Anti-infection and bone repair promoting preparation for sequentially regulating immune microenvironment of macrophages and preparation method thereof

Abstract

The application provides a preparation method of an anti-infection bone repair promoting preparation for sequentially regulating a macrophage immune microenvironment, which comprises the following steps: uniformly mixing 1-10% flavone modified hydroxyapatite, 2-10% sodium alginate hydrogel, 0.1-1% hydrogen sulfide sustained release carrier and 79-96.9% tricalcium phosphate in proportion by weight percentage, adding a calcium chloride aqueous solution, uniformly stirring, and freeze-drying. The flavone tricalcium phosphate, the calcium phosphate cement of the light crosslinking alginate hydrogel containing the hydrogen sulfide sustained release carrier, exhibits the function of regulating the macrophage immune microenvironment, and the preparation itself can better realize the function of sequentially inducing the macrophages in a bone damage microenvironment to be induced to M1 type polarization and then to M2 type polarization.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of immunological medical engineering, and particularly relates to an anti-infection bone repair preparation for sequentially regulating a macrophage immune microenvironment and a preparation method thereof. BACKGROUND

[0002] Host immune response and anti-infection are key factors determining the success or failure of biological material in repairing bone defects, and the immune system plays a crucial role in defending bone infection and maintaining bone homeostasis. Macrophages, as a kind of immune cells, are an important part of the bone injury microenvironment. According to the phenotype and function, activated macrophages can be divided into M1 and M2 types, and the polarization direction is regulated by the microenvironment. Macrophages not only play a key role in host defense and inflammatory response, but also have great research value in bone injury repair due to their heterogeneity and functional diversity. Based on the body's own immune system, the activity of macrophages at the implant-tissue interface is regulated to clear bacteria, which has become a research hotspot in anti-infection bone repair materials. At present, the regulation methods of macrophages involved in anti-infection bone repair materials include: enhancing the recruitment of macrophages around the implant, promoting the M1 phenotype polarization of macrophages, and sequentially guiding the M1→M2 phenotype polarization. M1 macrophages have pro-inflammatory effects and participate in host defense against infection, while M2 macrophages help tissue repair and inflammation resolution.

[0003] It has been reported that transition metals such as copper, zinc, iron, titanium, and manganese can regulate the polarization of M1 macrophages, and show an increase in the expression levels of cytokines IL-6, IL-1β and TNFα in different cell types such as RAW264.7 and J774.A1, that is, promote the transition of macrophages from M0 to M1, but there are also many reports that copper can promote the M0 to M2 polarization phenotype of cells. It can be seen that metal ions can have different effects on the activation of macrophages, but in biomedical applications, there are still challenges such as metal toxicity, low biocompatibility, cell metabolic ablation barrier, and narrow immune regulator window.

[0004] For bone injury repair, M1 over-activation will prolong the inflammatory response and cause delayed or poor bone tissue healing, and M2 early polarization or sustained polarization will also prolong intracellular infection of pathogens and cause tissue fibrosis and other negative effects. It can be seen that the immune balance of M1 / M2 macrophages is crucial for bone integration. Therefore, it is extremely difficult to clear infection and promote bone integration by only regulating the M1 polarization of host immune metabolism in one direction. Therefore, a multifunctional immune regulation implant is designed to cooperatively guide the M1 and M2 phenotype conversion of macrophages to establish an optimal immune microenvironment for bone integration, and to enhance the intracellular bactericidal capacity or phagocytic capacity of macrophages, which can provide a new research route for using the host's own immune capacity to clear infection and enhance bone integration around the implant. SUMMARY

[0005] The technical purpose of the present application is to guide the immune balance of M1 / M2 macrophages at the bone injury site to establish an optimal bone integration immune microenvironment, thereby providing an anti-infection and bone repair promoting preparation for sequentially regulating the macrophage immune microenvironment and a preparation method thereof.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following series of technical solutions to solve it.

