CN114404370A

CN114404370A - Nano-fat functionalized injectable super-lubricating microsphere and preparation method and application thereof

Info

Publication number: CN114404370A
Application number: CN202111521826.9A
Authority: CN
Inventors: 杨兴; 韩泽宇; 韩启斌; 崔文国; 郝跃峰
Original assignee: Suzhou Municipal Hospital
Current assignee: Suzhou Municipal Hospital
Priority date: 2021-12-13
Filing date: 2021-12-13
Publication date: 2022-04-29
Anticipated expiration: 2041-12-13
Also published as: CN114404370B

Abstract

The invention provides a nano-fat functionalized injectable super-lubricating microsphere and a preparation method and application thereof. The preparation method comprises the steps of firstly preparing PLGA porous microspheres by using a microfluidic technology, and then fixing nano fat rich in grease, stem cells and growth factors inside the microspheres through Schiff base condensation reaction, non-covalent interaction and a three-dimensional carrier physical network structure to obtain the nano fat functionalized injectable super-lubricating microspheres. After the microspheres are injected into the articular cavity, the cartilage surface adhesion targeted planting is realized, and meanwhile, the friction experiment verifies that the microspheres have super-lubricating property. In addition, it significantly reduced osteophyte formation in vivo, improved rat behavioral performance, and inhibited the progression of osteoarthritis. The invention provides a multifunctional platform with great potential for minimally invasive treatment of osteoarthritis and even alleviation of disease processes.

Description

Nano-fat functionalized injectable super-lubricating microsphere and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nano-fat functionalized products and preparation, and particularly relates to a nano-fat functionalized injectable super-lubricating microsphere and a preparation method and application thereof.

Background

Tonnard equal to 2013 first reported the preparation of a fine emulsion with the advantages of a liquid and a homogeneous liquid by mechanically emulsifying a micro-fat, called "nano-fat". Nano fat is an injectable viscous extract rich in lipid, growth factors and stem cells, and has been successfully used in the fields of scar repair, limb ischemia and vascular regeneration, cartilage defect repair and the like.

The nano-fat is an ideal stent-free graft due to its multiple components and versatility (angiogenesis, anti-fibrosis, analgesia, and anti-inflammation). However, the existing research shows that the nano-fat after injection has the problems of low bioactivity and low retention rate, and the fluid state of the nano-fat cannot well meet the requirement of precise transplantation. The above factors limit the maximum biological function of the nano fat to a certain extent.

The problems of low activity, low retention rate, incapability of field planting and the like caused by the injection of the nano fat cause the loss of biological functions. To remedy these drawbacks, several nano-fat based derivative products were developed in succession. Huang et al found that culturing nano-fat mixed with ceramic particles for 4 weeks induced cartilage formation, which was manifested by a significant increase in cell number and glycosaminoglycans in vitro. Li and the like obtain extracellular matrix/matrix vascular fraction gel (ECM/SVF-gel) by separating fatty oil and concentrating effective components, and find that the gel can effectively repair rabbit cartilage defects.

Unlike cartilage defects, osteoarthritis is a chronic disease characterized by the destruction of articular cartilage and inflammation of the joint capsule, which eventually manifests itself. Interruption of lubrication is considered to be a key factor in the disease progression, leading further to cartilage damage and failure of lubrication, eventually leading to aseptic osteoarthritis. However, the existing treatment modes have some disadvantages more or less, and oral non-steroidal anti-inflammatory drugs are difficult to escape from the body to clear barriers and cannot reach joint cavities to exert curative effects, and the digestive system can be damaged; the hyaluronic acid injected into the joint cavity can correspondingly increase the injection frequency due to the shear thinning effect, and the infection risk is improved; knee replacement is accompanied by a heavy economic burden while causing trauma to the body. In addition to the above mentioned drawbacks, the most critical factor affecting the clinical efficacy of osteoarthritis is that the existing methods do not effectively compromise both anti-inflammatory and lubrication supplementation aspects.

The emergence of nano-fat as a novel implant brings hope for breaking through the treatment bottleneck. It has been found that nano-fats have a complex cellular composition and, although the specific physiopathological mechanism thereof has not been elucidated, exhibit beneficial effects in the treatment of osteoarthritis, such as anti-inflammatory and analgesic effects, which appear to be mediated by the release of secreted cytokines. In addition, certain pretreatment of the nano-fat, such as preparation of a three-dimensional sphere, can further induce the secretion of growth factors, and various spherical biomaterials can meet the required three-dimensional space structure. However, whether the nano-fat can be easily combined with biological materials or not affects the biological functions of the biological materials, such as good hydrophilicity of hydrogel microspheres such as GelMA (methacrylic acid gelatin), HAMA (hyaluronic acid) and the like, and the nano-fat cannot be combined with nano-fat rich in grease. The solid polymer microspheres can meet the requirement of hydrophobicity, but the non-porous structure limits the biological application of the nano-fat. How to better exert the biological effect of nano fat in treating osteoarthritis becomes a technical problem at the present stage.

On the other hand, although nano-fat shows a certain therapeutic relief effect on osteoarthritis, the existing research shows that the lubrication effect for improving osteoarthritis is difficult to maintain continuously. After the nano fat is injected to reach the joint cavity, although the friction system can be obviously reduced in a short time and the lubricating effect is supplemented, the lubricating effect can be maintained for a short time, the lubricating effect brought by the nano fat is obviously reduced after a certain time, the friction coefficient is continuously increased, and the stability effect of the lubricating effect is poor. Therefore, the technical problem of how to improve the time for supplementing and lubricating the nano fat in the joint cavity and solve the problem that the lubricating effect is difficult to stably maintain for a long time is one of the technical problems in the prior art of adopting the nano fat to treat osteoarthritis.

Disclosure of Invention

The invention aims to solve the technical problems, and provides a nano-fat functionalized injectable super-lubricating microsphere and a preparation method and application thereof. The technical purpose of the invention is as follows: overcomes the problems of short maintenance time of the lubricating effect of the injectable nano fat, easy increase of the friction coefficient and difficult long-time stable maintenance of high lubricating performance in the prior art. The injectable super-lubricating microsphere capable of maintaining stable nanometer fat functionalization for a long time is provided, and not only can a better lubricating effect be realized, but also the lubricating effect on joints can be maintained stable for a long time, and the problem of obvious reduction can not occur.

One of the purposes of the invention is to provide a preparation method of nano-fat functionalized injectable super-lubricating microspheres, which comprises the following steps:

(1) preparing nano fat: physically emulsifying adipose tissues, filtering, and collecting a filtered product to obtain nano fat;

(2) preparing porous microspheres: preparing an aldehyde-based polylactic acid-glycolic acid copolymer into porous microspheres by adopting a microfluidic device;

(3) co-culturing the nano-fat obtained in the step (1) and the porous microspheres obtained in the step (2) according to the mass ratio of 1:3, and preparing the nano-fat functionalized injectable super-lubricating microspheres.

