oHA hydrogel containing AgSD possesses potent antimicrobial properties against both Streptococcus mutans and Enterococcus faecalis, with stronger activity against Streptococcus mutans. The CMCTS / oHA+0.25% AgSD hydrogel exhibited superior antimicrobial activity compared to the CMCTS / oHA+0.1% AgSD hydrogel. This suggests that increasing the AgSD content enhances the antimicrobial activity of the hydrogel, thus confirming the effective delivery of AgSD by the hydrogel matrix. Historically, Ag has been recognized as a potent antimicrobial agent against Streptococcus mutans and Enterococcus faecalis.

[0082] Therefore, Figure 7A shows 14 days of in vitro culture of CMCTS / oHA, CMCTS / oHA+0.1% AgSD, and CMCTS / oHA+0.25% AgSD hydrogels loaded with medium-density DPSCs. In these cultures, cells in the CMCTS / oHA and CMCTS / oHA+0.1% AgSD groups adhered well to the culture plates and formed 3D tissue aggregates after 14 days of culture. In the CMCTS / oHA+0.25% AgSD hydrogel group, DPSCs adhered to and proliferated on the culture plates, but did not form 3D tissue aggregates. Figure 7B shows the cell proliferation rate of the CMCTS / oHA, CMCTS / oHA+0.1% AgSD, and CMCTS / oHA+0.25% AgSD hydrogels at 7 days, and Figure 7C shows the live / dead cell assay and F-actin staining in Figure 7D at 7 days of culture. Mild cytotoxicity was observed in the CMCTS / oHA+0.1% AgSD hydrogel compared to the CMCTS / oHA hydrogel, but cell proliferation and 3D tissue formation were not impaired. Histological analysis using H&E and Alcian blue staining with nuclear solid red showed strong cell aggregation in the CMCTS / oHA and CMCTS / oHA+0.1% AgSD groups, but none in the CMCTS / oHA+0.25% AgSD group. Values ​​p*<0.05, p**<0.01, and p***<0.001 were considered statistically significant.

[0083] The results of the colony-forming unit (CFU) antimicrobial test are shown in Figures 9C and 9E. For Streptococcus mutans, all CMCTS / oHA conditions significantly reduced bacterial density compared to the negative control. Furthermore, the CMCTS / oHA hydrogel containing AgSD reduced bacterial colonies more than the CMCTS / oHA hydrogel alone.This indicates that AgSD significantly enhances the antimicrobial activity against *Streptococcus mutans*. This is consistent with the results of the inhibition test. For *Enterococcus faecalis* (Figs. 9D and 9F), the CMCTS / oHA hydrogel and CMCTS / oHA+0.1% AgSD hydrogel groups showed a slight increase in bacterial colonies compared to the negative control. Significant antimicrobial activity was observed only at the 0.25% AgSD concentration, which significantly reduced bacterial count. This suggests that 0.25% AgSD is necessary for antimicrobial activity against *Enterococcus faecalis*, as lower concentrations would promote bacterial proliferation. Interestingly, the CMCTS / oHA / AgSD hydrogel showed better performance against *Streptococcus mutans* compared to *Enterococcus faecalis*. To date, there has been no systematic comparative study of the characteristics of these two bacteria. However, we believe their characteristics are interrelated. The metabolic characteristics of *Streptococcus mutans* may make it more sensitive to AgSD. AgSD works by inhibiting folate synthesis in bacteria, therefore *Streptococcus mutans* may be more sensitive to this inhibition. On the other hand, *Enterococcus faecalis* has a more flexible metabolism with multiple metabolic pathways that allow it to utilize various carbon sources and amino acids as energy. For example, it can use arginine and sorbitol without requiring folate, making it less sensitive to AgSD. Furthermore, *Enterococcus faecalis* can withstand extreme environmental conditions such as high salt, high temperature, and low pH, enabling it to survive in a variety of environments. In contrast, *Streptococcus mutans* is better suited to acidic environments and sugar-rich conditions. This may explain why AgSD exhibits better antibacterial activity against *Streptococcus mutans*. Previous research by Sheng et al. established dose-response curves for silver nanoparticles (AgNPs) (Sheng Z, Liu Y. Potential impacts of silver nanoparticles on bacteria in the aquatic environment. Journal of environmental management. 2017;191:290-296). As the concentration of Ag+ ions increases, there is a narrow range that stimulates bacterial growth, but beyond this range, antibacterial properties are exhibited. It can be inferred that the Ag+ ions released by CMCTS / oHA+0.1% AgSD hydrogel are within the concentration range that stimulates bacterial proliferation.

