CN112618800A

CN112618800A - Mandibular condyle osteochondral repair scaffold material and preparation method thereof

Info

Publication number: CN112618800A
Application number: CN202011549911.1A
Authority: CN
Inventors: 孙勇; 王鸿哲; 徐扬; 王培磊; 樊渝江
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-04-09
Anticipated expiration: 2040-12-24
Also published as: CN112618800B

Abstract

The invention provides a mandible condylar process osteochondral repair scaffold material and a preparation method thereof, the scaffold material consists of a cartilage surface layer, a cartilage deep layer and a bone layer, wherein the cartilage surface layer consists of composite hydrogel of cross-linked sulfhydryl-hyaluronic acid and type I collagen and bone marrow mesenchymal stem cells distributed in the composite hydrogel, the cartilage deep layer consists of composite hydrogel of cross-linked sulfhydryl-hyaluronic acid and type I collagen and cartilage cells distributed in the composite hydrogel, the bone layer is porous biphase calcium phosphate ceramic, and the cartilage deep layer is positioned between the cartilage surface layer and the bone layer and in a porous structure of the bone layer to connect the cartilage surface layer and the bone layer into a whole. In vivo experiments prove that the scaffold material has excellent repair capability on the cartilage defect of the mandibular condyle, can solve the problems that the existing method and material for treating the cartilage defect of the mandibular condyle cannot repair the cartilage defect of the mandibular condyle, and provides new possibility for the treatment of the cartilage defect of the mandibular condyle.

Description

Mandibular condyle osteochondral repair scaffold material and preparation method thereof

Technical Field

The invention belongs to the field of mandible repairing materials, and relates to a mandible condylar process osteochondral repairing scaffold material and a preparation method thereof.

Background

Temporomandibular joint disorders are common clinically, often accompanied by osteochondral defects of mandibular condylar process, resulting in joint pain, restricted mandibular movement, concomitant osteoarthritis, even restricted mouth opening, and severe impact on the quality of life of the patient. The mandibular condyle lacks direct blood supply and innervation, has poor self-repair ability, and the condylar osteochondral defect caused by the temporomandibular joint disorder is hardly completely repaired. At present, clinically applied methods for treating mandibular condylar osteochondral injury all have obvious disadvantages: (1) non-invasive treatments such as oral medication, physical therapy and the like can only play a role in relieving symptoms and delaying the state of an illness, but cannot repair the existing mandibular condylar osteochondral defect; (2) minimally invasive treatment operations such as hyaluronic acid, trophic factors and the like injected into joint cavities are mainly used for relieving joint zone pain and increasing the degree of lubrication in joints so as to improve the mandibular movement function, but do not have the function of repairing the cartilage defect of the mandibular condylar bone; (3) open surgery treatment of temporomandibular joint area is mainly to perform condylar replacement surgery on patients who seriously affect the function of the mandible, and also can not repair the cartilage defect of the mandible condylar process.

For the bone cartilage injury, the ideal treatment effect is that a new cartilage layer and a bone layer which have similar structures with surrounding normal tissues and good biological properties are formed at the injury part and are tightly combined with the surrounding normal tissues. The tissue engineering technology can utilize allogenic cells to be compounded on a multi-layer scaffold with good biological performance to construct a tissue engineering osteochondral complex with proper size and shape, effectively overcomes the limitations of donor deficiency, donor part damage and poor cell amplification capability in autologous osteochondral transplantation, and provides a new hope for repairing the mandibular condylar osteochondral damage. Most synovial articular cartilage is mainly composed of hyaline cartilage, and mandibular condylar cartilage is composed of fibrocartilage, and the tissue composition and layering of the synovial articular cartilage are greatly different from those of most synovial articular cartilage, so that the applicability of the existing material suitable for repairing synovial articular cartilage to the repair of mandibular condylar cartilage is limited. At present, the construction research of the common synovial joint tissue engineering osteochondral complex tissue is deeper at home and abroad, but the osteochondral repair of the mandibular condyle is still in an initial stage. How to develop an ideal mandibular condyle osteochondral repair scaffold material to solve the problems of the current clinically applied methods for treating mandibular condyle osteochondral injury becomes one of the main research directions in the future.

Disclosure of Invention

The invention provides a mandibular condyle osteochondral repair scaffold material and a preparation method thereof, aiming at the problems that no scaffold material specially used for mandibular condyle osteochondral repair exists in the prior art, and a clinically applied method and material for treating mandibular condyle osteochondral injury can not realize mandibular condyle osteochondral defect repair.

In order to achieve the purpose, the invention adopts the following technical scheme:

a mandible condylar process osteochondral repair scaffold material comprises a cartilage surface layer, a cartilage deep layer and a bone layer, wherein the cartilage surface layer comprises a composite hydrogel of cross-linked sulfhydryl-hyaluronic acid and type I collagen and bone marrow mesenchymal stem cells distributed in the composite hydrogel, the cartilage deep layer comprises a composite hydrogel of cross-linked sulfhydryl-hyaluronic acid and type I collagen and cartilage cells distributed in the composite hydrogel, the bone layer is porous biphase calcium phosphate ceramic, the cartilage deep layer is positioned between the cartilage surface layer and the bone layer and in a porous structure of the bone layer to connect the cartilage surface layer and the bone layer into a whole, the cross-linked sulfhydryl-hyaluronic acid hydrogel is formed by self-crosslinking reaction of disulfide bonds formed between sulfydryl and sulfhydryl-hyaluronic acid with the structural formula shown as formula (I), the grafting rate of cysteine in the sulfhydryl-hyaluronic acid is 30-60%,

in the technical scheme of the mandibular condylar process osteochondral repair scaffold material, the content of cross-linked sulfhydryl-hyaluronic acid in the cartilage surface layer is 5-30 mg/mL, and the content of type I collagen is 15-25 mg/mL; in the cartilage deep layer, the content of the cross-linked sulfhydryl-hyaluronic acid is 5-30 mg/mL, and the content of the type I collagen is 15-25 mg/mL. Furthermore, the content of the type I collagen in the cartilage surface layer and the cartilage deep layer is 0.5-3 times, preferably 2-3 times of the content of the cross-linked sulfhydryl-hyaluronic acid. The content of the cross-linked sulfhydryl-hyaluronic acid in the cartilage surface layer and the cartilage deep layer is preferably 6-20 mg/mL, more preferably 6-10 mg/mL, and the content of the type I collagen is preferably 15-20 mg/mL. Further, the contents of the cross-linked thiol-hyaluronic acid in the surface layer and the deep layer of cartilage are preferably the same, and the contents of the type I collagen in the surface layer and the deep layer of cartilage are also preferably the same.