The preparation method comprises the steps of firstly preparing Nano Fat (NF) from an allogeneic source through physical chyle, then preparing porous polylactic acid-glycolic acid copolymer microspheres (PLGA porous microspheres, pore microspheres, abbreviated as PMs) through a microfluidic device, then adopting 3D co-culture, and fixing the nano fat rich in grease, stem cells and growth factors inside the microspheres through Schiff base condensation reaction, non-covalent interaction and a three-dimensional carrier physical network structure to prepare the nano fat functionalized injectable super-lubricating microspheres (abbreviated as PMs @ NF).

The inventors initially studied the lubricating properties of nano-fats as shown in fig. 4 and 5 in the examples, and as a result, found that: although the nano fat can play a good lubricating role at first and show a low friction coefficient, the nano fat is poor in lubricating stability, and the friction coefficient is greatly increased in a short period of time, so that the lubricating performance is poor and the trend of extremely unstable lubricating performance is shown. The phenomenon of poor lubricating stability of the nano fat is difficult to meet the requirements of continuous lubrication and accurate and efficient drug delivery of joints. In order to improve the stability of the nano-fat, the inventor has conducted a great deal of research on the functionalization of the nano-fat, and attempts to search for a preparation process of the nano-fat functionalized super-lubricating microsphere are made to find that the technical problems are difficult to solve. Finally, the inventors have conducted a great deal of research on the ratio between the porous microspheres and the nano-fat, and have unexpectedly found that when the mass ratio of the porous microspheres to the nano-fat is controlled to be 1:3, injectable super-lubricating microspheres capable of stably maintaining the lubricating effect for a long time are obtained. When the nano-fat or the porous microspheres are used for lubrication in other proportions or independently used, the phenomenon that the lubricating performance is greatly reduced after a period of time occurs, and the microspheres which stably maintain the high lubricating performance for a long time are difficult to obtain.

In addition, as shown in the embodiment of the invention, after the MPs @ NF is injected into the articular cavity, the cartilage surface adhesion targeted planting is also realized, and meanwhile, a friction experiment verifies that the MPs @ NF has the super-lubricating property. In vitro experiments show that MPs @ NF can up-regulate the expression of cartilage anabolic substances such as type II collagen, glycosaminoglycan and the like, and down-regulate the expression of matrix metalloproteinase-1 (MMP1), interleukin 1 beta (IL1 beta) and tachykinin-1 (TAC 1). Simultaneously, osteophyte formation is obviously reduced in vivo, the behavioral performance of rats is improved, and the progress of osteoarthritis is inhibited. The results show that the nano-fat functionalized injectable super-lubricating microsphere provided by the invention can show an excellent effect of treating osteoarthritis. The invention provides a multifunctional platform with great potential for minimally invasive treatment of osteoarthritis and even alleviation of disease processes.

Further, the physical emulsification in the step (1) is mechanical emulsification of fat tissue by using a fat chyle device.

Further, the animal fat tissue is washed clean and sheared in the step (1), then filtered under the aseptic condition, and then the physical emulsification process is carried out.

Further, when the micro-fluidic device is used for preparing the porous microspheres in the step (2), gelatin and aldehyde polylactic acid-glycolic acid copolymer dichloromethane solution are used as a water phase, and polyvinyl alcohol solution is used as an oil phase.

Further, the flow rate ratio of the oil phase to the aqueous phase in step (2) was 16: 1.

Further, the co-cultivation conditions in step (3) are: the cells were co-cultured at 37 ℃ and 90rpm for 24 hours in three dimensions.

The invention also aims to provide a nano-fat functionalized injectable super-lubricating microsphere prepared by the method.

Specifically, the microspheres have an average diameter of 324 μm and an average pore diameter of 27 μm.

The invention also aims to provide application of the nano-fat functionalized injectable super-lubricating microspheres, which comprises application in preparing medicines for treating orthopedic diseases.

In particular, the bone disease comprises osteoarthritis.

The invention has the following beneficial effects:

(1) according to the invention, the PLGA porous microspheres are prepared by adopting a microfluidic technology, and then nano-fat rich in grease, stem cells and growth factors is fixed in the PLGA porous microspheres through Schiff base condensation reaction, non-covalent interaction and a three-dimensional carrier physical network structure, so that the nano-fat functionalized injectable super-lubricating microspheres are obtained, and the nano-fat functionalized injectable super-lubricating microspheres can stably maintain excellent lubricating property of the nano-fat for a long time, and can not greatly reduce the lubricating property after being injected for a period of time.

(2) The nano-fat functionalized injectable super-lubricating microsphere provided by the invention not only effectively improves the loading efficiency and the capacity of inducing stem cell secretion, but also improves the concentration of local cytokines, realizes the accurate delivery of effective components by targeting adhesion to the cartilage surface, and enhances the lubrication of the articular cartilage surface.

(3) The nano fat functional injectable super-lubricating microsphere provided by the invention can up-regulate the expression of cartilage anabolic substances, down-regulate and degrade cartilage catabolic enzymes, inflammation-related and pain-related genes, obviously improve the lubrication of joint cavities of rats with arthritis, reduce osteophyte generation and improve the behavioral performance, thereby inhibiting the generation of osteoarthritis.

Drawings

FIG. 1 is a flow chart; (a) nano-fat (NF), PLGA Porous Microspheres (PMs) and nano-fat loaded PLGA porous microspheres (PMs @ NF); (b) the multi-biological functional micro-fluidic PMs have the effective components of enhancing lubrication, targeting cartilage surface adhesion, conveying anti-inflammation and the like and are clinically applied.

FIG. 2 is a representation of nano-fat functionalized injectable super-lubricious microfluidic microspheres; (a) 1H NMR spectrum of PLGA; (b) NF prepared by emulsification; (c) nf (i) and PMs @ nf (ii) images injected (0.45mm i.d.) through a syringe needle; (d) mirror images of PMs; (e) SEM images of PMs; (f) analyzing the particle size of PMs; (g) pore size analysis of PMs; (h) BSA-FITC stained PMs (I) and common PMs (II) images under fluorescent microscopy; (i) NF (I) and PMs @ NF (II) images under a light microscope; (j) SEM images of nf (i) and PMs @ nf (ii) after critical point drying, yellow triangles: lipid droplet, red arrow: and (4) PMs.

FIG. 3 shows the in vitro degradation analysis and Calcein-AM/DAPI staining of rat articular cavity injection and cartilage surface adhesion surface and PMs cultured in PBS (pH 7.4) for 7 weeks; (a) injecting different substances into joint cavities of rats; (b) after the injection of the joint cavity, fully exposing the knee joint of the rat, and observing the condition of the cartilage surface; (c) surface SEM images of PMs degradation; (d) the pH value of the buffer solution; (e) residual mass change of PMs; (f) Calcein-AM staining NF under a fluorescence microscope; (g) Calcein-AM fluorescence stained images of PMs @ NF at different time points of 1, 6, 12 and 24h in three dimensions.

