A tissue repair filler based on adipose stem cells and a preparation method and application thereof

Abstract

The application provides a kind of tissue repair filler based on adipose-derived stem cells and its preparation method and application, belong to tissue repair technical field. Autologous adipose tissue is mixed with cerium dioxide nanozyme armed adipose-derived stem cells to obtain a mixed stem cell / fat system. Hyaluronic acid microspheres loaded with zinc ions and magnesium ions are added to a sodium carboxymethyl cellulose hydrogel matrix, and the mixed stem cell / fat system is added and mixed uniformly. After standing and defoaming, a tissue repair filler based on adipose-derived stem cells is prepared. The filler has good anti-inflammatory, antioxidant and microenvironment improvement effects, low rejection reaction, good autologous adaptability, high safety, and suitable mechanical properties.

Description

Technical Field

[0001] This invention relates to the field of tissue repair technology, specifically to a tissue repair filler based on adipose-derived stem cells, its preparation method, and its application. Background Technology

[0002] Repairing and reconstructing soft tissue defects has always been a challenging problem in plastic and reconstructive surgery. Facial soft tissues include muscles, skin, and fat. Defects are primarily caused by iatrogenic injuries such as tumor resection or improper injections, traumatic injuries such as severe lacerations and burns, congenital conditions such as cleft lip and palate, cleft nasal ala, and subcutaneous fat atrophy, as well as aging. Some facial soft tissue defects can lead to organ dysfunction, causing inconvenience to patients' daily activities such as chewing, speaking, and swallowing. They can also cause appearance anxiety and psychological problems. Therefore, soft tissue repair not only helps patients restore the physiological function of their original tissues but also helps overcome psychological problems caused by appearance, which is of great significance for improving their quality of life.

[0003] Currently, clinical treatment for facial soft tissue depressions of varying degrees primarily relies on surgery, including traumatic and minimally invasive procedures. Traumatic surgery has several drawbacks. For example, using autologous tissue flaps for filling can easily lead to defects at the donor site, and the recovery period is long, with a high risk of infection and necrosis. Using prostheses results in poor biocompatibility, a high risk of rejection, and subsequent tissue fibrosis and infection. Autologous fat grafting is a minimally invasive surgical method that has gained considerable attention in recent years. However, this therapy currently faces challenges such as significant fluctuations in the absorption rate of biomaterials, poor survival rates of transplanted fat cells, and unpredictable long-term treatment outcomes, which limit its development. Injecting soft tissue fillers is a minimally invasive procedure with minimal damage, rapid recovery, and a low risk of infection. This not only provides convenience for doctors and patients but also offers high cost-effectiveness, making it highly favored in plastic and reconstructive surgery.

[0004] Currently used injectable fillers in clinical practice are mainly collagen and hyaluronic acid fillers, which have low compressive strength and rapid absorption. Microsphere fillers can provide a longer-lasting filling effect, but have lower shape retention. Microsphere-reinforced hydrogel fillers can achieve immediate filling while stimulating collagen regeneration. However, with the extension of application time, adverse clinical events caused by solid microsphere fillers have been increasing in recent years. Therefore, the development of novel fillers is particularly urgent and important. Summary of the Invention

[0005] The purpose of this invention is to propose a tissue repair filler based on adipose stem cells, its preparation method and application, which has good anti-inflammatory, antioxidant and microenvironment-improving effects, low rejection reaction, good autologous adaptability, high safety and suitable mechanical properties.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for preparing a tissue repair filler based on adipose-derived stem cells. Autologous adipose tissue is mixed uniformly with cerium dioxide nanozyme-enhanced adipose-derived stem cells to obtain a mixed stem cell / fat system. Hyaluronic acid microspheres loaded with zinc and magnesium ions are added to a sodium carboxymethyl cellulose hydrogel matrix, followed by the mixed stem cell / fat system. The mixture is then mixed uniformly and allowed to stand to remove bubbles, thus obtaining the tissue repair filler based on adipose-derived stem cells.

[0008] As a further improvement to the present invention, the following steps are included:

[0009] S1. Aspirate the autologous blood lipid mixture, centrifuge at low temperature to obtain autologous adipose tissue;

[0010] S2. Cerium dioxide nanozymes were prepared into a suspension and added to stem cell culture medium to obtain a culture medium containing cerium dioxide nanozymes. Adipose stem cells were cultured in stem cell culture medium. When the fusion rate reached 60-70%, the culture medium was replaced with one containing cerium dioxide nanozymes. The cells were then washed, cultured in stem cell culture medium, and separated to obtain cerium dioxide nanozyme-armed adipose stem cells. These cells were then added to autologous adipose tissue to obtain a mixed stem cell / adipose tissue system.

[0011] Autologous fat, derived from the patient's own body, eliminates the risk of rejection. However, simple autologous fat transplantation has a high absorption rate, often requiring multiple surgeries, and the inability to extract large amounts of fat at once limits its application. This invention combines autologous fat with adipose-derived stem cells. Adipose-derived stem cells possess multi-directional differentiation potential, reducing complications such as fat particle absorption, liquefaction, and infection, and improving the survival of fat particles. Furthermore, it exhibits low rejection rates and good autologous adaptability.