In the technical scheme of the mandibular condyle osteochondral repair scaffold material, the porous biphasic calcium phosphate ceramic is composed of hydroxyapatite and beta-tricalcium phosphate, and the content of the hydroxyapatite in the porous biphasic calcium phosphate ceramic is 10 wt% -40 wt%, preferably 20 wt% -40 wt%. Further, the porosity of the porous biphasic calcium phosphate ceramic is 60-80%.

In the technical scheme of the mandibular condyle osteochondral repair scaffold material, the thickness ratio of the cartilage surface layer, the cartilage deep layer and the bone layer is 1 (1.5-2) to 2-3. In practical applications, the specific thicknesses of the cartilage surface layer, the cartilage deep layer, the bone layer and the shape of the bone layer are determined according to actual repair objects, wherein the thickness of the cartilage surface layer is usually 0.2-1 mm, and further the thickness of the cartilage surface layer can be 0.2-0.5 mm. The thickness of the cartilage deep layer refers to the thickness of the cartilage deep layer between the cartilage surface layer and the bone layer.

In the technical scheme of the mandibular condyle osteochondral repair scaffold material, the chondrocyte group in the deep cartilage layer is primary chondrocyte of allogeneic source or autologous source, and the mesenchymal stem cells in the surface cartilage layer are primary mesenchymal stem cells of allogeneic source or autologous source.

In the above technical solution of the mandibular condyle osteochondral repair scaffold material, the thiol-hyaluronic acid is obtained by cysteine modification based on hyaluronic acid, and the molecular weight of the hyaluronic acid based on the modification is preferably 0.1MDa to 4.0MDa, and more preferably, the molecular weight of the hyaluronic acid based on the modification is preferably 0.2MDa to 0.5 MDa.

In the technical scheme of the mandibular condyle osteochondral repair scaffold material, the grafting rate of cysteine in thiol-hyaluronic acid is preferably 30-40%.

The invention also provides a preparation method of the mandibular condyle osteochondral repair scaffold material, which comprises the following steps:

(1) placing the formed porous biphase calcium phosphate ceramic in a mould;

(2) uniformly mixing a sulfhydryl-hyaluronic acid solution and a type I collagen solution, adjusting the pH value to 7.3-7.5, then adding a chondrocyte suspension, uniformly mixing, injecting the obtained mixed solution containing cells above the molded porous biphase calcium phosphate ceramic, and standing to a gel state to form a cartilage deep layer;

(3) uniformly mixing a sulfhydryl-hyaluronic acid solution and a type I collagen solution, adjusting the pH value to 7.3-7.5, then adding a bone mesenchymal stem cell suspension, uniformly mixing, injecting the obtained mixed solution containing cells above the deep layer of cartilage, standing to a gel state to form a cartilage surface layer, and taking out from a mold to obtain a mandibular condylar osteochondral repair scaffold material;

and (3) performing the operations of the steps (2) to (3) under an ice bath condition, dissolving the sulfhydryl-hyaluronic acid by using an alpha-MEM culture medium to obtain sulfhydryl-hyaluronic acid, dissolving the type I collagen by using an acetic acid solution to obtain a type I collagen solution, and uniformly mixing the sulfhydryl-hyaluronic acid solution and the type I collagen solution to form a mixed solution, wherein the concentration of the cross-linked sulfhydryl-hyaluronic acid is 5-30 mg/mL, and the concentration of the type I collagen is 15-25 mg/mL.

In the technical scheme of the preparation method of the mandibular condylar process osteochondral repair scaffold material, the addition amount of the chondrocyte suspension in the step (2) is such that the content of the chondrocytes in the mixed solution containing the cells obtained in the step (2) at least reaches 1 x 10⁵cells/mL, for example, may be 1X 10⁶～5×10⁸cells/mL; the addition amount of the mesenchymal stem cell suspension in the step (3) is that the content of the mesenchymal stem cells in the mixed solution containing the cells obtained in the step (2) at least reaches 1 x 10⁵cells/mL, for example, may be 1X 10⁶～5×10⁸cells/mL。

In the steps (2) to (3) of the preparation method of the mandibular condylar process osteochondral repair scaffold material, in the mixed solution formed by uniformly mixing the thiol-hyaluronic acid solution and the type I collagen solution, the concentration of the cross-linked thiol-hyaluronic acid is preferably 6 to 20mg/mL, more preferably 6 to 10mg/mL, and the concentration of the type I collagen is preferably 15 to 20 mg/mL.

According to the technical scheme of the preparation method of the mandibular condylar process osteochondral repair scaffold material, if the mandibular condylar process osteochondral repair scaffold material prepared in the step (3) cannot be used immediately, the mandibular condylar process osteochondral repair scaffold material is placed in a cartilage induction culture medium to be cultured in vitro, the cartilage induction culture medium is replaced every 2-3 days during the in vitro culture period, and the mandibular condylar process osteochondral repair scaffold material is taken out when being used. Furthermore, the cartilage induction culture medium is obtained by adding transforming growth factor beta 1, non-essential amino acid, L-proline, dexamethasone, ascorbic acid 2-phosphate and penicillin-streptomycin mixed solution on the basis of the D-MEM culture medium. Further, 5% CO at 37 ℃₂Under the conditions of (1) performing in vitro culture.

According to the technical scheme of the preparation method of the mandibular condylar process osteochondral repair scaffold material, when the type I collagen solution is prepared, the concentration of the adopted acetic acid solution is 0.25-1.0 mol/L.

In the technical scheme of the preparation method of the mandibular condylar osteochondral repair scaffold material, the type I collagen can be extracted from bovine tendon.

Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects:

1. the invention provides a mandibular condyle osteochondral repair scaffold material, which consists of a cartilage surface layer, a cartilage deep layer and a bone layer, wherein the cartilage structure of the mandibular condyle is simulated by layer design, selection of high molecular materials of the cartilage surface layer and the cartilage deep layer and loading of different cells on the cartilage surface layer and the cartilage deep layer, the cartilage surface layer and the cartilage deep layer provide original cells for a defect part through loading cells, in addition, the layer repair of a target part is realized by designing the bone layer and the cartilage layer and loading different types of cells in the cartilage surface layer and the cartilage deep layer, and simultaneously, the crosslinked sulfhydryl-hyaluronic acid-I type collagen composite hydrogel and the porous biphase calcium phosphate ceramic can effectively promote the adhesion, proliferation and differentiation of the cells. The stent material provided by the invention can solve the problem that the cartilage defect of the mandibular condyle bone cannot be repaired in the current clinically applied method for treating the mandibular condyle cartilage damage, and provides a new possibility for the treatment of the mandibular condyle cartilage damage.

2. A cytotoxicity test shows that the mandibular condyle osteochondral repair scaffold material provided by the invention has no cytotoxicity. Through the detection of the cell morphology in the scaffold material and the layering state between the cartilage surface layer and the cartilage deep layer, the scaffold material is found that the bone marrow mesenchymal stem cells on the cartilage surface layer are fusiform and the cartilage cells on the cartilage deep layer are circular along with the increase of the culture time during in-vitro culture, and the bone marrow mesenchymal stem cells on the cartilage surface layer and the cartilage cells on the cartilage deep layer are obviously layered when the in-vitro culture is carried out to the 14 th day, so that the structure of the natural mandibular condyle cartilage layer is well simulated. The layered structure is inosculated with the natural structure of the natural mandible condylar bone cartilage, and the problem that the existing scaffold material for repairing the synovial articular cartilage is not suitable for repairing the mandible condylar bone cartilage due to the structural difference can be solved.

3. Through in vitro chondrogenic capacity test, it is further found that the expressions of cartilage related genes AGG, ACAN, SOX9, COL1A2, COL2A1 and COL10A1 in the cartilage superficial layer and cartilage deep layer loaded cells are increased along with the prolonging of culture time, and the bone marrow mesenchymal stem cells which are wrapped by the cartilage superficial layer material are highly expressed with COL1A2, and the cartilage cells which are loaded by the cartilage deep layer are highly expressed with COL2A1 and COL10A1, which is beneficial for the layering of the cartilage deep layer to correspond to a fibrous layer for repairing natural condylar cartilage, a proliferation layer (highly expressed type I collagen) and a mature layer, a hypertrophic layer (highly expressed type II and type X collagen), meanwhile, along with the increasing of in vitro culture time, the secretory GAG content of the cells loaded by the cartilage superficial layer and the cartilage deep layer is increased, wherein the cartilage cells loaded by the cartilage deep layer are secreted with more GAG, and histological staining shows that the bone marrow mesenchymal stem cells loaded by the superficial layer are in a fusiform, and the cartilage deep layer is in a round, the superficial layer of cartilage and the deep layer of cartilage have distinct layering. The above further shows that the scaffold material provided by the invention can well simulate the layered structure of the mandibular condylar osteochondral and has good capability of promoting chondrogenesis.

4. The in vivo bone regeneration capability repair experiment shows that the mandibular condyle cartilage repair scaffold material provided by the invention basically fills up the defect area when implanted in vivo for 6 weeks, and can well simulate the natural mandibular condyle cartilage structure when implanted in vivo for 24 weeks, thereby constructing the condyle cartilage surface layer and the cartilage deep layer in a layered manner. The scaffold material shows good cartilage repair capability after being implanted into a body for 6 weeks and 24 weeks, and respectively well simulates type I collagen on the surface layer of repaired cartilage and type II collagen in the deep layer of cartilage. In-vivo regeneration capability repair experiments further prove that the scaffold material provided by the invention has excellent repair capability on the mandibular condylar osteochondral, is very suitable for repairing the mandibular condylar osteochondral defect, and fills the blank that no scaffold material specially used for repairing the mandibular condylar osteochondral defect is reported in the prior art.

Drawings

FIG. 1 is the nuclear magnetic spectrum of HA-SH and HA in example 1.

FIG. 2 is a photograph showing the mixture of HA-SH, Col I and HA-SH/Col I in example 2 after forming a gel.

FIG. 3 is a photograph of the scaffold prepared in example 2, a photograph of the scaffold, and scanning electron micrographs of the porous BCP ceramic after in vitro culture of a mesenchymal stem cell group, wherein a is a photograph of the scaffold, b is a photograph of a longitudinal section of the scaffold, c is a photograph of the porous BCP ceramic after in vitro culture of a mesenchymal stem cell group, and d-e are photographs of cartilage and a deep cartilage surface layer of the scaffold, respectively.

FIG. 4 shows the results of the rheological mechanical properties, swelling rate, degradation rate and mechanical properties of the HA-SH hydrogel, the Col I hydrogel and the HA-SH/Col I composite hydrogel, wherein a is a rheological mechanical property diagram, b is a swelling rate diagram, c is a degradation rate diagram, d is a storage modulus curve, and e is a loss modulus curve.

FIG. 5 is a confocal scanning laser micrograph of the osteochondral repair scaffold of the mandibular condyle after live-dead staining of the bone layer, the cartilage deep layer and the cartilage surface layer after 1, 3 and 7 days of in vitro culture in example 4.

FIG. 6 is a confocal scanning laser micrograph of cytoskeleton after culturing chondrocytes and mesenchymal stem cells in the deep layer and the surface layer of cartilage of the scaffold material in example 5 for different periods of time.

FIG. 7 is a photograph showing ALP and ARS staining after culturing human mesenchymal stem cells for 14 days with shaped porous BCPs in example 6.