[0084] In dentistry, where oral bacteria are a problem, Streptococcus mutans is one of the main pathogens causing dental caries. It forms a biofilm on the tooth surface and metabolizes carbohydrates to produce organic acids, leading to tooth demineralization and caries formation. When caries are not treated in time, the bacteria can penetrate the dentin and reach the pulp, leading to pulpitis and infection. Enterococcus faecalis is a common pathogen associated with failed root canal treatment. Clinically,The main methods for removing biofilms of infection within root canals involve mechanical and chemical cleaning and disinfection. However, complete eradication is challenging due to the complexity of the root canal system and the robust biofilm-forming ability and multidrug resistance of Enterococcus faecalis. Enterococcus faecalis can survive in extreme environments, including high pH and low oxygen conditions, which enhances its survival within the root canal. Its biofilm not only protects the bacteria from antimicrobial agents but also causes persistent inflammation in the pulp and periapical tissues. In these cases, the CMCTS / oHA / AgSD hydrogel, which has strong antimicrobial properties against both bacteria, is easy to inject and can also carry cells, and is considered to have a significant impact on the development of dentistry.

[0085] As explained above, Figure 9A shows the results of in vitro antimicrobial tests of CMCTS / oHA and CMCTS / oHA / AgSD hydrogels against Streptococcus mutans and Enterococcus faecalis. In the experiment, the antimicrobial agent was used as a positive control (1), empty culture as a negative control (2), CMCTS / oHA hydrogel as a sample (3), CMCTS / oHA+0.1% AgSD hydrogel as a sample (4), and CMCTS / oHA+0.25% AgSD hydrogel as a sample (5). They were characterized by an inhibition zone test (a) and their quantitative results were provided by measuring the diameter of the antimicrobial zone (b). The same experimental groups without a positive control were also characterized by a CFU test (c) and its quantitative results (d). The amount and area of ​​newly formed blood vessels were quantified from ImageJ software (NIH) based on slide staining. The count of Streptococcus mutans colonies was significantly reduced in all experimental groups. However, only the CMCTS / oHA+0.25% AgSD hydrogel group showed a reduction in the number of Enterococcus faecalis colonies. A p*** < 0.001 value was considered statistically significant.

[0086] To evaluate the biocompatibility and degradability of CMCTS / oHA / AgSD in vivo, the hydrogel was subcutaneously injected into mice. Figure 10 shows the appearance of the injected mice immediately after hydrogel application. The hydrogel was subcutaneously injected into the back of the mice without any prior incision. The injected hydrogel maintained its volume integrity. Figure 11 shows well-integrated AgSD (red arrows) found in the 0.25% and 0.5% AgSD groups. No adverse tissue reactions such as necrosis or edema were observed in any of the groups including CMCTS / oHA and CMCTS / oHA / AgSD. H&E staining showed normal muscle and adipose tissue morphology in all groups, with well-preserved connective tissue. Alcian blue staining with nuclear solid red showed similar normal tissue morphology without abnormal features. Even in the 0.25% and 0.5% AgSD groups, which exhibited cytotoxicity in vitro, all tissues showed normal and good integration. This indicates thatThe in vivo environment, with its greater complexity and hydrodynamics, can mitigate some of the cytotoxic effects observed in vitro. Therefore, higher AgSD concentrations may be feasible for animal and future clinical applications. However, immune responses and foreign body reactions require further in vivo and in vitro biological validation.

[0087] Safranin O staining with Fast Green indicated that the CMCTS / oHA hydrogel was completely degraded in vivo, confirming its function in promoting tissue integration while locally delivering the drug (e.g., AgSD) as described on pages 15 / 19 of the instruction manual (CN 121588279 A). No significant differences or abnormalities were observed in major organ tissues compared to the sham-operated group (data not shown), as observed by H&E staining (Figure 12).