[0012] Cerium dioxide nanozymes are a novel rare-earth oxide nanomaterial that has attracted particular attention due to their excellent antioxidant and anti-inflammatory properties. They also possess multifunctional enzyme-like activity, effectively scavenging reactive oxygen species and thus alleviating cell damage caused by oxidative stress. Furthermore, they play a crucial role in regulating inflammatory responses, significantly improving tissue repair processes by influencing the secretion of inflammatory factors and the microenvironment. Therefore, this invention co-cultures cerium dioxide nanozymes with adipose-derived stem cells to obtain cerium dioxide nanozyme-enhanced adipose-derived stem cells, thereby improving stem cell function and significantly enhancing their anti-inflammatory, antioxidant, and microenvironment-improving capabilities.

[0013] The hybrid stem cell / fat system obtained by combining cerium dioxide nanozymes-armed adipose stem cells with autologous adipose tissue has a good effect on promoting angiogenesis, reducing tissue inflammation and oxidation. At the same time, adipose stem cells can also directly differentiate into adipocytes, which can reduce complications such as fat particle absorption, liquefaction, and infection, and improve the survival of fat microparticles, so that the target effect can be achieved in a single operation.

[0014] S3. Add zinc citrate and magnesium citrate to a methacrylated hyaluronic acid solution, add a photoinitiator, homogenize, and then heat and sonicate to obtain a precursor solution;

[0015] S4. Using the precursor solution as the dispersed phase and paraffin containing emulsifier as the continuous phase, the two phases are injected into the microfluidic chip channel using a precision injection pump. The droplet diameter is controlled at 70-100μm by adjustment. The emulsion droplets are collected, cured by ultraviolet light, centrifuged, and washed to remove unreacted monomers, initiators, and emulsifiers, resulting in reinforced microspheres.

[0016] This invention utilizes reinforcing particles to improve the mechanical properties of tissue filler materials while reducing adipose tissue concentration. On one hand, it can load magnesium and zinc ions with high physiological relevance, promoting vascular endothelial cell differentiation, increasing fat survival rate, promoting adipogenic differentiation of stem cells, and reducing post-transplant inflammatory responses. On the other hand, it can improve the compressive strength of tissue repair fillers, promote connective tissue regeneration, and provide long-term stable filling. Using sodium hyaluronate as the main material, it exhibits good safety.

[0017] Macrophages, with a diameter of approximately 25-30 μm, can engulf about 25% of their own volume per hour. To avoid acute inflammatory reactions triggered by macrophage phagocytosis and ensure filling effectiveness, the microsphere particle size must meet specific requirements. Therefore, when the microsphere particle size is less than 20 μm, it is easily engulfed by macrophages, inducing a foreign body macrophage response, accelerating degradation, and causing a rapid reduction in filling volume, thereby weakening tissue regeneration and shaping effects. Conversely, when the particle size is greater than 20 μm, the likelihood of microspheres being engulfed by macrophages is significantly reduced, thus improving the stability of the filling material. However, it is important to note that if the particle size is too large, it may cause tissue overstimulation, inducing chronic inflammatory proliferation and affecting biocompatibility. Therefore, the particle size of the enhanced microspheres is prepared to be 70-100 μm, greater than 20 μm, to avoid phagocytosis and degradation, while being smaller than the inner diameter of clinical injection needles (usually <100 μm) to ensure smooth injection.

[0018] S5. Dissolve sodium carboxymethyl cellulose in PBS solution, stir to mix evenly, let stand to remove bubbles, and obtain sodium carboxymethyl cellulose hydrogel matrix;

[0019] Sodium carboxymethyl cellulose hydrogel scaffolds feature interconnected pore structures and high specific surface area, mimicking the natural fibrous architecture of the extracellular matrix and providing an ideal microenvironment for cell infiltration, angiogenesis, and collagen deposition.

[0020] S6. Add the enhanced microspheres to the PBS solution and disperse them. After mixing evenly, add them to the sodium carboxymethyl cellulose hydrogel matrix and repeatedly push and mix. Add the mixed stem cell / adipose system, mix evenly, and let stand to remove bubbles to obtain an adipose stem cell-based tissue repair filler.

[0021] Ideal injectable tissue fillers should have good biocompatibility and biodegradability, possess mechanical properties similar to the target tissue after filling, have suitable compressive strength, plasticity and morphological stability, effectively resist the pressure generated by facial skin and muscle activity, stimulate collagen regeneration through slow degradation to maintain body volume, and ensure that degradation products are non-toxic and non-immunogenic. In addition, the material should have suitable porosity and pore size to provide sufficient space for cell activity and the exchange of nutrients and oxygen.

[0022] As a further improvement of the present invention, the low-temperature centrifugation in step S1 is carried out at a temperature of 3-6℃, a rotation speed of 1000-1500 r / min, and a time of 3-10 min. The present invention uses low-speed centrifugation (1000-1500 r / min), which does not damage adipocytes and improves the survival rate of adipocytes.

[0023] As a further improvement of the present invention, the concentration of the suspension in step S2 is 5-15 mg / mL, the concentration of cerium dioxide nanozyme in the culture medium containing cerium dioxide nanozyme is 50-80 μg / mL, the stem cell culture medium is selected from at least one of DMEM-L, M-199, and K-SFM, the culture time is 5-8 h, and the concentration of cerium dioxide nanozyme-armed adipose stem cells in the mixed stem cell / adipose tissue system is 10 μg / mL. 4-6 cell / mL.

[0024] As a further improvement of the present invention, the mass ratio of zinc citrate, magnesium citrate, methacrylated hyaluronic acid, and photoinitiator in step S3 is 2.87-5.74:4.51-6.77:4-7:0.3-0.5, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonite, the homogenization speed is 10000-15000 r / min, the time is 10-15 min, the heating and ultrasonic treatment temperature is 50-60℃, the ultrasonic power is 150-250 W, and the time is 20-30 min.