[0007] The first purpose of the present application is to provide a preparation method of an anti-infection and bone repair promoting preparation for sequentially regulating the macrophage immune microenvironment, which comprises the following steps: uniformly mixing 1-10% flavone modified hydroxyapatite, 2-10% sodium alginate hydrogel, 0.1-1% hydrogen sulfide sustained release carrier and 79-96.9% tricalcium phosphate paste according to the proportion of weight percentage, adding calcium chloride aqueous solution containing Na2HPO4, stirring uniformly, and freeze-drying.

[0008] Preferably, in the flavone modified hydroxyapatite, the concentration of Ca ions is in the range of 0.1-0.5 mol / L, and the molar ratio of flavone compound / Ca is 0.002-0.01:1.

[0009] Preferably, the preparation method of the flavone modified hydroxyapatite comprises: simultaneously dropping (NH4)2HPO4 aqueous solution and flavone compound water-alcohol solution into Ca(NO3)2·4H2O aqueous solution at a molar ratio of Ca / P of 1.67, stirring while dropping, keeping stirring at room temperature for 3-5 days; then heating to 70-90℃, adjusting the pH to 10 with dilute ammonia water and keeping it, and continuously stirring at constant temperature for 3-5 hours, then centrifuging at 10000 rpm for 10 minutes, and repeatedly washing with deionized water; finally, freeze-drying into powder, grinding and passing through a 100-200 mesh sieve, to obtain flavone modified hydroxyapatite powder.

[0010] Preferably, the flavone is 3-hydroxyflavone, 5-hydroxyflavone, 6-hydroxyflavone, 3,7-dihydroxyflavone, chrysin or galangin.

[0011] Preferably, the preparation method of the sodium alginate hydrogel comprises: irradiating an aqueous solution containing 2% (w / v) methacrylated sodium alginate and 0.1% (w / v) photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure-2959) with a wavelength of 365 nm and an intensity of 1.7 mW / cm2 for 20 min.

[0012] Preferably, the hydrogen sulfide sustained release carrier is GYY4137, ADT-OH or AP39; and the concentration of Na2HPO4 is 2% (mass / volume).

[0013] Preferably, the preparation method of the copper tricalcium phosphate paste comprises: adding tricalcium phosphate into a 0.2 mol / L CuCl2·2H2O aqueous solution, and stirring uniformly, wherein the ratio of solid phase to liquid phase is 1:(0.3-0.6) g / mL; the tricalcium phosphate contains 5% amorphous tricalcium phosphate, 8% β-tricalcium phosphate and 87% α-tricalcium phosphate in mass fraction.

[0014] A second object of the present application is to provide an anti-infection and bone repair promoting preparation for sequentially regulating the immune microenvironment of macrophages prepared by the above method.

[0015] Preferably, the sequentially regulated immune microenvironment of macrophages is to enhance the polarization of macrophages to M1 type for 0-4 weeks, and to induce the polarization to M2 type for 4-8 weeks.

[0016] A third object of the present application is to provide the use of the anti-infection and bone repair promoting preparation for sequentially regulating the immune microenvironment of macrophages in promoting bone repair, macrophage polarization, intravenous endothelial vascular cell proliferation, osteoblast proliferation or osteogenic differentiation of bone marrow mesenchymal stem cells.

[0017] The anti-infection and bone repair promoting preparation for sequentially regulating the immune microenvironment of macrophages has the action principle that the copper calcium phosphate cement paste releases copper ions in the aqueous solution, the copper ions are cross-linked with the photo-crosslinked sodium alginate to wrap the tricalcium phosphate and the flavone modified hydroxyapatite, and the hydrogen sulfide carrier releases hydrogen sulfide after being dissolved. 2+ The copper ions and the hydrogen sulfide generate a mixed system with Cu + , Cu 2+ , S 2- , S 0 , which can maintain stable Cu(I) to enhance the polarization of macrophages to M1 type; and the degradation of the flavone hydroxyapatite can continuously improve the proliferation ability of macrophages to enhance the polarization to M2 type, so that the macrophages play the roles of anti-infection and promoting repair.

[0018] Advantages of the present application:

[0019] The present application constructs a bone repair preparation capable of adjusting the behavior of local macrophages in the microenvironment of bone injury by loading the hydrogen sulfide slow-release carrier and the bimetal onto the tricalcium phosphate covered by the cross-linked photo-crosslinked sodium alginate hydrogel layer.