[0088] As mentioned above, Figure 11 shows the optical images and histological analysis of CMCTS / oHA, +0.1% AgSD, +0.25% AgSD, and +0.5% AgSD hydrogels injected subcutaneously into mice, and 2 weeks after injection. The optical images showed no hydrogel residue or inflammatory pus. Typical mouse subcutaneous tissues (such as fat, muscle, and connective tissue) were visualized in all stainings, with no residual hydrogel.

[0089] In Figure 12, H&E staining of representative organs in the CMCTS / oHA+0.1% AgSD hydrogel group is shown 2 weeks after injection. No abnormal features were observed, and the tissues appeared similar to organs from normal mice. The developed CMCTS / oHA and CMCTS / oHA / AgSD hydrogels exhibit excellent injectability, making them promising candidates for a variety of tissue engineering applications. Their injectability allows for minimally invasive administration, which is advantageous for clinical procedures. For example, the hydrogel can be precisely delivered to target sites such as periodontal pockets, root canals, or wound beds using a standard syringe. This convenient injection technique simplifies the clinical application process, reduces the need for complex surgical procedures, and improves patient comfort. Ease of application is particularly beneficial in outpatient settings, where rapid and effective treatment is crucial. As a means of utilizing our system in a hybrid approach, the CMCTS / oHA hydrogel shows promise for the local delivery of dental stem cells, such as DPSCs and periodontal ligament stem cells, thereby enhancing the regeneration of damaged tooth tissue. DPSCs, crucial for pulp remodeling, can be derived from the patient's own wisdom teeth. When combined with the CMCTS / oHA hydrogel, the system can be effectively used for applications after pulp capping and endodontic procedures (such as fillings or restorative materials) aimed at achieving optimal pulp regeneration. Maintaining sterility during and after endodontic surgery is crucial for prognosis, especially in the microbiologically rich oral environment. In this context, the incorporation of antimicrobial agents such as AgSD helps reduce the risk of infection at the postoperative site, promoting faster healing and better pulp recovery outcomes. Furthermore,In the context of wound healing—particularly for craniofacial injuries—the ability of hydrogels to promote cell aggregation while maintaining a moist microenvironment can significantly accelerate the repair of soft and hard tissue injuries. Their injectable formulation allows for minimally invasive application, which is beneficial for managing chronic wounds or burns where conventional treatments may be less effective.

[0090] Furthermore, long-term biocompatibility is crucial for therapeutic applications, particularly in dental and craniofacial wound healing. Typically, hydrogels remain in contact with tissue for extended periods. We recognize the need for thorough in vivo studies to assess potential inflammatory responses and adverse effects over time. Our studies have shown that the CMCTS / oHA hydrogel degrades rapidly, with no residual hydrogel observed in vivo (Figures 11 and 14). Therefore, we propose addressing concerns about the presence of residual hydrogel by fully utilizing its rapid self-degradation properties, which facilitates efficient in vivo breakdown and reduces the risk of long-term accumulation for clinical wound healing applications. Scalability associated with hydrogel manufacturing for clinical or commercial applications is also a concern. While our formulation has shown promise in a laboratory setting, translating it into a clinically viable product requires careful optimization of manufacturing technologies to ensure consistent quality and efficacy. Our method, utilizing minimal chemical modifications, enhances reproducibility and aligns with regulatory requirements. Through our simple manufacturing process, we have established a robust framework for transitioning from the laboratory to the clinical setting, enabling the widespread use of our hydrogels in a variety of therapeutic applications. By incorporating these considerations, our goal is to provide a balanced perspective that recognizes the potential benefits and challenges associated with the clinical use of our CMCTS / oHA hydrogel system.