FIG. 4 shows cytokine secretion and lubrication performance of super-lubricated MPs @ NF; (a1-a6) ELISA was performed to detect the concentration of VEGF, HGF, TGF-. beta.1, BDNF, IL-4, IL-10 in 6 different PMs @ NF samples, with the symbols indicating the samples from different sources; (b1) a plot of coefficient of friction versus time for various concentrations of PMs @ NF; (b2) a plot of coefficient of friction versus time for PMs @ NF at different reciprocation frequencies; (b3) coefficient of friction versus time plot of PMs @ NF at different loads; (b4) schematic diagram of tribology testing apparatus.

FIG. 5 is a graph of coefficient of friction versus time for PMs @ NF of various constituent mass ratios.

FIG. 6 is a plot of coefficient of friction versus time for PBS, PMs, NF, and PMs @ NF.

FIG. 7 is the biocompatibility of super-lubricated PMs @ NF; (a) fluorescence images of live/dead staining after coculture of leachate of PMs, NF, and PMs @ NF with chondrocytes; (b) quantification of viable cells in live/dead experiments; (c) CCK-8 to detect cytotoxicity; n is 3, NS: meaningless, represents P < 0.001.

FIG. 8 is a graph demonstrating the anti-inflammatory effects of the in vitro environment super-lubricated PMs @ NF; (a1) the expression level of Col2 α mRNA after TNF α treatment for 0, 3, 6, 12 and 24h, n-3, representing P < 0.01 compared to 0 h; (a2-a6) TNF- α intervenes in chondrocytes, and after 24h co-culture with PMs, NF and PMs @ NF leachate, the mRNA expression levels of Col2 α (a2), AGG (a3), MMP1(a4), IL-1 β (a5), TAC1(a 6); (b) TNF-alpha intervenes in chondrocytes, and after the chondrocytes are co-cultured with leachate of PMs, NF and PMs @ NF for 12 hours, Col2 alpha representative fluorescence images of the chondrocytes are obtained; green: a Col2 α protein; blue color: cell nucleus; red: cell actin, n-3, NS: it is not significant that the values of "and" # "indicate that P is less than 0.05 when compared with the control group and the blank group, respectively, and that P is less than 0.01 when compared with the control group and the blank group, respectively.

FIG. 9 is a representation of the in vivo model of super lubricated PMs @ NF treatment of osteoarthritis; (a) lateral slice images of rat knee joints at 1 week and 8 weeks after surgery; (b) CT representative image of rat knee joint at 8 weeks post-surgery, red arrow and red shading both represent osteophytes; (c) relative width of knee joint gap of rat 1 week after operation; (d) relative width of knee joint gap of rats 8 weeks after operation; (e) the relative volume of total knee joint osteophytes of rats is quantified after 8 weeks of operation; n is 3, NS: it is meaningless that a and # indicate P < 0.05 when compared with the control group and the blank group, respectively, and a and # indicate P < 0.01 when compared with the control group and the blank group, respectively.

FIG. 10 is a diagram: (a) the medial meniscus of the rat knee was exposed, the yellow circle showing the medial meniscus; (b) the joint space behind the medial meniscus of the rat was removed and the yellow circle shows the space between the femur and tibia; (c) osteoarthritis is induced by postoperative motor intervention in rats.

FIG. 11 is a post-operative 8-week rat footprint collection; (a) rat footprints of 8 weeks post-surgery; blue color: a healthy side; red: a molding side; (b) quantifying the step length of the footprint; (c) supporting and quantifying the footprint base; (d) quantifying the footprint area; n is 3, NS: it is meaningless that a and # indicate P < 0.05 when compared with the control group and the blank group, respectively, and a and # indicate P < 0.01 when compared with the control group and the blank group, respectively.

FIG. 12 super-lubricated PMs @ NF protected cartilage invariance; (a) h & E stained images of the sections; (b) safranin O-fast green staining images of the sections; (c) section Col2 α immunohistochemical staining images, each group n ═ 6; (d) relative aggrecan content; (e) an OARSI score; (f) quantification of Col 2. alpha. positive cells.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments, and it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Experimental method example 1

First, experimental material

1. Major drugs and reagents

Aldehyde-based polylactic-co-glycolic acid (PLGA-CHO, M)_w10KDa) from guangzhou carbohydrate science co ltd (china); TNF- α (tumor necrosis factor) was purchased from Peprotech (USA); bovine Serum Albumin (BSA) -FITC was purchased from Solarbio technologies ltd, beijing (china); sodium pentobarbital was purchased from Sigma (usa); hematoxylin staining solution, eosin staining solution, primary antibody dilution solution, and secondary antibody dilution solution were obtained from Shanghai Biyun TiantianScience and technology limited (china); the 10% neutral formalin fixing solution was purchased from Beijing Lankangkoc science and technology Co., Ltd. (China); other reagents were purchased from Aladdin reagents, Inc. (China), unless otherwise noted.

2. Main apparatus and equipment

High speed centrifuges were purchased from beckmann coulter co ltd (usa); the electric heating constant temperature water bath kettle is purchased from Shanghai-Hengshi Co., Ltd (China); paraffin embedding machines, slicers, baking tables, freezing tables are available from come corp (germany); the optical microscope was purchased from Yongxin optics Limited (China) in south Jing, Jiangnan; microscope imaging systems are available from olympus corporation (japan).

3. Laboratory animal

12-week-old healthy male SD rats were purchased from the university of Suzhou laboratory animal center 30. Rats were housed 6 per cage with free access to water. The temperature of the animal room is constant at 25 ℃, the ventilation is good, and the illumination and the dark environment are alternately carried out for 12 h.

Second, Experimental methods

Preparation of main solution

Decalcification solution (10% EDTA) preparation (2L system):

TABLE 1' 10% EDTA2L System configuration method

Preparation of (II) nano fat functionalized injectable super-lubricating microspheres

1. Preparation of NF (nano fat):

collecting adipose tissues of the groin and the abdominal cavity of the rat, washing, shearing by using ophthalmic scissors, and filtering by using sterile nylon cloth. The filtered adipose tissues were transferred to two 20mL screw-top syringes, between which the adipose tissues were mechanically emulsified by passing through a fat chyle (kepa, Jiangsu), and then by moving back and forth between the two syringes 30 times. After the emulsification process, the adipose tissue was again filtered through the sterile nylon cloth described above and the filtered product was collected in a sterile collector for use.