FIG. 8 is the expression of the cartilage related genes of the surface layer and the deep layer of cartilage of the scaffold material cultured in vitro for 7, 14 and 21 days in example 7, the glycosaminoglycan (GAG)/DNA values of the surface layer and the deep layer of cartilage of the scaffold material cultured in vitro for 7 and 21 days, and the histological staining results of the scaffold material cultured in vitro for 28 days, wherein, a is a PCR quantitative detection analysis chart obtained by culturing the deep layer of cartilage, chondrocytes in the surface layer of cartilage and mesenchymal stem cells for different time periods of the scaffold material, b is a GAGs quantitative detection analysis chart, in the a and b, a dark bar chart represents the surface layer of cartilage, a light bar chart represents the deep layer of cartilage, and c is the histological staining results of the section of the cartilage surface layer sample and the deep layer of cartilage of the scaffold material after 28 days of culture.

Fig. 9 shows the results of the bone regeneration ability test of the scaffold material in example 8 in vivo after 6 weeks and 24 weeks of implantation, wherein, a is the gross view and Micro CT analysis of the repair of cartilage defect of rabbit mandibular condyle bone cartilage of blank control group, no-load scaffold group and experimental group, b is the histological staining result of the section of the defect, and c is the immunohistochemical staining result of the section of the defect.

Detailed Description

The mandibular condyle osteochondral repair scaffold material and the preparation method thereof provided by the present invention are further described by the following examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make certain insubstantial modifications and adaptations of the present invention based on the above disclosure and still fall within the scope of the present invention.

Example 1

Thiol-hyaluronic acid (HA-SH) was prepared in this example by the following steps:

(1) dissolving 2g of sodium Hyaluronate (HA) with the molecular weight of 0.3MDa in deionized water, adding 10mmol of N-hydroxysuccinimide (NHS), stirring in the deionized water at room temperature until the sodium hyaluronate is completely dissolved, then adding 25mmol of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC & HCl) to fully dissolve, adjusting the pH value of the mixed solution to 4.75-5.0 by using 1mol/L NaOH solution and 1mol/L HCl solution, reacting at room temperature for 2h, then adding 25mmol of cysteine hydrochloride (CSH & HCl), reacting at room temperature for 24h, then transferring the solution into a dialysis bag with the molecular weight cutoff of 8000-14000KDa, dialyzing at room temperature for 72h, and finally freeze-drying the dialyzate to obtain solid HA-SH.

HA and HA-SH were detected by NMR spectroscopy, and the NMR spectra are shown in FIG. 1. from FIG. 1, it can be seen that new peaks of HA-SH at 2.45ppm and 2.63ppm compared to HA, which demonstrates the successful synthesis of HA-SH.

The degree of thiol substitution in HA-SH is determined by the modified Ellman method, and the grafting rate of cysteine in HA-SH is about 30%.

The improved Ellman method is adopted to measure the substitution degree of the mercapto in HA-SH, and the molecular weight of HA is changed by changing the molar ratio of EDCI to CSA & HCl, so that the substitution degree of the mercapto in HA-SH can be changed, namely the grafting rate of cysteine in HA-SH is changed, the substitution degree of the mercapto is gradually increased along with the increase of the molar ratio of EDCI to CSA & HCl, and the substitution degree of the mercapto is lower as the molecular weight of HA is higher, and the grafting rate of the cysteine in HA-SH can be adjusted to be within the range of 30% -60% by adjusting the molar ratio of EDCI to CSA & HCl and the molecular weight of HA.

Example 2

In this embodiment, the preparation of the mandibular condyle osteochondral repair scaffold material includes the following steps:

(1) the method comprises the steps of placing formed porous biphase calcium phosphate ceramic (porous BCP ceramic) in a cylindrical die with the inner diameter of 8mm and the height of 3mm and one closed end, wherein the porous BCP ceramic consists of hydroxyapatite and beta-tricalcium phosphate, the mass ratio of the hydroxyapatite to the beta-tricalcium phosphate is 3:7, the porosity is about 75%, the formed porous BCP ceramic is a cylindrical block with the diameter of 8mm and the height of 1.5mm, and the formed porous BCP ceramic is placed at the closed end of the cylindrical die in a mode of being coaxial with the die.

(2) Obtaining primary culture bone marrow mesenchymal stem cells and chondrocytes: selecting 5-day-old suckling rabbits, injecting sodium pentobarbital for killing, centrifuging and enriching extracts of two-side femoral marrow cavities to obtain mesenchymal stem cells, and culturing by using an alpha-MEM culture medium. Cartilage tissue at knee joint part was minced, trypsinized for 30min, treated with 2.5mg/mL collagenase II at 37 ℃ for 4h, and then the suspension was centrifuged and enriched with a 70mm cell filter to obtain chondrocytes, which were cultured in alpha-MEM medium.

(3) Dissolving the HA-SH prepared in example 1 in an alpha-MEM medium to obtain an HA-SH solution with the concentration of 25 mg/mL; type I collagen (Col I) was dissolved in 0.25mol/L acetic acid solution in an ice bath to obtain a Col I solution at a concentration of 25 mg/mL.

Uniformly mixing an HA-SH solution and a Col I solution in a volume ratio of 3:7 in an ice bath, adjusting the pH value to 7.4 by using a 1mol/L NaOH solution to obtain an HA-SH/Col I mixed solution, adding a chondrocyte suspension, and uniformly mixing to obtain a mixed solution containing cells, wherein the chondrocytesIs 5X 10⁶cells/mL. Injecting the mixed solution containing the cells above the molded porous BCP ceramic in the mold, and standing to a gel state to form a cartilage deep layer which is about 1mm higher than the upper surface of the molded BCP ceramic.

(4) Dissolving the HA-SH prepared in example 1 in an alpha-MEM medium to obtain an HA-SH solution with the concentration of 25 mg/mL; dissolving Col I with 0.25mol/L acetic acid solution under ice bath to obtain Col I solution with concentration of 25 mg/mL.

Uniformly mixing an HA-SH solution and a Col I solution in a volume ratio of 3:7 in an ice bath, adjusting the pH value to 7.4 by using a 1mol/L NaOH solution, then adding a mesenchymal stem cell suspension and uniformly mixing to obtain a mixed solution containing cells, wherein the content of the mesenchymal stem cells is 5 multiplied by 10⁶cells/mL. Injecting the mixed solution containing the cells above the cartilage deep layer of the mould, standing to a gel state to form a cartilage surface layer which is about 0.5mm higher than the upper surface of the cartilage deep layer, and taking out the cartilage surface layer from the mould to obtain the mandibular condyle osteochondral cartilage repair scaffold material.