[0025] As a further improvement of the present invention, the concentration of emulsifier in the paraffin containing emulsifier in step S4 is 3-5 wt%, the emulsifier is a Span series emulsifier, and the ultraviolet light wavelength for ultraviolet curing is 365 nm, and the time is 3-6 min.

[0026] As a further improvement of the present invention, the concentration of sodium carboxymethyl cellulose in the sodium carboxymethyl cellulose hydrogel matrix in step S5 is 1-3 wt%.

[0027] As a further improvement of the present invention, the mass ratio of the enhanced microspheres, sodium carboxymethyl cellulose hydrogel matrix, and mixed stem cell / adipose system in step S6 is 5-10:80-120:100-150.

[0028] The present invention further protects a tissue repair filler based on adipose stem cells prepared by the above-described preparation method.

[0029] This invention further protects the application of the above-mentioned adipose stem cell-based tissue repair filler in tissue repair.

[0030] The present invention has the following beneficial effects:

[0031] 1. This invention combines autologous fat with cerium dioxide nanoenzyme-enhanced adipose stem cells. Adipose stem cells have multi-directional differentiation potential, which can reduce complications such as fat particle absorption, liquefaction, and infection, and improve the survival of fat particles. Cerium dioxide nanoenzyme-enhanced treatment improves the function of stem cells, significantly enhances their anti-inflammatory, antioxidant, and microenvironment-improving abilities, and at the same time, it has low rejection reaction and good autologous adaptability.

[0032] 2. This invention mixes a hybrid stem cell / fat system with a sodium carboxymethyl cellulose hydrogel matrix, which reduces the amount of autologous fat added and has the advantages of being biodegradable, non-toxic, non-immunogenic, biocompatible, and having low rejection. At the same time, it can form a connected pore structure, mimicking the natural fibrous structure of the extracellular matrix, providing an ideal microenvironment for cell infiltration, angiogenesis, and collagen deposition.

[0033] 3. This invention improves the mechanical properties of tissue filling materials while reducing the concentration of adipose tissue by adding reinforcing microspheres. The loaded physiologically active metal ions can promote the differentiation of vascular endothelial cells, improve the survival rate of fat, promote the adipogenic differentiation of stem cells, reduce the inflammatory response after transplantation, and have high safety. The particle size is prepared between 70-100μm, which can avoid being phagocytosed by macrophages and has good injectability. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a TEM image of the enhanced microspheres obtained in step S5 of Example 1.

[0036] Figure 2 The image shows a SEM image of the sodium carboxymethyl cellulose hydrogel matrix obtained in step S6 of Example 1.

[0037] Figure 3 This is a diagram showing the morphological changes of different samples after subcutaneous implantation in Test Example 2. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The stem cell culture medium formula is as follows: DMEM-L: 10% FBS, 100 U / mL penicillin, 100 U / mL streptomycin.

[0040] Example 1

[0041] This embodiment provides a method for preparing a tissue repair filler based on adipose-derived stem cells, including the following steps:

[0042] S1. 500 mL of autologous blood lipid mixture was extracted by liposuction. 300 mL was taken for further processing and centrifuged at 5℃, 1000 r / min for 10 min to remove oil droplets and lower layer liquid, thus obtaining autologous adipose tissue.

[0043] S2. Take 200 mL of autologous blood lipid mixture to separate adipose-derived stem cells and obtain adipose-derived stem cells;

[0044] The separation method is as follows:

[0045] The autologous blood lipid mixture was allowed to stand, and the lower layer of blood fluid was discarded. Adipose tissue was added to 50 mL centrifuge tubes in portions, washed three times with PBS solution at pH 7.4, and an equal volume of 0.25% type I collagenase was added. The tubes were sealed and digested at 37°C and 120 rpm for 40 min on a shaker. The centrifuge tubes were removed and sterilized with 75% alcohol. The mixture was passed through an 80-mesh filter and centrifuged at 1000 rpm for 5 min, discarding the supernatant. The mixture was resuspended in PBS solution at pH 7.4, and then centrifuged at 1000 rpm for 5 min, discarding the supernatant. This process was repeated once, and the precipitate was collected to obtain adipose-derived stem cells.

[0046] S3. Cerium dioxide nanozyme was prepared into a suspension with a concentration of 5 mg / mL and added to DMEM-L stem cell culture medium to obtain a culture medium containing cerium dioxide nanozyme at a concentration of 50 μg / mL. Adipose-derived stem cells were cultured in DMEM-L stem cell culture medium. When the confluence reached 60%, the medium was replaced with cerium dioxide nanozyme-containing medium, and cultured for another 8 hours. The cells were then washed, replaced with DMEM-L stem cell culture medium, digested, and centrifuged at 300g for 3 min to obtain cerium dioxide nanozyme-armed adipose-derived stem cells. These were then added to autologous adipose tissue to obtain a mixed stem cell / adipose tissue system with a concentration of 10 μg / mL cerium dioxide nanozyme-armed adipose-derived stem cells. 5 cell / mL;

[0047] S4. Add 2.87g of zinc citrate and 4.51g of magnesium citrate to 100mL of methacrylated hyaluronic acid solution (concentration of 4wt%), add 0.3g of photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonite lithium, homogenize at 10000r / min for 15min, heat to 50℃, and sonicate at 150W for 30min to obtain the precursor solution;

[0048] S5. Using the precursor solution as the dispersed phase and paraffin containing 3 wt% emulsifier Span-85 as the continuous phase, the two phases were injected separately into the microfluidic chip channel using a precision injection pump. The droplet diameter was controlled to be below 100 μm by adjustment. The emulsion droplets were collected, cured under 365 nm UV light for 3 min, centrifuged, and washed to remove unreacted monomers, photoinitiators, and emulsifiers, yielding reinforced microspheres. Figure 1 The image shows a TEM image of the prepared reinforced microspheres, which indicates that the particle size is less than 100 micrometers.