2. Preparation of PMs (PLGA porous microspheres):

PMs were prepared using microfluidic technology, a microfluidic simple device as follows: a custom made coaxial needle (25G, 18G), an inner metal channel (0.26mm i.d. × 0.51mm o.d.), an outer metal channel (0.84mm i.d. × 1.27mm o.d.), a polyvinyl chloride tube connected to the needle to the product.

PMs are prepared by adopting the micro-fluidic device to disperse emulsion, and the specific method is as follows:

(1) dissolving gelatin in deionized water to serve as a water phase, dissolving PLGA-CHO in dichloromethane to be added into an oil phase, emulsifying the gelatin and the PLGA-CHO to form a W-O structure according to the mass ratio of 1:3, and preparing an initial stable emulsion through an ultrasonic crusher (ultrasonic treatment is carried out for 2s, and the interval is 3 s);

(2) the solution of gelatin-PLGA-CHO dichloromethane is used as a dispersed phase, and the solution of 1 wt% PVA (polyvinyl alcohol) is used as a continuous phase, so that the shearing stress is generated to form droplets. Introducing an initial emulsion into an inner channel of a discontinuous phase, introducing a PVA (polyvinyl alcohol) solution into an outer channel of the discontinuous phase, adjusting the flow rate ratio of the continuous phase to a dispersed phase to be 16:1, dispersing the emulsion in the PVA solution to form, arranging a polyvinyl chloride tube in a beaker containing 500ml of ice water, slightly stirring the mixture overnight, and then transferring the beaker into warm water at 45 ℃ to stir the mixture for 2 hours so as to remove gelatin;

(3) PMs are collected and are frozen and dried for 1 day, and then the PMs are harvested to obtain the final product.

(III) testing of microsphere Properties

1. The physical characterization method comprises the following steps:

(1) of PMs¹H NMR spectra were measured with an NMR instrument (Avance III, Bruker, Germany);

(2) PMs and NF were co-cultured for 24 hours to give super-lubricating microspheres PMs @ NF, samples were fixed with 0.25% glutaraldehyde and washed 3 times with PBS after overnight at 4 ℃. Then, the mixture was dehydrated with a gradient of 10%, 30%, 50%, 70%, 85%, 90%, 100% ethanol, and the morphology was observed by a scanning electron microscope (Hitachi, S-4800, Japan) after gold spraying.

2. The super-lubricating microsphere has the following adhesion performance:

(1) 5mg each of PMs prepared from aldehyde PLGA and ordinary PLGA (Sigma-Aldrich, USA) were conjugated to NF, followed by incubation with a PBS dilution of 1/200 of BSA-FITC for 1h at 25 ℃ followed by fluorescent microscopy.

(2) Rats were anesthetized and injected with equal amounts of PBS, PLGA microspheres, PMs, NF, and PMs @ NF into the articular cavities of the rats, respectively, and then the rats were sacrificed by excessive anesthesia to carefully separate the exposed articular surfaces and observe the adhesion on the cartilage surface.

3. Microsphere degradation experiments:

to simulate the degradation process of microspheres in physiological environment, the prepared PMs (30mg) were divided equally into three groups, each group of PMs was exposed to 10ml of PBS solution and cultured in a constant temperature shaking phase. At various time points (1-7 weeks), the microspheres were removed, washed with PBS, freeze-dried, and subjected to various assays. Including SEM observation of topography changes, changes in pH values of PBS (0-7 weeks), and calculation of the weight percent remaining using the formula Wt/Wo.

4. NF binding to PMs:

PMs and NF are subjected to 3D co-culture to obtain the nano-fat functionalized injectable super-lubricating microspheres (PMs @ NF), and the preparation and application processes are shown in figure 1.

PMs and NF in different mass ratios are mixed in a 50ml centrifugal tube, after co-culture for 1, 6, 12 and 24 hours at 37 ℃ and 90rpm, the PMs are gently extracted, and the structure and adhesion of the NF on the PMs are observed by a fluorescence microscope (LSM 900, Zeiss, Germany) after staining with Calcein-AM.

The NF extracted by the preparation and PMs are mixed and then the content of the cell factor in the mixture is quantified by ELISA. According to the instructions of ELISA kit manufacturers (MultiSciences, Zhejiang, China), the levels of Vascular Endothelial Growth Factor (VEGF), transforming growth factor-beta 1 (TGF-beta 1), Hepatocyte Growth Factor (HGF), brain-derived neurotrophic factor (BDNF), interleukin-4 (IL-4) and interleukin-10 (IL-10) are obtained.

5. And (3) tribology testing:

to evaluate the lubricating properties of PMs @ NF, tribological tests were performed using a universal material testing machine (UMT-3, Bruker Nano Inc., Germany). The upper sample is a polyethylene ball (PE) with the diameter of 8mm, the lower sample is a titanium alloy (Ti6AI4V), the joint physiological state is simulated by sliding the upper sample on the lower sample surface, and different PMs to NF ratios (the mass ratio of PMs to NF is 1:0, 3:1, 1:3 and 0:1 respectively), different substances, different concentrations (the concentration of PMs NF @ is 1mg/ml, 2mg/ml, 5mg/ml and 10mg/ml respectively), different reciprocating frequencies (1Hz, 3Hz, 5Hz and 10Hz), different loads (1N, 2N, 3N and 4N) are respectively tested in a reciprocating manner, and the suspension lasts for 20min each time. All samples were prepared as suspensions in PBS and added as a lubricant between the upper and lower sample contact surfaces and the time-coefficient of friction curve recorded during the test.

(IV) animal test methods

1. Establishment of animal model

The experimental use of 30 male SD rats purchased from the Experimental animals center of Suzhou university at an average week age of 12 weeks, rat osteoarthritis model approved by the ethical Committee of Suzhou municipal hospital affiliated to the Nanjing medical university, all procedures were performed according to the guidelines of the American national institute of health. Osteoarthritis models were prepared by DMM surgery. Briefly, 10% chloral hydrate (2ml/kg) was anesthetized intraperitoneally, the skin from the distal patella to the proximal tibial plateau of the right knee was incised, the right knee was fully exposed, the medial meniscal was removed after detachment of the medial meniscal tibial ligament using microangioforceps, and the surgical incision was sutured layer by layer. The sham group did not sever MMTL. Rats had free access to food and water and were allowed to perform unrestricted activities daily. Rats were randomized into 5 groups (n ═ 6), received running exercises on a horizontal treadmill at a rate of 20m/min for 3 days per week to induce knee osteoarthritis, and received joint cavity injections of 100 μ l PBS, PMs, NF, PMs @ NF (all substance concentrations 10mg/ml) every 2 weeks.

2. Grouping of laboratory animals

Experimental rats were selected and randomly grouped as follows, 6 rats/group. Articular cavities received 100 μ l PBS, PMs, NF, PMs @ NF (all concentrations 10mg/ml) every 2 weeks.