(5) Since the mandibular condyle osteochondral repair scaffold prepared in step (4) is not used immediately, the mandibular condyle osteochondral repair scaffold is immersed in a cartilage induction medium at 37 ℃ with 5% CO₂The cartilage induction culture medium is replaced every 2-3 days during the in vitro culture period, and the cartilage induction culture medium is taken out when the cartilage induction culture medium is used.

The cartilage induction culture medium is obtained by adding a transforming growth factor beta 1, an unnecessary amino acid, L-proline, dexamethasone, ascorbic acid 2-phosphate and a penicillin-streptomycin mixed solution on the basis of a D-MEM culture medium, wherein the adding amount of the transforming growth factor beta 1 is 10ng/mL, the adding amount of the unnecessary amino acid is 0.1mmol/L, L-proline is 40 mu g/mL, the adding amount of the dexamethasone is 100nmol/L, the adding amount of the ascorbic acid 2-phosphate is 91.5 mu g/mL, and the adding amount of the penicillin-streptomycin mixed solution is 1%.

The photographs of the above-mentioned HA-SH, Col I and HA-SH/Col I mixed solutions after forming a gel are respectively shown in the three panels (A), (B) and (C) of FIG. 2, the HA-SH hydrogel is transparent and colorless, the Col I hydrogel is opaque and milk white, and the HA-SH/Col I hydrogel is translucent and milk white.

The mandible condylar osteochondral repair scaffold material prepared in the embodiment is characterized by using a camera and a scanning electron microscope, and meanwhile, bone marrow mesenchymal stem cells are added into the single porous BCP ceramic for in vitro culture, and the scanning electron microscope is adopted for characterization after the culture is carried out for a period of time. The side of the stent material was photographed using a camera, and as a result, as shown in a of fig. 3, it was confirmed that the layers of the stent material were closely connected and structurally stabilized. The longitudinal section of the scaffold material was observed using a scanning electron microscope, and the result is shown in b of fig. 3, from which it can also be seen that the bone layer of the scaffold material is closely connected to the cartilage layer. The surface layer of the porous BCP ceramic added with the mesenchymal stem cells for in vitro culture is observed by using a scanning electron microscope, and the result is shown in a c picture of figure 3, so that the mesenchymal stem cells can be seen to be adhered to the surface layer of the porous BCP ceramic to grow, and the osteogenesis effect of the porous BCP ceramic is facilitated. The deep layer of cartilage in the bone layer of the scaffold material was observed by scanning electron microscopy, and the results are shown in d of fig. 3, which shows that chondrocytes proliferate between materials and secrete a large amount of extracellular matrix, which is favorable for the formation of new cartilage. The surface layer of cartilage in the bone layer of the scaffold material was observed by scanning electron microscopy, and as a result, as shown in fig. 3 e, it was seen that mesenchymal stem cells proliferated well between the materials.

Comparative example 1

In this example, a scaffold material not loaded with any cells was prepared by the following steps:

(1) same as in step (1) of example 2.

(2) Dissolving the HA-SH prepared in example 1 in an alpha-MEM medium to obtain an HA-SH solution with the concentration of 25 mg/mL; dissolving Col I with 0.25mol/L acetic acid solution under ice bath to obtain Col I solution with concentration of 25 mg/mL.

Uniformly mixing the HA-SH solution and the Col I solution in a volume ratio of 3:7 in an ice bath, adjusting the pH value to 7.4 by using 1mol/L NaOH solution, then injecting the mixture above a molded porous BCP ceramic in a mold, and standing the mixture until the mixture is in a gel state to form a cartilage deep layer which is about 1mm higher than the upper surface of the molded BCP ceramic.

Uniformly mixing the HA-SH solution and the Col I solution in a volume ratio of 3:7 in an ice bath, adjusting the pH value to 7.4 by using 1mol/L NaOH solution, then injecting the mixture to the upper part of the deep layer of the cartilage of a mould, and standing the mixture to a gel state to form a cartilage surface layer, wherein the cartilage surface layer is about 0.5mm higher than the upper surface of the deep layer of the cartilage, so as to obtain the scaffold material without any cells.

Example 3

In this example, the physicochemical properties of a crosslinked thiol-hyaluronic acid and type I collagen composite hydrogel (HA-SH/Col I) were tested, while comparing HA-SH hydrogel alone and Col I hydrogel as controls.

Dissolving the HA-SH prepared in example 1 in an alpha-MEM medium to obtain an HA-SH solution with the concentration of 25 mg/mL; dissolving Col I with 0.25mol/L acetic acid solution under ice bath to obtain Col I solution with concentration of 25 mg/mL. Respectively sucking the HA-SH solution and the Col I solution by using a syringe, uniformly mixing the HA-SH solution and the Col I solution according to the volume ratio of 3:7, adjusting the pH to 7.4 by using a NaOH solution, and standing for a period of time at 37 ℃ to form the composite hydrogel HA-SH/Col I. The HA-SH prepared in example 1 was dissolved in an α -MEM medium to obtain an HA-SH solution having a concentration of 25mg/mL, the pH was adjusted to 7.4 with an NaOH solution, and the solution was allowed to stand at 37 ℃ for a while to form an HA-SH hydrogel. Dissolving Col I in 0.25mol/L acetic acid solution in ice bath to obtain Col I solution with concentration of 25mg/mL, adjusting pH to 7.4 with NaOH solution, and standing at 37 deg.C for a while to obtain Col I hydrogel.