[0091] In vivo studies have confirmed the complete degradation of CMCTS / oHA hydrogels, a key feature for tissue engineering applications. The degradation of the hydrogel ensures that it does not require surgical removal after achieving its therapeutic purpose. This property is highly desirable in regenerative medicine, where temporary scaffolds are needed to support tissue growth, followed by natural degradation to allow for the formation of new tissue. The ability of CMCTS / oHA hydrogels to form 3D cell aggregates and support cell proliferation makes them suitable for 3D tissue engineering. These hydrogels can be used to create 3D tissue constructs to repair or replace damaged tissues and organs. For example, the hydrogel's instructions (pages 16 / 19, CN 121588279 A) state that it can be used to engineer cartilage, bone, or skin tissue, providing a scaffold that promotes cell growth and tissue regeneration. The incorporation of AgSD provides antimicrobial properties, which are particularly useful in preventing infection during the healing process. Therefore, the CMCTS / oHA hydrogel, with its excellent injectability, controlled degradation, and ability to support 3D tissue engineering, represents a significant advancement in bioengineering. Its potential applications in clinical practice are vast, and in the near future…Providing improved treatment outcomes and patient experience across a range of medical disciplines.

[0092] In summary, the present invention provides AgSD-incorporated CMCTS / oHA hydrogels as promising biomaterials for a variety of tissue engineering applications. The excellent injectability of this hydrogel allows for minimally invasive administration, thereby enhancing applicability and hand-comfort. In vitro evaluation identified 0.1% AgSD as the optimal concentration for balancing cell proliferation and antimicrobial efficacy. Antimicrobial tests showed significant inhibition against Streptococcus mutans and Enterococcus faecalis, with antimicrobial activity increasing with increasing AgSD concentration. In vivo evaluation confirmed the biocompatibility and complete degradability of the CMCTS / oHA hydrogel, ensuring no adverse tissue reactions and supporting tissue integration. These findings highlight the hydrogel's suitability for 3D tissue engineering, enabling the generation of 3D cell aggregation. The CMCTS / oHA hydrogel exhibits self-crosslinking, self-degradation, antimicrobial activity, and the ability to support 3D tissue formation without the need for additional crosslinking agents, making it a valuable tool in regenerative medicine.

[0093] Furthermore,A hydrogel system loaded with high-density HepG2 / Hhu-7 cells was tested. HepG2 cells (catalog number HB-8065, ATCC, Manassas, Virginia, USA) were cultured in Eagle Minimum Essential Medium (EMEM, catalog number 130-2003, ATCC, Manassas, Virginia, USA) containing 10% fetal bovine serum (FBS, catalog number 10270106, Life Technologies, Carlsbad, California, USA) and 1× penicillin-streptomycin (catalog number 15140-122, Gibco, Waltham, Massachusetts, USA) in 150 mm cell culture dishes (catalog number CS016-0129, ExCell Bio Inc., Shanghai, China). Huh-7 cells (catalog number CVCL_0336, Cellosaurus, Geneva, Switzerland) were cultured in Duchenne Modified Eagle Medium (DMEM, catalog number 11995065, Life Technologies, Carlsbad, California, USA) containing 10% FBS (catalog number 10270106, Life Technologies, Carlsbad, California, USA) and 1× penicillin-streptomycin (catalog number 15140-122, Gibco, Waltham, Massachusetts, USA) in 150 mm cell culture dishes (catalog number CS016-0129, ExCell Bio Inc., Shanghai, China).

[0094] The medium was changed twice a week to maintain optimal growth conditions. Cell clusters were obtained when the culture reached approximately 80-90% confluence. After aspirating culture medium and washing cells once with PBS (catalog number 20012027, Life Technologies, Carlsbad, California, USA), trypsin-EDTA (0.25%) (catalog number 25200072, Gibco, Waltham, Massachusetts, USA) was added to the culture flask, and the cells were incubated at 37°C for 8 minutes until they were completely detached from the plate. The trypsin and cell suspension were neutralized by adding an equal volume of complete culture medium.Then centrifuged at 1800 rpm for 5 minutes. The cells were then used in the CMCTS / oHA hydrogel assembly.

[0095] To encapsulate the cells in the CMCTS / oHA hydrogel, HepG2 cell clusters and Huh-7 cell clusters were thoroughly mixed with the CMCTS macromonomer solution at a density of 150 x 106 cells / ml and then bound with oHA. The detailed hydrogel preparation procedure was the same as that described above in the hydrogel system loaded with high-density DPSCs.