(1) Sham group: rats were not excised for the medial meniscus and injected intra-articular with 100 μ l of sterile saline;

(2) PBS group: after rat anesthesia, 100 μ l PBS solution was injected into the joint cavity;

(3) group PMs: after rat anesthesia, 100 μ l PMs suspension solution was injected intra-articular;

(4) NF group: after rat anesthesia, 100 mul NFs suspension solution was injected intra-articular;

(5) PMs @ NF set: after anesthetizing the rats, 100. mu.l of PMs @ NFs suspension was injected intra-articularly.

3. Rat knee joint gap measurement

And after 1 week and 8 weeks of operation, the rats are anesthetized and then taken out of the prone position, and the right knee joint is placed on the molybdenum target camera. X-ray exposure parameters: the distance from the collimator to the film is 5cm, the exposure time is 55mAs, and the voltage is 30 kV. The knee joint gap width is measured according to the X-ray film result.

4. Paw print collection

A dark narrow footpath with the length of 150cm and the width of 10cm is manufactured, white paper with the same size is laid at the bottom of the footpath, and a light hole is formed at the terminal point, so that a rat can walk to the light position at the terminal point in a straight line. The rat hind paw was dipped evenly with ink (blue on the normal side and red on the model side) and placed in the walkway for the rat to pass through, and the white paper was printed with the rat hind paw blot for statistical analysis after being photographed by a camera.

5. Micro-CT evaluation

At 8 weeks post-surgery, the knee joints were removed after sacrifice and analyzed by Micro-CT (SkyScan 1176, SkyScan, Aartselaar, belgium). The software calculates the osteophyte volume after performing a three-dimensional reconstruction.

6. Specimen collection

After the rats die in anesthesia, the skin on the front side of the knee joint is longitudinally cut, the skin is peeled, soft tissues and muscles around the joint are removed, the femur and the tibiofibula are cut off, the knee joint is left, the PBS is cleaned, the rat knee joint specimen of each group is fixed in 10% neutral formalin for 48h, 10% EDTA is decalcified for 30 days, and the rat knee joint specimen is embedded in paraffin and subjected to histological detection.

7. Histological staining

The knee joints were decalcified with 10% EDTA and then embedded in paraffin by conventional methods. Square sections of the knee joint were cut in the coronal plane at a thickness of 5 μm using a microtome, and were subjected to Hematoxylin-Eosin staining (Hematoxylin and Eosin Stain, H & E) and immunohistochemical staining, respectively.

8. In vitro chondrocyte isolation and culture

(1) And (3) chondrocyte separation: chondrocytes were isolated from the joints of SD rats by cutting large pieces of cartilage tissue into small pieces using an ophthalmologic scissors, then digesting the pieces of cartilage tissue for 5 hours under shaking conditions of a constant temperature with 0.25% collagenase, followed by filtering the undigested cartilage tissue with a 70 μm cell sieve, collecting chondrocytes released from the filtrate, and culturing in Nutirent mix F-12(F12) medium supplemented with 10% fetal bovine serum and 1% double antibody. Chondrocytes within the third generation for subsequent experiments.

(2) Preparing a leaching solution: to explore the effect of PMs, NF on cartilage, leachate was prepared according to ISO third edition guidelines. The method comprises the following steps: PMs, NF and PMs @ NF were suspended in the medium at 1mg/ml for 24 hours at 37 ℃ and the cultured chondrocytes were taken out from the extract for subsequent experiments.

9. Cytotoxicity, proliferation

(1) CCK 8: CCK8 was used to explore the cytotoxicity of PMs, NF on chondrocytes. The specific method comprises the following steps: cells were cultured at 2X 10³The extract is cultured in 96-well plates containing 5% CO in a humid environment₂After incubation in an incubator at 37 ℃ for 1, 3, and 5 days, the old medium was removed, 10. mu.l of CCK8 solution and 100. mu. l F12 medium were added to each well, incubation was continued at 37 ℃ for 2 hours, and absorbance of the solution was measured at a wavelength of 450nm using a plate reader (Thermo Scientific Varioskan LUX, USA).

(2) Cell live and dead staining: chondrocyte activity was measured using a live/dead cell kit (invitrogen, usa). Briefly, cells were plated at 1 × 10⁴The culture medium is collected in 24-well plates at a density of one ml, cultured with the above-mentioned leaching solution for 1, 3, and 5 days, then aspirated, thoroughly washed with PBS, and incubated with live/dead cell dye solution (200. mu.l) per well at room temperature for 30 min. Stained cells were examined with a fluorescence microscope (Carl Zeiss inc., zeissaxioviert 200, usa) and live cells were green and dead cells were red.

10. qRT-PCR experiment

The inoculation density of each hole of the 6-hole plate is 5 multiplied by 10⁴Chondrocytes/ml, followed by TNF-alpha treatment at 10ng/ml, leaching as described aboveThe fluid was co-cultured with chondrocytes for 24 hours. Total RNA was extracted using TRIzol reagent (Invitrogen, USA), and concentration and purity were determined by measuring absorbance at 260nm and 280 nm. cDNA was synthesized using 1. mu.g of RNA and the RevertAId First Strand cDNA Synthesis Kit (TaKaRa, Japan), and then qRT-PCR was performed using SYBR Premix Ex Tag Kit (TaKaRa, Japan) and ABI 7500 sequencing assay System (Applied Biosystems, USA) to amplify the cDNA. mRNA expression levels of type II collagen (Col 2 α), Aggrecan (AGG), matrix metalloproteinase 1(MMP1), interleukin-1 β (IL-1 β), tachykinin 1(TAC1), and actin (β -actin) were quantified using specific primers, and β -actin was normalized, with the primer sequences shown in Table 1.

Table 1: primer sequences

11. Immunofluorescence staining

The inoculation density of each hole of the 24-hole plate is 0.5 multiplied by 10⁴The chondrocytes were plated in a cell-slide at a concentration of/ml, treated with 10ng/ml TNF-. alpha.and then co-cultured with the above-mentioned leachate for 12 hours, after which the cells were fixed with 4% paraformaldehyde for 10 minutes and then blocked with 0.3% Triton X-100(Solarbio, Beijing, China) for 10 minutes and 5% bovine serum albumin (BSA, Biosharp, Shanghai, China) for 1 hour. Thereafter, the cells were incubated overnight in a refrigerator at 4 ℃ with rat anti-type II collagen antibody (invitrogen, USA, 1: 200). Subsequently, Alexa Fluor conjugated secondary antibody (Life Tech, USA, 1:400) was used for incubation at 37 ℃ for 1 hour. Thereafter, the cytoskeleton was stained with phalloidin-rhodamine (Yearsen, shanghai, china) for 20 minutes and the nucleus was stained with 4', 6-diamino-2-phenylindole (DAPI, Life Tech, usa) for 5 minutes at room temperature, followed by observation with a fluorescence microscope.