[0020] As shown in the examples, the flavone tricalcium phosphate, the calcium phosphate cement containing the photo-crosslinked sodium alginate hydrogel slow-release carrier of hydrogen sulfide, has the function of regulating the immune microenvironment of macrophages, and the preparation itself can better realize the function of sequentially inducing the polarization of macrophages in the microenvironment of bone injury to M1 type and then to M2 type, while the existing scaffold materials are difficult to realize the above function.

[0021] On the other hand, by loading the calcium phosphate / hydrogel scaffold with a hydrogen sulfide carrier, the slowly released hydrogen sulfide interacts with the low-dose Cu(II) released by the tricalcium phosphate to generate Cu(I) with good antibacterial effect, and the system can maintain a dynamic balance between Cu(I) and Cu(II) in the early 0-4 weeks. As shown in the examples, it is found that the combination of the two plays a very good mutual promotion effect, which can greatly reduce the dosage of the inhibitor used, not only greatly reduces the toxic side effects of copper on macrophages, which is more than 10 times, but also significantly polarizes the M1 of macrophages, thereby achieving intracellular bactericidal effect.

[0022] The tricalcium phosphate in the application can self-cure to form calcium phosphate cement (CPC) in an aqueous solution, which has been applied to clinical bone repair and has shown good bone integration performance, and the metal tricalcium phosphate pre-mixed paste (PCPC) not only has good bone integration function, but also can improve the defects of manual operation of self-curing calcium phosphate cement. The PCPC hydrogel preparation obtained by combining sodium alginate hydrogel, hydrogen sulfide carrier, copper(II) ion and flavonoid compound can not only provide the degradation rate of CPC, improve its water absorption, significantly enhance the M2 polarization of macrophages in 4-8 weeks, and show the characteristics of promoting the osteogenic differentiation of bone marrow mesenchymal stem cells. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 To characterize the bone repair promoting preparation; the scanning electron microscope results show the surface morphology of the preparation.

[0024] Figure 2 Qualitative and quantitative detection of Cu(I) in the leaching solution of the preparation in examples 1-3 at different times. a-d are the ultraviolet absorption spectra of 3h, 6h, 9h, 24h respectively, e: comparison of ultraviolet absorption spectra of 1d-14d of example 3, f: Cu(I) reduction rate graph of different time periods in three examples.

[0025] Figure 3 Flow analysis of M1 and M2 polarization of macrophages induced by example 3 for 1 week and 4 weeks.

[0026] Figure 4 Promotion of bone repair preparation at different times on intravenous endothelial vascular cells (HUVES) and osteoblasts (MCT3T3-E1). DETAILED DESCRIPTION

[0027] The following examples are further illustrations of the application and are not intended to limit the application.

[0028] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this application belongs. The test reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods described are all conventional methods unless otherwise specified.

[0029] Example 1:

[0030] (1) Preparation of flavone-modified hydroxyapatite powder

[0031] 20 ml of (NH4)2HPO4 aqueous solution and 20 ml of 3-hydroxyflavone 50% ethanol aqueous solution were simultaneously added dropwise into 20 ml of Ca(NO3)2·4H2O aqueous solution at a Ca / P molar ratio of 1.67, and stirred at room temperature for 3 days; then the temperature was raised to 70°C, and the pH was adjusted to 10 with 50% dilute ammonia water and kept constant for 3 hours of constant temperature stirring, then centrifuged at 10,000 rpm for 10 minutes, and washed repeatedly with deionized water; finally, freeze-dried into powder, ground and passed through a 100-mesh sieve to obtain the flavone-modified hydroxyapatite powder. The concentration of Ca ions was 0.5 mol / L, and the molar ratio of flavone compound / Ca was 0.002:1.

[0032] (2) Photocrosslinked sodium alginate hydrogel

[0033] Methacrylated sodium alginate (MA) and photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure-2959) were dissolved in deionized water at concentrations of 2.5% (w / v) and 0.15% (w / v), respectively, and mixed thoroughly to prepare the hydrogel precursor solution. Then the precursor solution was irradiated with a UV lamp (wavelength 365 nm, intensity 2.0 mW / cm 2 ) for 30 min to prepare the photocrosslinked sodium alginate hydrogel (PMA).