[0096] Next, the hydrogels encapsulating HepG2 were characterized. As shown in Figures 15A and 15B, HepG2 cells were detected in the CMCTS / oHA hydrogel (red arrows), while the cell-free CMCTS / oHA appeared transparent (yellow arrows). CMCTS / oHA loaded with high-density HepG2 cells was fabricated using a tunnel mixing system (Figure 15C) and then smoothly extruded onto a well plate, as shown in Figures 15D and 15E.

[0097] Prior to culturing, the rheology of cell-free and cell-loaded CMCTs / oHA was characterized (Fig. 15F). The transition of the hydrogel from sol to gel was demonstrated by dynamic frequency scanning of the relative values ​​between G' and G''. According to the frequency scanning curves on pages 17 / 19 of the dynamic specification (CN 121588279 A), the values ​​of G' and G'' remained relatively stable, and below 5 Hz, the energy storage modulus was higher than the loss modulus, indicating that the gel behaved primarily elastically in this range. However, as the frequency increased to 5 Hz, the G'' of the hydrogel gradually became higher than G', indicating that the system was closer to a solution state. The CMCTS / oHA hydrogels as a whole exhibited a stable energy storage modulus, and no single G'' value exceeded G' by more than two orders of magnitude, indicating that they had weak gelation behavior. The cell-free hydrogel appeared to be slightly stronger than the cell-containing gel, which was due to the weakening of the hydrogel chains when cells were incorporated. However, the slight physical changes did not affect the extrusion of cells and hydrogels via syringe injection.

[0098] On day 0, HepG2 cells were encapsulated in a CMCTS / oHA hydrogel matrix (Fig. 15G). Over time, starting from day 1, cells began to aggregate (Fig. 15H), and the 3D tissue matrix became increasingly compact, resulting in highly aggregated HepG2 tissue aggregates (Fig. 15I). Rhodamine-labeled HepG2 cells were also shown to exhibit strong aggregate formation on day 7 of culture (Fig. 16). On day 3, the morphology of the cell matrix was confirmed by H&E staining and F-actin nuclear staining (Figs. 15J and 15K). This indicated that cells were tightly connected and aggregated together in aggregates. Furthermore, on day 7, all cell aggregates were maintained, and all cells remained viable in an aggregated state (Fig. 15L).

[0099] In addition,Huh-7 cells were further encapsulated in CMCTS / oHA hydrogels and cultured for 7 days. Optical camera images showed that the CMCTS / oHA hydrogels loaded with high-density Huh-7 cells were initially in a paste-like state, but became cell aggregates after 1 day of culture (Fig. 17A and 17B). The CMCTS / oHA hydrogels loaded with HepG2 cells showed a similar trend during a continuous 7-day culture (Fig. 17C). On day 3 of culture, their cell aggregates were well formed, which was confirmed by H&E and F-actin staining (Fig. 17D and 17E). Similarly, on day 7 of culture, most cells remained viable while maintaining their aggregates (Fig. 17F).

[0100] To assess the in vivo degradability of the CMCTS / oHA hydrogels encapsulating HepG2 cells, the cell-loaded hydrogels were directly injected subcutaneously into Balb / C mice. Grafts were obtained at 1 and 2 weeks after injection (Fig. 18). Histological analysis showed that in OD, HepG2 cells were embedded as single cells in CMCTS / oHA and they did not form cell-to-cell junctions (red arrows). One week after injection, staining results showed that the connective tissue in the natural host group was well preserved. The transplanted cells showed aggregation (blue arrows) but were tightly connected to the natural host tissue (green arrows). However, the cells did not form extracellular matrix, which was attributed to the immune response in Balb / C mice. This was more clearly visible two weeks after injection, with high-density HepG2 aggregates showing strong association with the host tissue in H&E and Alcian blue staining.

[0101] Interestingly, Safranin O staining with Fast Green showed that the CMCTS / oHA hydrogel appeared strongly red in the OD group. However, one week after injection, the red color from the transplantation area completely disappeared. The results indicate that the hydrogel spontaneously and completely degrades in vivo, promoting integration between the transplanted cells and the host's natural tissue. Although no strong cell-to-cell junctions and vascular systems were found in the tumor region, high-density HepG2 cells established junctions with the host's native tissue (Fig. 19).