Gel forming time is measured by a rheometer, a graph a in FIG. 4 is a rheological mechanical property graph of the HA-SH/Col I composite hydrogel, in the graph, the junction of two curves represents material gel forming, and the gel forming time of the HA-SH/Col I composite hydrogel is about 1 minute. The HA-SH hydrogel, the Col I hydrogel and the HA-SH/Col I composite hydrogel are freeze-dried, then are immersed in the PBS solution, the weight of the solution is measured at regular intervals, and the swelling characteristics of the solution are measured, so that the result is shown in a b diagram of figure 4, the swelling rate of the HA-SH/Col I composite hydrogel is between that of the HA-SH hydrogel and that of the Col I hydrogel, and the requirements of experimental design are met. And (3) freeze-drying the HA-SH hydrogel, the Col I hydrogel and the HA-SH/Col I composite hydrogel, then placing the dried hydrogel in a dithiothreitol solution of 1mmol/L, placing the dithiothreitol solution in a constant-temperature shaking table to oscillate at 37 ℃ and a rotating speed of 90rpm, taking out hydrogel samples at intervals, freeze-drying and weighing the samples, and measuring the dissolution characteristics of the samples, wherein the result is shown in a c diagram of figure 4, and the dissolution rate of the HA-SH/Col I composite hydrogel is between that of the HA-SH hydrogel and that of the Col I hydrogel, so that the requirements of experimental design are met. The storage modulus (G ') and the loss modulus (G') of the HA-SH hydrogel, the Col I hydrogel and the HA-SH/Col I composite hydrogel are measured by using a dynamic mechanical analyzer, and the results are shown in a d-e diagram of fig. 4, wherein the storage modulus (G ') and the loss modulus (G') of the HA-SH/Col I composite hydrogel are both between the HA-SH gel and the Col I gel, and the HA-SH/Col I composite hydrogel HAs good mechanical properties.

Example 4

In this example, cytotoxicity test was performed on the mandibular condyle osteochondral repair scaffold material.

The living death condition of cells carried in the bone layer, the cartilage deep layer and the cartilage surface layer of the mandibular condyle osteochondral repair scaffold material cultured in vitro in the cartilage induction culture medium for 1, 3 and 7 days in example 2 is detected by using a living death staining kit. The laser confocal scanning microscope photograph of the mandibular condyle osteochondral repair scaffold cultured in vitro for 1, 3 and 7 days after live-dead staining of the bone layer, cartilage deep layer and cartilage surface layer is shown in fig. 5. Wherein, green represents the survival bone marrow mesenchymal stem cells (bone layer and cartilage surface layer) and cartilage cells (cartilage deep layer), red represents the death bone marrow mesenchymal stem cells and cartilage cells, and BCP represents the bone layer. Substantially all of the green cells in fig. 5, rare red cells, indicate high survival of the cells in the scaffold material, and the scaffold material is not cytotoxic.

Example 5

In this example, the cellular morphology and the layering state between the cartilage surface layer and the cartilage deep layer in the mandibular condyle osteochondral repair scaffold material were examined.

Using phalloidin and DAPI to mark example 2, cytoskeleton and nucleus of cells carried in the cartilage deep layer and cartilage surface layer of mandibular condyle osteochondral repair scaffold material were cultured in vitro using cartilage induction medium for 1, 14 and 21 days, respectively, and cell morphology in the cartilage deep layer and cartilage surface layer was observed at different time points. As shown in fig. 6, it is understood from the graph a in fig. 6 that the mesenchymal stem cells in the cartilage surface layer became spindle-shaped and the chondrocytes in the cartilage deep layer appeared circular as the culture time increased. As can be seen from the b-picture in fig. 6, after the culture time was increased to day 14, it was observed that bone marrow mesenchymal stem cells in the cartilage surface layer and chondrocytes in the cartilage deep layer were significantly layered, and the structure of the cartilage layer of the natural mandibular condylar process bone was well simulated.

Example 6

In this example, the bone layer porous BCP material used in example 2 was separately cultured in vitro, and the in vitro osteogenic differentiation ability was verified.

Bone marrow mesenchymal stem cells were seeded on the molded porous BCP (the mass ratio of hydroxyapatite to beta-tricalcium phosphate was 3:7, the porosity was about 75%, and the molded porous BCP ceramic was a cylindrical block having a diameter of 8mm and a height of 1.5 mm) used in example 2 as an experimental group in an amount of 1X 10 for each molded porous BCP⁴cells). Control group was a molded porous BCP that was not inoculated with any cells. Then, the experimental group and the control group were cultured in vitro using an osteogenic induction medium for 14 days. Then alkaline phosphatase expression (ALP) staining and Alizarin Red (ARS) staining are used for detecting the osteogenic differentiation inducing capacity of the bone layer. The osteogenesis induction culture medium is obtained by adding dexamethasone, beta-glycerophosphate, ascorbic acid and a penicillin-streptomycin mixed solution on the basis of an alpha-MEM culture medium, wherein the addition amount of the dexamethasone is 100nmol/L, the addition amount of the beta-glycerophosphate is 10mmol/L, the addition amount of the ascorbic acid is 0.05mmol/L, and the addition amount of the penicillin-streptomycin mixed solution is 1%.

As shown in fig. 7, it can be seen from fig. 7 that the molded porous BCP ceramic of the experimental group showed deeper blue (ALP) and red (ARS) staining than the control group, demonstrating that the mesenchymal stem cells on the porous BCP were osteogenically differentiated, indicating that the bone layer material had good osteogenic induction properties.

Example 7

In this example, the in vitro chondrogenic capacity of the cartilage surface layer and the cartilage deep layer of the mandibular condyle osteochondral repair scaffold material was tested.

Scaffold materials cultured in vitro for 7, 14 and 21 days using the cartilage induction medium of example 2 were examined for the expression of cartilage-related genes in the surface layer and deep layer of cartilage using Polymerase Chain Reaction (PCR), scaffold materials cultured in vitro for 7 and 21 days were examined for glycosaminoglycan (GAG)/DNA values in the surface layer and deep layer of cartilage, and scaffold materials cultured in vitro for 28 days were histologically stained. The results are shown in fig. 8, wherein a is a PCR quantitative detection analysis chart obtained by culturing the chondrocytes in the cartilage deep layer and cartilage surface layer of the scaffold material at different times, b is a GAGs quantitative detection analysis chart, c is a histological staining result of a section of a specimen in the cartilage deep layer and cartilage surface layer of the scaffold material after 28 days of culture, and the results are hematoxylin-eosin staining result, safranin staining result and toluidine blue staining result in sequence from left to right.