[0034] (3) Copper tricalcium phosphate paste

[0035] Tricalcium phosphate (containing 5% amorphous, 8% β-tricalcium phosphate and 87% α-tricalcium phosphate) solid powder was added to 0.2 mol / L CuCl2·2H2O aqueous solution and stirred uniformly to form a copper tricalcium phosphate paste. The solid-to-liquid ratio was 1:0.6 (g / mL).

[0036] (4) The anti-infection and bone repair preparation for sequentially regulating the immune microenvironment of macrophages was prepared by mixing 5.5% flavone-modified hydroxyapatite powder, 5% photocrosslinked sodium alginate hydrogel, 0.5% GYY4137 and 89% tricalcium phosphate paste according to the mass ratio, and then adding 1 g of the mixture into 0.6 mL of a 2 mol / L CaCl2 aqueous solution (containing 2% Na2HPO4) to form a paste. The paste was freeze-dried and ground into powder.

[0037] The scanning electron microscope results show that the surface morphology of the preparation is as follows Figure 1 .

[0038] Example 2:

[0039] (1) Preparation of flavone-modified hydroxyapatite powder

[0040] According to the Ca / P molar ratio of 1.67, 20 mL of (NH4)2HPO4 aqueous solution and 20 mL of homoeriodictyol water-alcohol solution (1 / 1 V / V) were simultaneously added dropwise into 20 mL of Ca(NO3)2·4H2O aqueous solution, and stirred during the dropwise addition. The stirring was maintained at room temperature for 5 days. Then the temperature was increased to 90℃, and the pH was adjusted to 10 with 50% dilute ammonia water and maintained for 5 hours of constant temperature stirring. Then, the mixture was centrifuged at 10,000 rpm for 10 minutes and washed repeatedly with deionized water. Finally, the mixture was freeze-dried into powder, ground and passed through a 200 mesh sieve to obtain the flavone-modified hydroxyapatite powder. The concentration of Ca ions was 0.1 mol / L, and the molar ratio of flavone compound / Ca was 0.005:1.

[0041] (2) Photocrosslinked sodium alginate hydrogel

[0042] Methacrylated sodium alginate (MA) and photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure-2959) were dissolved in deionized water at concentrations of 2.5% (w / v) and 0.15% (w / v), respectively, and mixed uniformly to prepare the hydrogel precursor solution. Then, the precursor solution was irradiated with a UV lamp (wavelength 365 nm, intensity 2.0 mW / cm 2 ) for 30 min to prepare the photocrosslinked sodium alginate hydrogel (PMA).

[0043] (3) Tricalcium phosphate paste

[0044] Tricalcium phosphate (containing 5% amorphous, 8% β-tricalcium phosphate and 87% α-tricalcium phosphate) solid powder was added into a 0.2 mol / L CuCl2·2H2O aqueous solution and stirred uniformly to form a tricalcium phosphate paste. The solid-liquid ratio was 1:0.5 (g / mL).

[0045] (4) The mixture of 1% flavone-modified hydroxyapatite powder, 2% photo-crosslinked sodium alginate hydrogel, 0.1% AP39, and 95% tricalcium phosphate paste is mixed uniformly according to the mass ratio, 1 g of the mixture is added into 0.5 mL of 2 mol / L CaCl2 aqueous solution (containing 2% Na2HPO4), and stirred uniformly to form a paste. The paste is freeze-dried and ground into powder, which is an anti-infection and bone repair preparation for sequentially regulating the immune microenvironment of macrophages.

[0046] Example 3:

[0047] (1) Preparation of flavone-modified hydroxyapatite powder

[0048] 50 mL of (NH4)2HPO4 aqueous solution and 50 mL of 3,7-dihydroxyflavone water-alcohol solution (1 / 1 V / V) are simultaneously added dropwise into 50 mL of Ca(NO3)2·4H2O aqueous solution according to the Ca / P molar ratio of 1.67, and stirred while dropping. The stirring is maintained at room temperature for 4 days, then the temperature is increased to 80°C, and the pH is adjusted to 10 with 50% dilute ammonia water and maintained for 4 hours of constant temperature stirring. Then, centrifugation is performed at 10,000 rpm for 10 minutes, and repeated washing with deionized water is performed. Finally, freeze-drying is performed to obtain a powder, which is ground and passed through a 200-mesh sieve to obtain the flavone-modified hydroxyapatite powder. The concentration of Ca ions is 0.3 mol / L, and the molar ratio of flavone compound / Ca is 0.01:1.