[0102] To investigate neovascularization in tumor tissue, CMCTS / oHA hydrogels loaded with high-density HepG2 cells were subcutaneously injected into SCID mice, and histological analysis was performed 2 weeks post-injection (Fig. 20). Histological staining of the in situ hydrogel encapsulating HepG2 cells before injection (OD) showed that the cells remained as single, free-floating solids within the hydrogel (Fig. 20A, at OD). 2 weeks post-injection, solid tumors were observed at the injection site, showing vascular interconnection (Fig. 20A, at 2W). H&E and Alcian blue staining showed that HepG2 cells in the solid tumors formed large clusters with a high nucleus-to-cytoplasm ratio and exhibited irregular nuclear shapes, confirming tumor formation. Safranin O staining showed no residual red in the tumor tissue.This indicates that the hydrogel has completely degraded. Notably, a “blood lake” morphology was observed in all stainings, characterized by clusters of erythrocytes resembling vascular buds (red and black asterisks in Fig. 20A). Wright-Giemsa staining (asterisks) further confirmed the presence of erythrocytes. GPC3 and CD31 immunostaining also confirmed the presence of tumor capillaries, with CD31-positive staining indicating vascular structures in the tumor tissue (GPC3-positive staining) (Fig. 20B-D). The tumor tissue was surrounded by host adipose tissue, and the vascular system infiltrated the solid tumor. It was clear that the blood vessels originated from the mouse host tissue, as CD31 expression was not detected in the surrounding tissue prior to transplantation (Fig. 21).

[0103] After confirming the possibility of intratumoral vascularization, CMCTS / oHA loaded with HepG2 and Huh-7 cells were implanted into mice, and the formed blood vessels were quantified 2 weeks post-surgery (Fig. 22A). To validate the function of the hydrogel, commercial Matrigel® and gel-free cell suspension / PBS were used as controls. Matrigel® was injected in a cell-containing suspension before gelation. As expected, cells in the CMCTS / oHA group integrated well with the surrounding tissue and exhibited abundant and good angiogenesis within the tumor. In the PBS group, solid tumor masses formed; however, relatively fewer and smaller vessels were observed. This is thought to be due to rapid cell aggregation in vivo, leading to reduced vascular infiltration as cell aggregation begins. In the Matrigel® group, the number and area of ​​vessels were smaller than those in the CMCTS / oHA group when HepG2 cells were injected (Figs. 22B and 22C). However, when Huh-7 cells were injected, the number and area of ​​vessels were relatively increased compared to CMCTS / oHA. This is thought to be due to the functional response of cells to Matrigel®, resulting in improved integration with the surrounding tissue and increased tumor cell proliferation.

[0104] Notably, although Matrigel® did not solidify during the temperature-sensitive phase prior to implantation and was implanted immediately, it remained in situ, hindering vascular infiltration and integration with surrounding tissue (blue asterisk). In Figure 22D, CD31 staining shows the presence of blood vessels around the residual Matrigel® (white asterisk), but no vascular or surrounding tissue infiltration occurred within the Matrigel® itself. When Matrigel® containing fixed cells was compared to CMCTS / oHA hydrogel, the fully biodegradable CMCTS / oHA hydrogel exhibited easier injection capability and self-degradation, promoting integration with surrounding tissue of implanted cells and contributing to angiogenesis. Compared to Matrigel® gelled on day 0,The CMCTS / oHA hydrogel appears to have completely degraded and disappeared (Figure 23).