As shown in FIG. 8 a, the expressions of the cartilage-related genes AGG, ACAN, SOX9, COL1A2, COL2A1 and COL10A1 in the cells carried in the surface layer and deep layer of cartilage were increased with the lapse of culture time. The HA-SH/Col I composite hydrogel is shown to have good chondrogenic capacity for the cells entrapped therein. And bone marrow mesenchymal stem cells coated by the cartilage surface layer material are high-expression COL1A2, and chondrocytes coated by the cartilage deep layer are high-expression COL2A1 and COL10A1, which are beneficial to layered corresponding repair of fibrous layer, proliferation layer (high-expression type I collagen), mature layer and hypertrophic layer (high-expression type II collagen and type X collagen) of natural condylar cartilage. From the b-diagram of fig. 8, with the increase of the culture time, the content of GAG secreted by the cells encapsulated by the surface layer and the deep layer of cartilage increases, wherein more GAG is secreted by the chondrocytes encapsulated by the deep layer of cartilage. As can be seen from the c-diagram in fig. 8, histological staining revealed that the cartilage surface layer was clearly layered with the cartilage deep layer, the bone marrow mesenchymal stem cells carried in the cartilage surface layer became spindle-shaped, and the chondrocytes carried in the cartilage deep layer appeared fat round-like.

Example 8

In this example, the in vivo bone regeneration capacity of the mandibular condyle osteochondral repair scaffold material was tested, the mandibular condyle osteochondral repair scaffold material prepared in example 2 was used as the experimental group, the scaffold material prepared in comparative example 1 without any cell loading was used as the no-load scaffold group, and no material was implanted into the body in the blank control group.

A mandible condylar process osteochondral defect model is established in a New Zealand white rabbit body, two scaffold materials loaded with cells and unloaded with cells are respectively implanted into the defect to form an experimental group and an idle-load scaffold group, and no material is implanted into a blank control group. The samples were taken 6 weeks and 24 weeks after implantation, and the samples were photographed, analyzed by Micro CT, sectioned and histologically stained for hard tissue, and compared for bone regeneration in vivo, with the results shown in fig. 9. Fig. 9 a is a general view and Micro CT analysis results of repair of cartilage defects of rabbit mandibular condyle bone cartilage of a blank control group, an unloaded stent group and an experimental group, b is a histological staining result of a section of the defect, wherein the upper view is a hematoxylin-eosin staining result, the lower view is a safranin/fast green staining result, and c is an immunohistochemical staining result of the section of the defect.

As can be seen from the a-graph of fig. 9, the scaffold material of the experimental group showed good repair appearance and bone repair ability at 6 weeks and 24 weeks after implantation in vivo, compared to the blank control group and the unloaded scaffold group. As can be seen from the b-diagram of fig. 9, compared to the blank control group and the empty stent group, the stent material of the experimental group shows good cartilage repair ability after being implanted for 6 weeks and 24 weeks, and basically fills up the defect area after being implanted for 6 weeks, and the stent material of the experimental group well simulates the natural mandibular condylar cartilage structure after being implanted for 24 weeks, thereby hierarchically constructing the superficial condylar cartilage layer and the deep cartilage layer. As can be seen from the c-graph of fig. 9, the scaffold material of the experimental group showed good cartilage repair ability at 6 weeks and 24 weeks after implantation, compared to the blank control group and the empty scaffold group, and well stimulated the type I collagen in the surface layer of the repaired cartilage and the type II collagen in the deep layer of the cartilage, respectively.

Example 9

(1) HA-SH with the cysteine grafting rate of about 35-60% is prepared by controlling the molar ratio of EDCI to CSA & HCl according to the method of example 1 by using sodium hyaluronate with the molecular weight of 0.5 MDa.

(2) The method comprises the steps of placing formed porous BCP ceramic in a cylindrical die with the inner diameter of 8mm and the height of 4.5mm and one closed end, wherein the porous BCP ceramic consists of hydroxyapatite and beta-tricalcium phosphate, the mass ratio of the hydroxyapatite to the beta-tricalcium phosphate is 4:6, the porosity is about 80%, the formed porous BCP ceramic is a cylindrical block with the diameter of 8mm and the height of 2mm, and the formed porous BCP ceramic is placed at the closed end of the cylindrical die in a coaxial mode with the die.

(3) Same as in step (2) of example 2.

(4) Dissolving the HA-SH prepared in the step (1) by using an alpha-MEM culture medium to obtain an HA-SH solution; dissolving Col I with 0.25mol/L acetic acid solution under ice bath to obtain Col I solution. Mixing HA-SH solution and Col I solution at a proper volume ratio under ice bath, adjusting pH to 7.4 with 1mol/L NaOH solution to obtain HA-SH/Col I mixed solution, adding chondrocyte suspension, and mixing to obtain mixed solution containing cells, wherein the content of chondrocytes is 1 × 10⁶cells/mL. Injecting the mixed solution containing the cells above the molded porous BCP ceramic in the mold, and standing to a gel state to form a cartilage deep layer which is about 1.5mm higher than the upper surface of the molded BCP ceramic.

(5) Dissolving the HA-SH prepared in the step (1) by using an alpha-MEM culture medium to obtain an HA-SH solution; dissolving Col I with 0.25mol/L acetic acid solution under ice bath to obtain Col I solution. Mixing HA-SH solution and Col I solution uniformly in a proper volume ratio under ice bath, adjusting the pH value to 7.4 by using 1mol/L NaOH solution to obtain HA-SH/Col I mixed solution, wherein the concentration of HA-SH in the HA-SH/Col I mixed solution is 10mg/mL, the concentration of Col I in the HA-SH/Col I mixed solution is 20mg/mL, then adding bone marrow mesenchymal stem cell suspension and uniformly mixing to obtain mixed solution containing cells, wherein the content of bone marrow mesenchymal stem cells is 1 multiplied by 10⁶cells/mL. Injecting the mixture containing cells into the mold above the cartilage deep layer, standing to gel state to form cartilage surfaceAnd (3) taking the cartilage out of the mould, wherein the surface layer of the cartilage is higher than the upper surface of the cartilage deep layer by about 1mm, and obtaining the mandibular condyle osteochondral repair scaffold material.

(6) Same as in step (5) of example 2.

Example 10

(1) HA-SH with cysteine grafting rate of about 40% was prepared by controlling the molar ratio of EDCI to CSA & HCl according to the method of example 1 using sodium hyaluronate with molecular weight of 0.2 MDa.

(2) Same as in step (1) of example 2.