[0049] (2) Photo-crosslinked sodium alginate hydrogel

[0050] Methacrylated sodium alginate (MA) and photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure-2959) are dissolved in deionized water at concentrations of 2.5% (w / v) and 0.15% (w / v), respectively, and mixed uniformly to prepare a hydrogel precursor solution. Then, the precursor solution is irradiated with a UV lamp (wavelength 365 nm, intensity 2.0 mW / cm 2 ) for 30 min to prepare photo-crosslinked sodium alginate hydrogel (PMA).

[0051] (3) Tricalcium phosphate paste

[0052] Tricalcium phosphate (containing 5% amorphous, 8% β-tricalcium phosphate, and 87% α-tricalcium phosphate) solid powder is added into 0.2 mol / L CuCl2·2H2O aqueous solution and stirred uniformly to form a tricalcium phosphate paste. The solid-liquid ratio is 1:0.3 (g / mL).

[0053] (4) The anti-infection and bone repair preparation for sequentially regulating the immune microenvironment of macrophages was prepared by mixing 10% flavonoid-modified hydroxyapatite powder, 10% photocrosslinked sodium alginate hydrogel, 1% ADT-OH, and 79% tricalcium phosphate paste in a mass ratio, adding 1 g of the mixture to 0.3 mL of a 2 mol / L calcium chloride aqueous solution (containing 2% Na2HPO4), stirring uniformly to form a paste, freeze-drying the paste, and grinding the paste into powder.

[0054] Example 4:

[0055] 1) Cu(I) quantitative analysis

[0056] 1.0 g of the anti-infection and bone repair preparation for sequentially regulating the immune microenvironment of macrophages in Examples 1-3 was soaked in 10 mL of simulated body fluid SBF buffer for an 8-week degradation experiment. During the degradation process, 0.2, 0.15, 0.1, and 0.05 mL of the degradation solution were taken and 1 mL of bathocuproin sulfonate disodium salt (BCS) was added, the mixture was uniformly mixed and then left to stand for half an hour, and Cu(I) was qualitatively and quantitatively detected using an ultraviolet spectrophotometer. Figure 2 )。

[0057] 2) Macrophage M1 / M2 polarization analysis

[0058] 1. One hour before the experiment, 20 ng / mL CD86 or 20 ng / mL IL-4 was added to the DMEM medium to prepare macrophage M1 and M2 type polarization induction medium. By adding 1 g of the bone repair preparation in Example 3 to 10 mL of DMED medium, incubating at 37°C, preparing 1-week and 4-week preparation extracts, and filtering with a 0.22 μm filter, 200 ul of macrophage Raw264.7 was uniformly inoculated in each well of a 6-well plate at a cell number of 5×10 5 After 24 hours of culture at 5% CO2 and 37°C, the cells were collected, stained for specific markers CD86 and IL-4, and further detected for M1 and M2 macrophage phenotype proportions using a flow cytometer. Figure 3 )。

[0059] 3) Biocompatibility analysis:

[0060] The bone repair preparation (0.5 mg / mL) was soaked in 37°C DMEM medium for 4 weeks, and 3 tubes were taken out every week to collect the extracts by centrifugation (8000 g, 15 minutes) and sterilized with a 0.22 μm filter. HUVEC and MC3T3-E1 cells were inoculated in 6-well plates at a cell number of 1×10 4Cell density of 1 cell / well was seeded in 96-well plates, supplemented with DMEM medium (containing 1% double antibody + 10% calf serum) to 200 μL and incubated at 37℃, 5% CO2 for 24 hours for cell adhesion. Replace the medium with an equal volume of sterile extract. After 24 hours of culture (37℃, 5% CO2), analyze cell proliferation using a CCK8 kit (Bi Yun Tian, C0038).