[0105] The above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications or substitutions that are obvious to those skilled in the art fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. Instruction Manual 19 / 19 Page 21 CN 121588279 A Figure 1A Figure 1B Figure 1C Instruction Manual Drawings 1 / 20 Page 22 CN 121588279 A Figure 2C Figure 2D Instruction Manual Drawings 2 / 20 Page 23 CN 121588279 A Figure 2E Figure 3A Instruction Manual Drawings 3 / 20 Page 24 CN 121588279 A Figure 3B Figure 4A Instruction Manual Drawings 4 / 20 Page 25 CN 121588279 A Figure 4B Instruction Manual Drawings 5 / 20 Page 26 CN 121588279 A Instruction Manual Drawings 6 / 20 Page 27 CN 121588279 A Instruction Manual Drawings 7 / 20 Page 28 CN 121588279 A Figure 8 Instruction Manual Drawings 8 / 20 Page 29 CN 121588279 A Figure 10 Figure 9A Figure 9B Instruction Manual Drawings 9 / 20 Page 30 CN 121588279 A, ​​Instruction Manual Drawings 10 / 20 Page 31 CN 121588279 A, ​​Figures 9E and 9F, Instruction Manual Drawings 11 / 20 Page 32 CN 121588279 A, ​​Figures 11 and 12, Instruction Manual Drawings 12 / 20 Page 33 CN 121588279 A, ​​Figures 13 and 14, Instruction Manual Drawings 13 / 20 Page 34 CN 121588279 A, ​​Figures 15 and 16, Instruction Manual Drawings 14 / 20 Page 35 CN 121588279 A, ​​Figure 17, Instruction Manual Drawings 15 / 20 Page 36 CN 121588279 A, ​​Figure 18, Instruction Manual Drawings 16 / 20 Page 37 CN 121588279 A, ​​Figure 19, Instruction Manual Drawings 17 / 20 Page 38 CN 121588279 A, ​​Figures 20 and 21, Instruction Manual Drawings 18 / 20 Page 39 CN 121588279 A Figure 22 Appendix to the Specification 19 / 20 Page 40 CN 121588279 A Figure 23 Appendix to the Specification 20 / 20 Page 41 CN 121588279 A Abstract,

Claims

1. An injectable hydrogel comprising a carboxymethyl chitosan (CMCTS) / oxidized hyaluronic acid (oHA) hydrogel bound to silver sulfadiazine (AgSD), wherein the hydrogel has a uniform porous structure on its surface.

2. The injectable hydrogel of claim 1, comprising about 0.1% AgSD.

3. The injectable hydrogel of claim 1, wherein high-density dental pulp stem cells (DPSCs) are encapsulated within a carboxymethyl chitosan (CMCTS) and oHA hydrogel system reinforced with antibacterial silver sulfadiazine (AgSD) material.

4. The injectable hydrogel of claim 1, in the form of 4% CMCTS / 3% oHA+0.x% AgSD, wherein x is one of 0.1%, 0.25% and 0.5%.

5. The injectable hydrogel of claim 4, wherein x equals 0.1% AgSD.

6. A method for preparing an injectable hydrogel, comprising the following steps: Carboxymethyl chitosan (CMCTS), oxidized hyaluronic acid (oHA), human cells, and silver sulfadiazine (AgSD) were directly combined to form a hydrogel construct at room temperature.

7. The method of claim 6, wherein AgSD is incorporated into a CMCTS macromonomer solution and then combined with oHA to produce a CMCTS / oHA / AgSD hydrogel.

8. The method of claim 6, wherein oHA is synthesized by oxidatively cleaving the cyclic structure of hyaluronic acid (HA) into an open chain using sodium periodate, thereby generating two aldehyde groups from the vicinal diol unit of the HA backbone.

9. The method of claim 6, wherein the oHA is prepared by the following steps: Add HA to deionized water and stir at room temperature until completely dissolved; NaIO4 pre-dissolved in Milli-Q water was added to the HA solution and stirred vigorously; Leave the solution in a dark place overnight; Then, excess ethylene glycol was added to the solution to terminate the reaction by maintaining the solution at room temperature for 30 minutes. The solution was dialyzed using a dialysis bag for 7 days, with the deionized water changed every 12 hours. The purified solution was freeze-dried for one week to obtain a clear oHA composite material, wherein the freeze-dried composite material was stored at -20 °C until use.

10. The method of claim 6, wherein the CMCTS / oHA is manufactured by a tunnel mixer or pipette mixing method.

11. The method of claim 6, wherein the human cell is a dental pulp stem cell (DPSC).

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