(3) Same as in step (2) of example 2.

(4) Dissolving the HA-SH prepared in the step (1) by using an alpha-MEM culture medium to obtain an HA-SH solution; dissolving Col I with 0.25mol/L acetic acid solution under ice bath to obtain Col I solution. Mixing HA-SH solution and Col I solution at a proper volume ratio under ice bath, adjusting pH to 7.4 with 1mol/L NaOH solution to obtain HA-SH/Col I mixed solution, adding chondrocyte suspension, and mixing to obtain mixed solution containing cells, wherein the content of chondrocytes is 1 × 10⁸cells/mL. Injecting the mixed solution containing the cells above the molded porous BCP ceramic in the mold, and standing to a gel state to form a cartilage deep layer which is about 1mm higher than the upper surface of the molded BCP ceramic.

(5) Dissolving the HA-SH prepared in the step (1) by using an alpha-MEM culture medium to obtain an HA-SH solution; dissolving Col I with 0.25mol/L acetic acid solution under ice bath to obtain Col I solution. Mixing HA-SH solution and Col I solution uniformly in a proper volume ratio under ice bath, adjusting the pH value to 7.4 by using 1mol/L NaOH solution to obtain HA-SH/Col I mixed solution, wherein the concentration of HA-SH in the HA-SH/Col I mixed solution is 6mg/mL, the concentration of Col I in the HA-SH/Col I mixed solution is 15mg/mL, then adding bone marrow mesenchymal stem cell suspension and uniformly mixing to obtain mixed solution containing cells, wherein the content of bone marrow mesenchymal stem cells is 1 multiplied by 10⁸cells/mL. Injecting the mixed solution containing cells into the mold above the deep layer of cartilageAnd standing to a gel state to form a cartilage surface layer which is higher than the upper surface of the cartilage deep layer by about 1.5mm, and taking out the cartilage surface layer from the die to obtain the mandibular condyle osteochondral repair scaffold material.

(6) Same as in step (5) of example 2.

Claims

1. The mandibular condyle osteochondral repair scaffold material is characterized by comprising a cartilage surface layer, a cartilage deep layer and a bone layer, wherein the cartilage surface layer comprises a composite hydrogel of cross-linked sulfhydryl-hyaluronic acid and type I collagen and mesenchymal stem cells distributed in the composite hydrogel, the cartilage deep layer comprises a composite hydrogel of cross-linked sulfhydryl-hyaluronic acid and type I collagen and chondrocytes distributed in the composite hydrogel, the bone layer is porous biphase calcium phosphate ceramic, the cartilage deep layer is positioned between the cartilage surface layer and the bone layer and in a porous structure of the bone layer to connect the cartilage surface layer and the bone layer into a whole, the cross-linked sulfhydryl-hyaluronic acid hydrogel is formed by self-crosslinking reaction of sulfhydryl-hyaluronic acid shown in a structural formula (I) through disulfide bond formation between sulfhydryl groups, the grafting rate of cysteine in the sulfhydryl-hyaluronic acid is 30-60 percent,

2. the mandibular condyle osteochondral repair scaffold material according to claim 1, wherein the content of cross-linked thiol-hyaluronic acid in the cartilage surface layer is 5-30 mg/mL, and the content of type I collagen is 15-25 mg/mL; in the cartilage deep layer, the content of the cross-linked sulfhydryl-hyaluronic acid is 5-30 mg/mL, and the content of the type I collagen is 15-25 mg/mL.

3. The mandibular condyle osteochondral repair scaffold material of claim 1, wherein said porous biphasic calcium phosphate ceramic is composed of hydroxyapatite and β -tricalcium phosphate, and the content of hydroxyapatite in the porous biphasic calcium phosphate ceramic is 10 wt% to 40 wt%.

4. The mandibular condyle osteochondral repair scaffold material of claim 3, wherein said porous biphasic calcium phosphate ceramic has a porosity of 60% to 80%.

5. The mandibular condyle osteochondral repair scaffold material of any one of claims 1 to 4, wherein the thickness ratio of the cartilage surface layer, the cartilage deep layer and the bone layer is 1 (1.5-2) to (2-3).

6. The mandibular condyle osteochondral repair scaffold material of claim 5, wherein the thickness of said cartilage surface layer is 0.2-1 mm.

7. The mandibular condyle osteochondral repair scaffold material of any one of claims 1 to 4, wherein the group of chondrocytes in the deep layer of cartilage are primary chondrocytes of xenogenic or autologous origin, and the mesenchymal stem cells in the superficial layer of cartilage are primary mesenchymal stem cells of xenogenic or autologous origin.

8. The method for preparing the mandibular condyle osteochondral repair scaffold material of any one of claims 1 to 7, comprising the steps of:

(1) placing the formed porous biphase calcium phosphate ceramic in a mould;

and (3) performing the operations of the steps (2) to (3) under an ice bath condition, dissolving the sulfhydryl-hyaluronic acid by using an alpha-MEM culture medium to obtain sulfhydryl-hyaluronic acid, dissolving the type I collagen by using an acetic acid solution in an ice bath to obtain a type I collagen solution, and uniformly mixing the sulfhydryl-hyaluronic acid solution and the type I collagen solution to form a mixed solution, wherein the concentration of the cross-linked sulfhydryl-hyaluronic acid is 5-30 mg/mL, and the concentration of the type I collagen is 15-25 mg/mL.

9. The method for preparing a mandibular condylar bone cartilage repair scaffold according to claim 8, wherein the chondrocyte suspension of step (2) is added in such an amount that the chondrocyte content in the cell-containing mixture obtained in step (2) is at least 1 x 10⁵cells/mL; the addition amount of the mesenchymal stem cell suspension in the step (3) is that the content of the mesenchymal stem cells in the mixed solution containing the cells obtained in the step (2) at least reaches 1 x 10⁵cells/mL。

10. The method for preparing a mandibular condylar osteochondral repair scaffold according to claim 8 or 9, wherein if the mandibular condylar osteochondral repair scaffold prepared in step (3) cannot be used immediately, the mandibular condylar osteochondral repair scaffold is placed in a cartilage induction medium for in vitro culture, and the cartilage induction medium is replaced every 2 to 3 days during the in vitro culture.