(1) H & E staining

The procedure for the H & E staining procedure is shown in Table 2.

TABLE 2H & E staining procedure

(2) Immunohistochemical staining

The immunohistochemical staining protocol is shown in Table 3.

TABLE 3 immunohistochemical staining procedure

12. X-ray, Micro-CT assessment and gait acquisition

And after 1 week and 8 weeks of operation, the rats are anesthetized and then taken out of the prone position, and the right knee joint is placed on the molybdenum target camera. X-ray exposure parameters: the distance from the collimator to the film is 5cm, the exposure time is 55mAs, the voltage is 30kV, and the knee joint gap width is measured according to the X-ray film result. At week 8 post-surgery, rats were dipped in blue ink on the left foot and red ink on the right foot, followed by natural walking on collection paper to collect footprints. The knee joints were then removed after sacrifice and analyzed by Micro-CT (SkyScan 1176, SkyScan, Aartselaar, belgium). The software calculates the osteophyte volume after performing a three-dimensional reconstruction.

13. Histological analysis

Knee joint specimens were collected 8 weeks post-surgery after overdose of anesthetized sacrificed rats, fixed in 4% paraformaldehyde for 24 hours, followed by decalcification in 10% EDTA for 1 month. Specimens were dehydrated and paraffin-embedded, cut into 5mm thick paraffin sections, and stained with H & E and safranin O-fast green. Safranin O-fast green sections were evaluated independently according to the osteoarthritis international institute of research (OARSI) scoring criteria and quantitation of glycosaminoglycan content was performed using Image J software. To observe the expression of type II collagen, the sections were incubated with rat anti-type II collagen antibodies, followed by DAB substrate development, and after Image acquisition, quantitative analysis of positive expression was also performed using Image J software.

14. Data analysis

Statistical analysis and statistical mapping were performed using GraphPad Prism 8 software. Data are presented as Mean ± standard deviation (Mean ± SD). Adopting single-factor variance analysis and unpaired t-test statistical analysis, adopting independent t-test for comparison between two groups of samples, adopting single-factor variance analysis for multiple groups of samples, and carrying out pairwise comparison by LSD-t test.^*Represents that P is less than 0.05,^**represents that P is less than 0.01,^#represents that P is less than 0.05,^##represents P < 0.01. The process was analyzed using GraphPad software (version 8.0, GraphPad software inc, usa).

Experimental results example 2

Preparation and characterization of (I) NF and PMs

The characterization results are shown in FIG. 2, section a-j.

The NF obtained after emulsification was a homogeneous fine emulsion as shown in part b of FIG. 2, and the injectability of the NF was subsequently demonstrated by using a needle having an inner diameter of 0.45mm (shown in part c of FIG. 2, upper panel (I)). In FIG. 2, the upper graph (I) of the section I shows that the NF after removing the connective tissue by filtration is rich in lipid droplets when observed under an optical microscope, and the condensed lipid droplets in the loose fibers of the NF (shown in the upper graph (I) of the section j in FIG. 2) can be seen by SEM. Lipid droplets are all indicated by yellow triangles.

Successful synthesis of the polymer was confirmed using aldehyde modified PLGA, 1HNMR spectra (part a in fig. 2). PMs are prepared by dispersing emulsion in a microfluidic device, taking gelatin-PLGA dichloromethane solution with the mass ratio of 1:3 as a dispersion phase, and emulsifying the two to form a W-O structure, wherein gelatin is taken as a pore-forming agent. The 1% PVA solution served as the continuous phase and was subjected to shear stress to form droplets. One of the main advantages of the microfluidic technology for preparing microspheres is uniform particle size, and the flow ratio between the continuous phase and the dispersed phase is adjusted (Qc: Qd ═ 16:1), and the d part and the e part in fig. 2 can be seen to obtain PMs with smooth and porous surfaces, the average diameter is about 324 μm (f part in fig. 2), and the average pore diameter is about 27 μm (g part in fig. 2). The porous through network structure makes the carrier more penetrable, and can increase the carrying capacity and provide the carrier with shelter from physical barrier.

Preparation and characterization of (II) PMs @ NF

NF coupled with three-dimensional culture of PMs, also confirmed the injectability of the mixture system, no needle clogging was observed after PMs addition, and the lines of PMs @ NF injection were more filled than the images after NF injection, which was probably caused by more NF adsorbed by PMs addition (fig. 2, panel (II) below section c). Subsequently, after the prepared aldehyde group modified PMs @ NF and common PMs @ NF were mixed with BSA-FITC, respectively, as shown in the upper graph (I) of the h part in fig. 2, it was observed by confocal scanning microscopy that PMs chemically coupled to the aldehyde group modified exhibited uniform green fluorescence, whereas ordinary PMs observed extremely weak green fluorescence signal (shown in the lower graph (II) of the h part in fig. 2), BSA-FITC chemically coupled to the microspheres was significantly higher than physisorption, again confirming the presence of aldehyde groups on PLGA. As shown in the lower part (II) of the section i in FIG. 2, good bonding of lipid droplets surrounded by PMs can be seen under a light mirror, and the surface of microspheres covered with loose fibers can be seen by SEM observation, wherein the lipid droplets are included (shown in the lower part (II) of the section j in FIG. 2). Lipid droplets are all indicated by yellow triangles and PMs by red arrows.

Subsequently, as shown in part a of fig. 3, PBS, PLGA microspheres (Ordinary PMs), PMs, NF, and PMs @ NF were injected into the joint cavities of rats, respectively. Then the joint is fully exposed, as shown in part b in fig. 3, it can be seen that a small amount of common PLGA microspheres exist on the cartilage surface, the adhesion force is reduced due to the lack of aldehyde groups, the articular cartilage surface can be distributed in a dispersed and unformed manner after NF injection, compared with the condition that PMs are adhered to the cartilage surface in a large amount, PMs @ NF consumes a part of aldehyde groups, the adhesion rate is reduced, but the NF distribution is more uniform.

(III) in vitro degradation of PMs, binding of PMs to NF

The degradation of PMs in Phosphate Buffered Saline (PBS) was studied and as shown in part c of fig. 3, the degradation appeared more pronounced with the passage of incubation time, in particular, as micropores were visible on the PMs surface from week 3 and as the bonds between PMs micropores were partially broken from week 5. Furthermore, the pH of PBS (part d in FIG. 3) and the residual mass of PMs (part e in FIG. 3) decreased significantly with the duration of the incubation, and the decrease in both trends appeared to be generally in positive correlation.

Calcein-AM/DAPI staining shows the presence of small clusters of active adipose tissue in the NF as shown in section f in FIG. 3. Subsequently, Calcein-AM/DAPI staining observed the binding of PMs and NF. The g portion of FIG. 3 shows that as the culture time is prolonged, NF binds tightly to PMs, and is embodied in that more stem cells are carried, and NF penetrates deeper into PMs.