[0049] S6. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 1 wt%. Figure 2The image shows the SEM image of the prepared sodium carboxymethyl cellulose hydrogel matrix. As can be seen from the image, the matrix has the characteristics of interconnected pore structure and high specific surface area, which simulates the natural fibrous structure of the extracellular matrix and provides an ideal microenvironment for cell infiltration, angiogenesis and collagen deposition.

[0050] S7. Disperse 5g of enhanced microspheres in PBS solution at pH 7.4, mix well, add to 80g of sodium carboxymethyl cellulose hydrogel matrix, repeatedly push and mix, add 100g of mixed stem cell / adipose system, mix well, let stand to remove bubbles, and obtain tissue repair filler based on adipose stem cells.

[0051] Example 2

[0052] This embodiment provides a method for preparing a tissue repair filler based on adipose-derived stem cells, including the following steps:

[0053] S1. 500 mL of autologous blood lipid mixture was extracted by liposuction. 300 mL was taken for further processing and centrifuged at 5℃, 1500 r / min for 3 min to remove oil droplets and lower liquid to obtain autologous adipose tissue.

[0054] S2. Take 200 mL of autologous blood lipid mixture to separate adipose-derived stem cells and obtain adipose-derived stem cells;

[0055] The separation method is as follows:

[0056] The autologous blood lipid mixture was allowed to stand, and the lower layer of blood fluid was discarded. Adipose tissue was added to 50 mL centrifuge tubes in portions, washed three times with PBS solution at pH 7.4, and an equal volume of 0.25% type I collagenase was added. The tubes were sealed and digested at 37°C and 120 rpm for 40 min on a shaker. The centrifuge tubes were removed, sterilized with 75% alcohol, and the mixture was passed through an 80-mesh filter and centrifuged at 1500 rpm for 5 min. The supernatant was discarded. The mixture was resuspended in PBS solution at pH 7.4, and then centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and this process was repeated once. The precipitate was collected to obtain adipose-derived stem cells.

[0057] S3. Cerium dioxide nanozyme was prepared into a suspension with a concentration of 15 mg / mL and added to DMEM-L stem cell culture medium to obtain a culture medium containing cerium dioxide nanozyme at a concentration of 80 μg / mL. Adipose-derived stem cells were cultured in DMEM-L stem cell culture medium. When the confluence reached 70%, the medium was replaced with cerium dioxide nanozyme-containing medium, and cultured for another 5 hours. The cells were then washed, replaced with DMEM-L stem cell culture medium, digested, and centrifuged at 300g for 5 minutes to obtain cerium dioxide nanozyme-armed adipose-derived stem cells. These were then added to autologous adipose tissue to obtain a mixed stem cell / adipose tissue system with a concentration of 10 μg / mL cerium dioxide nanozyme-armed adipose-derived stem cells. 5 cell / mL;

[0058] S4. Add 5.74g of zinc citrate and 6.77g of magnesium citrate to 100mL of methacrylated hyaluronic acid solution (concentration 7wt%), add 0.5g of photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonite lithium, homogenize at 15000r / min for 10min, heat to 60℃, and sonicate at 250W for 20min to obtain the precursor solution;

[0059] S5. Using the precursor solution as the dispersed phase and paraffin containing 5 wt% emulsifier Span-85 as the continuous phase, the two phases are injected into the microfluidic chip channel using a precision injection pump. The droplet diameter is controlled to be below 100 μm by adjustment. The emulsion droplets are collected, cured under 365 nm ultraviolet light for 6 min, centrifuged, and washed to remove unreacted monomers, photoinitiators, and emulsifiers to obtain reinforced microspheres.

[0060] S6. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 3 wt%.

[0061] S7. Disperse 10g of enhanced microspheres in PBS solution at pH 7.4, mix well, add to 120g of sodium carboxymethyl cellulose hydrogel matrix, repeatedly push and mix, add 150g of mixed stem cell / adipose system, mix well, let stand to remove bubbles, and obtain tissue repair filler based on adipose stem cells.

[0062] Example 3

[0063] This embodiment provides a method for preparing a tissue repair filler based on adipose-derived stem cells, including the following steps:

[0064] S1. 500 mL of autologous blood lipid mixture was extracted by liposuction. 300 mL was taken for further processing and centrifuged at 1200 r / min for 5 min at 5℃ to remove oil droplets and lower liquid to obtain autologous adipose tissue.

[0065] S2. Take 200 mL of autologous blood lipid mixture to separate adipose-derived stem cells and obtain adipose-derived stem cells;

[0066] The separation method is as follows:

[0067] The autologous blood lipid mixture was allowed to stand, and the lower layer of blood fluid was discarded. Adipose tissue was added to 50 mL centrifuge tubes in portions, washed three times with PBS solution at pH 7.4, and an equal volume of 0.25% type I collagenase was added. The tubes were sealed and digested at 37°C and 120 rpm for 40 min on a shaker. The centrifuge tubes were removed, sterilized with 75% alcohol, and the mixture was passed through an 80-mesh filter and centrifuged at 1200 rpm for 5 min. The supernatant was discarded. The mixture was resuspended in PBS solution at pH 7.4, and then centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and this process was repeated once. The precipitate was collected to obtain adipose-derived stem cells.