[0061] The bone repair promoting formulations in Examples 1-3 have good biocompatibility with both venous endothelial vascular cells and osteoblasts, with a range of 93.1%-183.9% for the proliferation promoting effect on venous endothelial vascular cells and a range of 95.8%-159.8% for the proliferation promoting effect on osteoblasts, having potential for promoting vascular regeneration and bone repair. Figure 4 )

Claims

1. A method for preparing an anti-infective pro-osteoremediative formulation that sequentially modulates the immune microenvironment of macrophages, characterized in that, The method comprises the following steps: 1-10% flavonoid-modified hydroxyapatite, 2-10% sodium alginate hydrogel, 0.1-1% hydrogen sulfide sustained-release carrier and 79-96.9% copper tricalcium phosphate paste are uniformly mixed in percentage by weight, a calcium chloride aqueous solution containing Na2HPO4 is added, stirring is uniformly carried out, and freeze-drying is carried out.

2. The method of claim 1, wherein, In the flavonoid-modified hydroxyapatite, the Ca ion concentration ranges from 0.1 to 0.5 mol / L, and the molar ratio of flavonoid compound / Ca is 0.002-0.01:

1.

3. The method of claim 1, wherein, The preparation method of the flavonoid-modified hydroxyapatite comprises the following steps: (NH4)2HPO4 aqueous solution and flavonoid compound hydroalcoholic solution are simultaneously and dropwise added into Ca(NO3)2·4H2O aqueous solution at a molar ratio of Ca / P of 1.67, stirring is carried out during dropping, stirring is maintained at room temperature for 3-5 days, then the temperature is increased to 70-90℃, the pH is adjusted to 10 by using dilute ammonia water, and constant-temperature stirring is continuously carried out for 3-5 hours, then centrifugation is carried out at 10,000 rpm for 10 minutes, and repeated washing is carried out with deionized water; finally, freeze-drying is carried out into powder, grinding is carried out and the powder is passed through a 100-200 mesh sieve, and the flavonoid-modified hydroxyapatite powder is obtained.

4. The method according to any of claims 1 to 3, characterized in that, The flavonoid is 3-hydroxyflavone, 5-hydroxyflavone, 6-hydroxyflavone, 3,7-dihydroxyflavone, chrysin or galangin.

5. The method of claim 1, wherein, The method for preparing the sodium alginate hydrogel comprises: irradiating an aqueous solution containing 2% (w / v) methacrylated sodium alginate and 0.1% (w / v) photoinitiator 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone with a UV light lamp with a wavelength of 365 nm and an intensity of 1.7 mW / cm 2 for 20 min.

6. The method of claim 1, wherein, The hydrogen sulfide sustained-release carrier is GYY4137, ADT-OH or AP39; and the concentration of Na2HPO4 is 2% by mass / volume.

7. The method of claim 1, wherein, The preparation method of the copper tricalcium phosphate paste comprises the following steps: tricalcium phosphate is added into 0.2 mol / L CuCl2·2H2O aqueous solution, and stirring is uniformly carried out, wherein the solid-liquid ratio is 1:(0.3-0.6) g / mL; the tricalcium phosphate contains 5% amorphous tricalcium phosphate, 8% β-tricalcium phosphate and 87% α-tricalcium phosphate by mass fraction.

8. The anti-infection and bone repair promoting preparation for sequentially regulating the immune microenvironment of macrophages prepared by the method of claim 1, 2, 3, 5, 6 or 7.

9. The anti-infective pro-osteoremediative formulation that sequentially modulates the immune microenvironment of macrophages according to claim 8, wherein, The sequential regulation of the immune microenvironment of macrophages is to enhance the polarization of macrophages to M1 type for 0-4 weeks and induce the polarization of macrophages to M2 type for 4-8 weeks.

10. The use of the anti-infection and bone repair promoting preparation for sequentially regulating the immune microenvironment of macrophages of claim 8 in the preparation of a product for promoting bone repair, macrophage polarization, intravenous endothelial vascular cell proliferation, osteoblast proliferation or osteogenic differentiation of bone marrow mesenchymal stem cells.

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