Cytokine detection and lubrication performance of (four) PMs @ NF

The three-dimensional space structure can stimulate adipose-derived stem cells to secrete cytokines, as shown in a1-a6 part in figure 4, and ELISA method detects high-level anti-inflammatory related cytokines, including VEGF (1682.0 +/-411.80 pg/ml), HGF (6135.0 +/-311.70 pg/ml), TGF-beta 1(910.1 +/-250.20 pg/ml), BDNF (1014.0 +/-167.50 pg/ml), IL-4(537.3 +/-107.60 pg/ml) and IL-10(341.0 +/-67.22 pg/ml).

Tribological tests showed the results of the lubricating properties of aqueous suspensions of PMs @ NF under different conditions. In order to bring the lubrication performance of PMs @ NF into the best state, the friction Coefficient (COF) of microspheres obtained in different composition ratios is firstly researched, and microsphere samples prepared according to the mass ratio of PMs to NF of 1:0, 3:1, 1:3 and 0:1 in the same batch are tested, and the results are shown in FIG. 5. As can be seen from FIG. 5, the resulting super-lubricated microspheres PMs @ NF had the lowest coefficient of friction (0.1293) and the best sustained stability when the mass ratio of PMs to NF was 1: 3; in other proportions, the microspheres initially had a low coefficient of friction but had poor sustained stability and a significant increase in coefficient of friction only in less than 5 min. Therefore, PMs @ NF was prepared in subsequent in vitro and in vivo experiments using PMs to NF in a mass ratio of 1: 3.

Another batch of samples, in which PMs @ NF was prepared at a PMs to NF mass ratio of 1:3, was tribologically tested against PBS, PMs, NF and PMs @ NF, and the resulting coefficient of friction-time curve is shown in FIG. 6. As can be seen from fig. 6, the coefficient of friction of PMs @ NF is significantly reduced (0.1006) and continues to remain low with excellent stability, as compared to PBS (0.5523) and PMs (0.4937) remaining at high levels for extended periods of time. It is noted that NF remains at an almost equal level at a COF value to composition ratio of 1:3 in the first part of the test, however, as the test time is prolonged, the COF value rises sharply showing a tendency of instability, which we believe is probably due to the correlation with the shear thinning effect. Furthermore, it is not desirable that the coefficient of friction of PMs be maintained continuously at a high level, and such a bio-bearing, although supported by rolling elements between the two contact surfaces, cannot exert its lubricating effect in the absence of bio-lubricating oil.

The b1 section in FIG. 4 shows the coefficient of friction versus time for PMs @ NF at various concentrations, showing a downward trend in coefficient of friction with increasing concentration, and COF values of 0.1364 and 0.1170 for 5mg/ml and 10mg/ml super-lubricated PMs @ NF, respectively. The coefficient of friction versus time at different reciprocation frequencies (1 Hz: 0.1260; 3 Hz: 0.1365; 5 Hz: 0.1311; 10 Hz: 0.2214) was then investigated (section b2 in FIG. 4). The different load-friction coefficient relationships (1N: 0.1393; 2N: 0.1390; 3N: 0.1365; 4N: 0.1837) are shown in section b3 of FIG. 4, indicating that the friction coefficient remains substantially low. The portion b4 in FIG. 4 is a schematic view of the tribology testing apparatus.

(V) in vitro biocompatibility and anti-inflammatory action

To examine the clinical application potential of PMs @ NF, the cytotoxicity of PMs, NF and PMs @ NF in vitro was studied. After 1, 3 and 5 days of co-culture with chondrocytes using the collected leachate, live/dead experiments and CCK8 experiments were performed. As shown in part a of fig. 7, live/dead staining revealed that chondrocytes were observed to be substantially viable at different time points, with only a few dead cells observed on day 5. In addition, counting the number of living cells showed that the number of living cells increased with the time of culture from day 1 to day 5, and that no significant difference was observed between all experimental groups and the control group at different time points (part b of fig. 7). The CCK8 test showed no significant difference in chondrocyte proliferation activity at each time point between all experimental groups and the control group (part c in fig. 7). The above results indicate that all NF, PMs have good cell compatibility with chondrocytes.

Studies have shown that multiple factors such as aging, obesity, sports injuries, inflammatory factors, genetic predisposition, and the like are involved in the pathogenesis of osteoarthritis, shifting healthy homeostasis to a catabolic state at the cellular and molecular level, specifically by regulating the synthesis of cartilage matrix, such as proteoglycans and collagen, and cartilage degradation-related enzymes, such as matrix metalloproteinases. TNF- α, a proinflammatory cytokine that plays a major role in osteoarthritis, is introduced in the present invention to mimic the specific inflammatory microenvironment in the course of osteoarthritis. Notably, Col2 α and aggrecan, as major components of the cartilage matrix, are present in large amounts in healthy chondrocytes. In a1 part of FIG. 8, it can be seen that the expression level of the gene Col2 alpha at different time points after TNF-alpha treatment is 50% lower after 12 hours of treatment and 80% lower after 24 hours of treatment as the expression level of the gene Col2 alpha is lower after treatment.

Subsequently, the anti-inflammatory and chondroprotective effects of PMs @ NF were examined. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the decrease in mRNA expression levels of Col2 α and aggrecan in chondrocytes was reversed to different extents after the addition of NF and PMs @ NF, and furthermore, there was no significant difference between the NF group and the PMs @ NF in the expression levels of the two genes (portions a2 and a3 in fig. 8). However, the levels of mRNA expression by the addition of PMs on Col2 a and aggrecan were similar to the blank group with minimal effect. In addition, mRNA expression levels of matrix metalloproteinase 1(MMP1), interleukin-1 beta (IL-1 β), and tachykinin-1 (TAC1) were also detected by qRT-PCR. MMP1 is one of the major degrading enzymes involved in the breakdown of cartilage matrix, IL-1 β is one of the cytokines mainly involved in anti-inflammation, and TAC1 is a neuropeptide involved in pain signaling. As shown in FIG. 8, in sections a4, a5, and a6, mRNA for MMP1, IL-1 β, and TAC1 were significantly elevated after TNF- α treatment. NF and PMs @ NF significantly reduced the mRNA expression levels of MMP1, IL-1 β, and TAC1 compared to the blank group, while no significant difference was observed for PMs.