[0068] S3. Cerium dioxide nanozyme was prepared into a suspension with a concentration of 10 mg / mL and added to DMEM-L stem cell culture medium to obtain a culture medium containing cerium dioxide nanozyme at a concentration of 65 μg / mL. Adipose-derived stem cells were cultured in DMEM-L stem cell culture medium. When the confluence reached 65%, the medium was replaced with cerium dioxide nanozyme-containing medium, and cultured for another 6 hours. The cells were then washed, replaced with DMEM-L stem cell culture medium, digested, and centrifuged at 300g for 4 minutes to obtain cerium dioxide nanozyme-armed adipose-derived stem cells. These were then added to autologous adipose tissue to obtain a mixed stem cell / adipose tissue system with a concentration of 10 mg / mL cerium dioxide nanozyme-armed adipose-derived stem cells. 5 cell / mL;

[0069] S4. Add 4.02g of zinc citrate and 5.41g of magnesium citrate to 100mL of methacrylated hyaluronic acid solution (concentration of 5wt%), add 0.4g of photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonite lithium, homogenize at 13000r / min for 12min, heat to 55℃, and sonicate at 200W for 25min to obtain the precursor solution;

[0070] S5. Using the precursor solution as the dispersed phase and paraffin containing 4 wt% emulsifier Span-85 as the continuous phase, the two phases are injected into the microfluidic chip channel using a precision injection pump. The droplet diameter is controlled to be below 100 μm by adjustment. The emulsion droplets are collected, cured under 365 nm ultraviolet light for 5 min, centrifuged, and washed to remove unreacted monomers, photoinitiators, and emulsifiers to obtain reinforced microspheres.

[0071] S6. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 2 wt%.

[0072] S7. Disperse 7g of enhanced microspheres in PBS solution at pH 7.4, mix well, add to 100g of sodium carboxymethyl cellulose hydrogel matrix, repeatedly push and mix, add 123g of mixed stem cell / adipose system, mix well, let stand to remove bubbles, and obtain tissue repair filler based on adipose stem cells.

[0073] Comparative Example 1

[0074] Compared with Example 3, the only difference is that step S3 did not involve arming with cerium dioxide nanozymes.

[0075] Includes the following steps:

[0076] S1. 500 mL of autologous blood lipid mixture was extracted by liposuction. 300 mL was taken for further processing and centrifuged at 1200 r / min for 5 min at 5℃ to remove oil droplets and lower liquid to obtain autologous adipose tissue.

[0077] S2. Take 200 mL of autologous blood lipid mixture to separate adipose-derived stem cells and obtain adipose-derived stem cells;

[0078] The separation method is as follows:

[0079] The autologous blood lipid mixture was allowed to stand, and the lower layer of blood fluid was discarded. Adipose tissue was added to 50 mL centrifuge tubes in portions, washed three times with PBS solution at pH 7.4, and an equal volume of 0.25% type I collagenase was added. The tubes were sealed and digested at 37°C and 120 rpm for 40 min on a shaker. The centrifuge tubes were removed, sterilized with 75% alcohol, and the mixture was passed through an 80-mesh filter and centrifuged at 1200 rpm for 5 min. The supernatant was discarded. The mixture was resuspended in PBS solution at pH 7.4, and then centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and this process was repeated once. The precipitate was collected to obtain adipose-derived stem cells.

[0080] S3. Add adipose-derived stem cells to autologous adipose tissue to obtain a mixed stem cell / fat system, with a concentration of adipose-derived stem cells of 10. 5 cell / mL;

[0081] S4. Add 4.02g of zinc citrate and 5.41g of magnesium citrate to 100mL of methacrylated hyaluronic acid solution (concentration of 5wt%), add 0.4g of photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonite lithium, homogenize at 13000r / min for 12min, heat to 55℃, and sonicate at 200W for 25min to obtain the precursor solution;

[0082] S5. Using the precursor solution as the dispersed phase and paraffin containing 4 wt% emulsifier Span-85 as the continuous phase, the two phases are injected into the microfluidic chip channel using a precision injection pump. The droplet diameter is controlled to be below 100 μm by adjustment. The emulsion droplets are collected, cured under 365 nm ultraviolet light for 5 min, centrifuged, and washed to remove unreacted monomers, photoinitiators, and emulsifiers to obtain reinforced microspheres.

[0083] S6. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 2 wt%.

[0084] S7. Disperse 7g of enhanced microspheres in PBS solution at pH 7.4, mix well, add to 100g of sodium carboxymethyl cellulose hydrogel matrix, repeatedly push and mix, add 123g of mixed stem cell / adipose system, mix well, let stand to remove bubbles, and obtain tissue repair filler based on adipose stem cells.

[0085] Comparative Example 2

[0086] Compared with Example 3, the only difference is that zinc citrate and magnesium citrate were not added in step S4.

[0087] Includes the following steps:

[0088] S1. 500 mL of autologous blood lipid mixture was extracted by liposuction. 300 mL was taken for further processing and centrifuged at 1200 r / min for 5 min at 5℃ to remove oil droplets and lower liquid to obtain autologous adipose tissue.

[0089] S2. Take 200 mL of autologous blood lipid mixture to separate adipose-derived stem cells and obtain adipose-derived stem cells;

[0090] The separation method is as follows:

[0091] The autologous blood lipid mixture was allowed to stand, and the lower layer of blood fluid was discarded. Adipose tissue was added to 50 mL centrifuge tubes in portions, washed three times with PBS solution at pH 7.4, and an equal volume of 0.25% type I collagenase was added. The tubes were sealed and digested at 37°C and 120 rpm for 40 min on a shaker. The centrifuge tubes were removed, sterilized with 75% alcohol, and the mixture was passed through an 80-mesh filter and centrifuged at 1200 rpm for 5 min. The supernatant was discarded. The mixture was resuspended in PBS solution at pH 7.4, and then centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and this process was repeated once. The precipitate was collected to obtain adipose-derived stem cells.

[0092] S3. Cerium dioxide nanozyme was prepared into a suspension with a concentration of 10 mg / mL and added to DMEM-L stem cell culture medium to obtain a culture medium containing cerium dioxide nanozyme at a concentration of 65 μg / mL. Adipose-derived stem cells were cultured in DMEM-L stem cell culture medium. When the confluence reached 65%, the medium was replaced with cerium dioxide nanozyme-containing medium, and cultured for another 6 hours. The cells were then washed, replaced with DMEM-L stem cell culture medium, digested, and centrifuged at 300g for 4 minutes to obtain cerium dioxide nanozyme-armed adipose-derived stem cells. These were then added to autologous adipose tissue to obtain a mixed stem cell / adipose tissue system with a concentration of 10 mg / mL cerium dioxide nanozyme-armed adipose-derived stem cells. 5 cell / mL;

[0093] S4. Add 0.4 g of photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonite lithium to 100 mL of methacrylated hyaluronic acid solution (concentration of 5 wt%), homogenize at 13000 r / min for 12 min, heat to 55 °C, and sonicate at 200 W for 25 min to obtain the precursor solution.

[0094] S5. Using the precursor solution as the dispersed phase and paraffin containing 4 wt% emulsifier Span-85 as the continuous phase, the two phases are injected into the microfluidic chip channel using a precision injection pump. The droplet diameter is controlled to be below 100 μm by adjustment. The emulsion droplets are collected, cured under 365 nm ultraviolet light for 5 min, centrifuged, and washed to remove unreacted monomers, photoinitiators, and emulsifiers to obtain reinforced microspheres.

[0095] S6. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 2 wt%.

[0096] S7. Disperse 7g of enhanced microspheres in PBS solution at pH 7.4, mix well, add to 100g of sodium carboxymethyl cellulose hydrogel matrix, repeatedly push and mix, add 123g of mixed stem cell / adipose system, mix well, let stand to remove bubbles, and obtain tissue repair filler based on adipose stem cells.

[0097] Comparative Example 3

[0098] Compared with Example 3, the only difference is that the autologous fat tissue was replaced by an equal mass of sodium carboxymethyl cellulose hydrogel matrix.

[0099] Includes the following steps:

[0100] S1. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 2 wt%.

[0101] S2. 200 mL of autologous blood lipid mixture was extracted using liposuction, and adipose-derived stem cells were separated to obtain adipose-derived stem cells;

[0102] The separation method is as follows:

[0103] The autologous blood lipid mixture was allowed to stand, and the lower layer of blood fluid was discarded. Adipose tissue was added to 50 mL centrifuge tubes in portions, washed three times with PBS solution at pH 7.4, and an equal volume of 0.25% type I collagenase was added. The tubes were sealed and digested at 37°C and 120 rpm for 40 min on a shaker. The centrifuge tubes were removed, sterilized with 75% alcohol, and the mixture was passed through an 80-mesh filter and centrifuged at 1200 rpm for 5 min. The supernatant was discarded. The mixture was resuspended in PBS solution at pH 7.4, and then centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and this process was repeated once. The precipitate was collected to obtain adipose-derived stem cells.

[0104] S3. Cerium dioxide nanozyme was prepared into a suspension with a concentration of 10 mg / mL and added to DMEM-L stem cell culture medium to obtain a culture medium containing cerium dioxide nanozyme at a concentration of 65 μg / mL. Adipose-derived stem cells were cultured in DMEM-L stem cell culture medium. When the confluence reached 65%, the medium was replaced with cerium dioxide nanozyme-containing medium, and cultured for another 6 hours. The cells were then washed, replaced with DMEM-L stem cell culture medium, digested, and centrifuged at 300g for 4 min to obtain cerium dioxide nanozyme-armed adipose-derived stem cells. This mixture was added to a sodium carboxymethyl cellulose hydrogel matrix to obtain a mixed system with a concentration of 10 mg / mL cerium dioxide nanozyme-armed adipose-derived stem cells. 5 cell / mL;

[0105] S4. Add 4.02g of zinc citrate and 5.41g of magnesium citrate to 100mL of methacrylated hyaluronic acid solution (concentration of 5wt%), add 0.4g of photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonite lithium, homogenize at 13000r / min for 12min, heat to 55℃, and sonicate at 200W for 25min to obtain the precursor solution;

[0106] S6. Using the precursor solution as the dispersed phase and paraffin containing 4 wt% emulsifier Span-85 as the continuous phase, the two phases are injected into the microfluidic chip channel separately using a precision injection pump. The droplet diameter is controlled to be below 100 μm by adjustment. The emulsion droplets are collected, cured under 365 nm ultraviolet light for 5 min, centrifuged, and washed to remove unreacted monomers, photoinitiators, and emulsifiers to obtain reinforced microspheres.