Immunofluorescence staining further examined the expression level of chondrocyte-specific protein-Col 2. alpha. protein (FIG. 8, panel b). After TNF-alpha treatment, the fluorescence signal intensity of the blank group is obviously lower than that of the control group, which indicates that the expression of the Col2 alpha protein is reduced. Compared with a control group, although the addition of NF and PMs @ NF has a certain limit on increasing the expression amount of the Col2 alpha protein, compared with a blank group, the addition of NF and PMs @ NF obviously increases the expression of the Col2 alpha protein. Proved by qRT-PCR and immunofluorescence staining, the super-lubricated multi-component delivered PMs @ NF developed by the invention has anti-inflammatory and cartilage protection effects, so that synthetic metabolic molecules such as Col2 alpha and aggrecan are up-regulated, and MMP1 catabolic protease and IL-1 beta anti-inflammatory cytokines are down-regulated. In addition, PMs @ NF also reduced TAC1 protein expression and alleviated pain in osteoarthritis patients. Since tribology experiments were not designed for in vitro experiments, there was no significant difference between the NF and PMs @ NF groups.

(VI) synergistic therapeutic Effect in vivo for osteoarthritis

Previous studies used a meniscal destabilization model to induce a rat osteoarthritis model, which was used in this study followed by motor intervention (see fig. 10), while SD rats received one week after DMM surgery were injected weekly intra-articular with PBS, PMs, NF, and PMs @ NF.

Section a in fig. 9 shows X-ray images of rat knee joints taken at

weeks

1 and 8 after DMM surgery. In addition, it was calculated from the X-ray film that the joint space widths of all experimental groups at 1 week after the operation were similar without any sign of narrowing (part c in fig. 9). Knee X-ray radiographic calculations at 8 weeks post-surgery showed that, although the joint spaces were reduced in all of the four experimental groups (PBS, PMs, NF, and PMs @ NF) that received DMM surgery, the PMs @ NF group joint space width was significantly higher than the PBS group (panel d in fig. 9). Subsequently, Micro CT scans were used to further analyze DMM postoperative knee osteophyte status. Section b in fig. 9 shows a 2D coronal scan of the knee joint with osteophytes marked with red arrows, followed by 3D modeling of the above obtained pictures to obtain stereoscopic pictures and distinguishing the osteophytes from normal bone tissue with red shading, and both types of pictures can be seen with the least amount of PMs @ NF macroscopic osteophytes in the experimental group.

As shown in section e of FIG. 9, the Total Osteophyte Volume (TOV) of each group was quantitatively analyzed, compared to the sham group (0.07. + -. 0.06 mm)³) Compared with the prior art, the TOV of each experimental group is remarkably increased and is respectively a PBS group (2.78 +/-0.36 mm)³) PMs group (2.53 + -0.46 mm)³) NF group (1.86 +/-0.35 mm)³) And PMs @ NF group (1.08. + -. 0.28 mm)³) However, TOV was significantly lower for the PMs @ NF group than for the PBS group.

In addition, as shown in part a of fig. 11, 8-week footprints after surgery were collected from each experimental group, and representative footprints were extracted from each group for comparison, where blue is healthy side and red is molded side footprints. Subsequently, static parametric analysis is performed on the collected footprints. As shown in fig. 11, sections b, c and d, the step lengths were increased to different extents for each of the experimental groups compared to the PBS group, with the PMs @ NF group increasing particularly significantly. In addition, the pedestal support (BOS) index, PMs @ NF, was reduced by 34.75% compared to the PBS group. Subsequently, the PMs @ NF group showed an increase of 216.13% over the PBS-molded side footprint area, as counted for footprint area.

Subsequently, the cartilage tissue was evaluated histologically by hematoxylin-eosin (H & E) staining and safranin O-fast green staining for 8 weeks after the operation. As shown in fig. 12, parts a and b, typical osteoarthritis features such as surface roughness, longitudinal fissures, erosive denudation, cell degeneration and disorganization were observed in the PBS group. The PMs, NF, and PMs @ NF morphologies were altered, matrix staining, and strio integrity were improved to different extents compared to the PBS group. In addition, it was shown from the safranin O-fast green staining that glycosaminoglycan of PMs @ NF group positively stained more strongly, next to NF group, indicating that PMs @ NF group could deposit glycosaminoglycan (GAG), maintaining cartilage matrix better during development of osteoarthritis (panel d in fig. 12).

The results of the OARSI scores are shown in fig. 12, section e, with the other treatment groups decreasing in magnitude compared to the score of the PBS group, and the PMs @ NF group again showed better treatment, a 59.01% decrease. In addition, the expression level of the chondrocyte marker type ii collagen was evaluated by immunohistochemical staining. As shown in section c of FIG. 12, all treatment groups including PBS, PMs, NF and PMs @ NF exhibited different levels of collagen type II (brown staining) expression than the sham group, with the PMs @ NF having the least decrease in the positive rate.

Subsequently, the proportion of statistically positive stained cells was quantified (panel f in fig. 12), and the PMs @ NF group was increased by about 142% in positive rate compared to the PBS group, followed by the NF group, which was increased by about 59.74%, with no statistical difference in the PMs group.

The results show that PMs @ NF has the characteristics of improving the lubricating property and continuously releasing the in-situ factor on the surface of the cartilage, and becomes an effective strategy for treating osteoarthritis.

Claims

1. A preparation method of nanometer fat functionalized injectable super-lubricating microspheres is characterized by comprising the following steps:

(3) co-culturing the nano-fat obtained in the step (1) and the porous microspheres obtained in the step (2) according to the mass ratio of the porous microspheres to the nano-fat of 1:3 to prepare the nano-fat functionalized injectable super-lubricating microspheres.

2. The method of claim 1, wherein the physical emulsification in step (1) is mechanical emulsification of adipose tissue using a fatty chylotor.

3. The method according to claim 1, wherein the animal adipose tissues are washed clean and sheared in step (1), and then filtered under aseptic conditions, followed by the physical emulsification process.

4. The method according to claim 1, wherein in the step (2), when the microfluidic device is used to prepare the porous microspheres, a solution of gelatin and an aldehyde-based polylactic acid-glycolic acid copolymer in dichloromethane is used as an aqueous phase, and a solution of polyvinyl alcohol is used as an oil phase.

5. The method according to claim 4, wherein the flow rate ratio of the oil phase to the aqueous phase in step (2) is 16: 1.

6. The production method according to claim 1, wherein the conditions of the co-culture in the step (3) are: the cells were co-cultured at 37 ℃ and 90rpm for 24 hours in three dimensions.

7. An injectable super-lubricious nanoparticle functionalized with nano-lipids prepared by the method of any of claims 1 to 6.

8. The injectable super-lubricious microsphere of claim 7 wherein the microsphere has an average diameter of 324 μm and an average pore size of 27 μm.

9. Use of nano-fat functionalized injectable super-lubricious microspheres prepared by the method of claims 1-6 or nano-fat functionalized injectable super-lubricious microspheres of claims 7-8, wherein the use comprises use in preparation of a medicament for treatment of orthopedic disorders.

10. Use according to claim 9, wherein the bone disease comprises osteoarthritis.