[0107] S7. Disperse 7g of enhanced microspheres in PBS solution at pH 7.4, mix well, add to 100g of sodium carboxymethyl cellulose hydrogel matrix, repeatedly push and mix, add 123g of mixed stem cell / adipose system, mix well, let stand to remove bubbles, and obtain tissue repair filler based on adipose stem cells.

[0108] Comparative Example 4

[0109] Compared to Example 3, the only difference is that no reinforcing microspheres were added.

[0110] Includes the following steps:

[0111] S1. 500 mL of autologous blood lipid mixture was extracted by liposuction. 300 mL was taken for further processing and centrifuged at 1200 r / min for 5 min at 5℃ to remove oil droplets and lower liquid to obtain autologous adipose tissue.

[0112] S2. Take 200 mL of autologous blood lipid mixture to separate adipose-derived stem cells and obtain adipose-derived stem cells;

[0113] The separation method is as follows:

[0114] The autologous blood lipid mixture was allowed to stand, and the lower layer of blood fluid was discarded. Adipose tissue was added to 50 mL centrifuge tubes in portions, washed three times with PBS solution at pH 7.4, and an equal volume of 0.25% type I collagenase was added. The tubes were sealed and digested at 37°C and 120 rpm for 40 min on a shaker. The centrifuge tubes were removed, sterilized with 75% alcohol, and the mixture was passed through an 80-mesh filter and centrifuged at 1200 rpm for 5 min. The supernatant was discarded. The mixture was resuspended in PBS solution at pH 7.4, and then centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and this process was repeated once. The precipitate was collected to obtain adipose-derived stem cells.

[0115] S3. Cerium dioxide nanozyme was prepared into a suspension with a concentration of 10 mg / mL and added to DMEM-L stem cell culture medium to obtain a culture medium containing cerium dioxide nanozyme at a concentration of 65 μg / mL. Adipose-derived stem cells were cultured in DMEM-L stem cell culture medium. When the confluence reached 65%, the medium was replaced with cerium dioxide nanozyme-containing medium, and cultured for another 6 hours. The cells were then washed, replaced with DMEM-L stem cell culture medium, digested, and centrifuged at 300g for 4 minutes to obtain cerium dioxide nanozyme-armed adipose-derived stem cells. These were then added to autologous adipose tissue to obtain a mixed stem cell / adipose tissue system with a concentration of 10 mg / mL cerium dioxide nanozyme-armed adipose-derived stem cells. 5 cell / mL;

[0116] S4. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 2 wt%.

[0117] S5. Add 123g of mixed stem cell / adipose system to 107g of sodium carboxymethyl cellulose hydrogel matrix, mix evenly, and let stand to remove bubbles to obtain adipose stem cell-based tissue repair filler.

[0118] Comparative Example 5

[0119] The only difference from Example 3 is that cerium dioxide nanozymes were not added to arm the adipose stem cells.

[0120] Includes the following steps:

[0121] S1. 300 mL of autologous blood lipid mixture was extracted by liposuction, centrifuged at 1200 r / min for 5 min at 5℃ to remove oil droplets and lower liquid to obtain autologous fat tissue;

[0122] S2. Add 4.02g of zinc citrate and 5.41g of magnesium citrate to 100mL of methacrylated hyaluronic acid solution (concentration of 5wt%), add 0.4g of photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonite lithium, homogenize at 13000r / min for 12min, heat to 55℃, and sonicate at 200W for 25min to obtain the precursor solution;

[0123] S3. Using the precursor solution as the dispersed phase and paraffin containing 4 wt% emulsifier Span-85 as the continuous phase, the two phases were injected into the microfluidic chip channel using a precision injection pump. The droplet diameter was controlled to be below 100 μm by adjustment. The emulsion droplets were collected, cured with 365 nm ultraviolet light for 5 min, centrifuged, and washed to remove unreacted monomers, photoinitiators, and emulsifiers to obtain reinforced microspheres.

[0124] S4. Dissolve sodium carboxymethyl cellulose in PBS solution at pH 7.4, stir to mix thoroughly, and allow to stand to remove bubbles to obtain a sodium carboxymethyl cellulose hydrogel matrix with a concentration of 2 wt%.

[0125] S5. Disperse 7g of enhanced microspheres in PBS solution at pH 7.4, mix well, add to 100g of sodium carboxymethyl cellulose hydrogel matrix, repeatedly push and mix, add 123g of autologous fat tissue, mix well, let stand to remove bubbles, and obtain tissue repair filler.

[0126] Test Example 1

[0127] The tissue repair fillers prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to performance tests, and the results are shown in Table 1.

[0128] 1. The compressive modulus of the prepared sample was determined using a universal testing machine in accordance with ISO 604 standard.

[0129] 2. The elastic modulus (G′) of the sample was tested using an MCR302e rotational rheometer with a shear strain of 0.1% and a frequency of 1Hz.

[0130] 3. At room temperature, the injection force of the sample was determined using a universal testing machine. A 1 mL syringe with a 23G needle was used. The injection force was tested by pushing the syringe at a constant injection rate of 30 mm / min in compression mode.

[0131] Table 1

[0132]

[0133] As can be seen from the table above, the tissue repair fillers based on adipose stem cells prepared in Examples 1-3 of this invention have good comprehensive performance.

[0134] Test Example 2

[0135] Healthy adult male SD rats (200-250g) were randomly divided into three groups: Examples 1-3, Comparative Examples 1-5, and a commercial control group (Ellansé®). Six rats were used in each group. During the experiment, the rats were anesthetized, and the back area was shaved and routinely disinfected. The samples were pre-sterilized by irradiation. 0.2 mL of sample was injected subcutaneously into the back using a 23G needle syringe. Two injection sites were set for each rat, without sutures, and the injection sites were at least 1 cm apart. The experiment lasted 12 weeks. Two rats from each group were euthanized at weeks 1, 4, and 12 post-operation. The rats were then dissected to observe the morphological changes of different samples after subcutaneous implantation. The results are shown in [Table data would be inserted here]. Figure 3The central white solid area is the implant. As shown in the figure, the total volume of the adipose-derived stem cell-based tissue repair filler prepared in Examples 1-3 decreased slightly over time, but was significantly better than the commonly used commercial control group (Ellansé®). The volume reduction was significant in Comparative Examples 1, 2, and 5, while the volume changes in Comparative Examples 3 and 4 were not significantly different from those in Example 3.

[0136] Twelve weeks later, after acquiring Masson stained section images, three locations were randomly selected at the junction of the implant and the tissue. The collagen fiber density (%) was analyzed using Image-J software (collagen fiber density is the ratio of collagen fiber area to total area).

[0137] The results are shown in Table 2.

[0138] Table 2

[0139]

[0140] As shown in the table above, the tissue repair fillers based on adipose stem cells prepared in Examples 1-3 of this invention can significantly increase the collagen fiber density in rats.

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a tissue repair filler based on adipose-derived stem cells, characterized in that, Autologous adipose tissue was mixed evenly with cerium dioxide nanozyme-armed adipose stem cells to obtain a hybrid stem cell / fat system. Hyaluronic acid microspheres loaded with zinc and magnesium ions were added to a sodium carboxymethyl cellulose hydrogel matrix, and the hybrid stem cell / fat system was added. The mixture was mixed evenly and allowed to stand to degas, thus obtaining a tissue repair filler based on adipose stem cells.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Aspirate the autologous blood lipid mixture, centrifuge at low temperature to obtain autologous adipose tissue; S2. Cerium dioxide nanozymes were prepared into a suspension and added to stem cell culture medium to obtain a culture medium containing cerium dioxide nanozymes. Adipose stem cells were cultured in stem cell culture medium. When the fusion rate reached 60-70%, the culture medium was replaced with one containing cerium dioxide nanozymes. The cells were then washed, cultured in stem cell culture medium, and separated to obtain cerium dioxide nanozyme-armed adipose stem cells. These cells were then added to autologous adipose tissue to obtain a mixed stem cell / adipose tissue system. S3. Add zinc citrate and magnesium citrate to a methacrylated hyaluronic acid solution, add a photoinitiator, homogenize, and then heat and sonicate to obtain a precursor solution; S4. Using the precursor solution as the dispersed phase and paraffin containing emulsifier as the continuous phase, the two phases are injected into the microfluidic chip channel using a precision injection pump. The droplet diameter is controlled at 70-100μm by adjustment. The emulsion droplets are collected, cured by ultraviolet light, centrifuged, and washed to remove unreacted monomers, initiators, and emulsifiers, resulting in reinforced microspheres. S5. Dissolve sodium carboxymethyl cellulose in PBS solution, stir to mix evenly, let stand to remove bubbles, and obtain sodium carboxymethyl cellulose hydrogel matrix; S6. Add the enhanced microspheres to the PBS solution and disperse them. After mixing evenly, add them to the sodium carboxymethyl cellulose hydrogel matrix and repeatedly push and mix. Add the mixed stem cell / adipose system, mix evenly, and let stand to remove bubbles to obtain an adipose stem cell-based tissue repair filler.

3. The preparation method according to claim 2, characterized in that, The low-temperature centrifugation in step S1 is carried out at a temperature of 3-6℃, a rotation speed of 1000-1500 r / min, and a time of 3-10 min.

4. The preparation method according to claim 2, characterized in that, The concentration of the suspension in step S2 is 5-15 mg / mL, the concentration of cerium dioxide nanozyme in the culture medium containing cerium dioxide nanozyme is 50-80 μg / mL, the stem cell culture medium is selected from at least one of DMEM-L, M-199, and K-SFM, the culture time is 5-8 h, and the concentration of cerium dioxide nanozyme-armed adipose stem cells in the mixed stem cell / adipose tissue system is 10 μg / mL. 4-6 cell / mL.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of zinc citrate, magnesium citrate, methacrylated hyaluronic acid, and photoinitiator is 2.87-5.74:4.51-6.77:4-7:0.3-0.

5. The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonite. The homogenization speed is 10000-15000 r / min, and the time is 10-15 min. The heating and ultrasonic treatment temperature is 50-60℃, the ultrasonic power is 150-250 W, and the time is 20-30 min.

6. The preparation method according to claim 2, characterized in that, In step S4, the concentration of the emulsifier in the paraffin containing the emulsifier is 3-5 wt%, the emulsifier is a Span series emulsifier, and the ultraviolet light wavelength for ultraviolet curing is 365 nm, and the time is 3-6 min.

7. The preparation method according to claim 2, characterized in that, The concentration of sodium carboxymethyl cellulose in the sodium carboxymethyl cellulose hydrogel matrix in step S5 is 1-3 wt%.

8. The preparation method according to claim 2, characterized in that, The mass ratio of the enhanced microspheres, sodium carboxymethyl cellulose hydrogel matrix, and mixed stem cell / adipose system in step S6 is 5-10:80-120:100-150.

9. A tissue repair filler based on adipose-derived stem cells prepared by the preparation method according to any one of claims 1-8.

10. The application of the tissue repair filler based on adipose-derived stem cells as described in claim 9 in tissue repair.

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