Application of ultrasonic response piezoelectric collagen scaffold

Abstract

The invention relates to the field of bone tissue engineering and the field of biomedical materials, in particular to application of an ultrasonic response piezoelectric collagen scaffold. According to the invention, full-thickness pigskin is used as a raw material, hair and subcutaneous adipose tissue are removed, and then decellularization is carried out so as to obtain the membrane-like scaffold. The membrane-like scaffold retains a complete three-stage structure of natural collagen, which is a source of the natural collagen having a piezoelectric effect; meanwhile, a piezoelectric signal detection result shows that periodic piezoelectric signals appear on the membrane-shaped stent, and then the collagen stent provided by the invention is proved to have an intrinsic piezoelectric effect. Experimental results show that mesenchymal stem cells can be attached to the surface of the collagen scaffold and can promote repair and regeneration of bone tissues, and in addition, rat subcutaneous ectopic bone regeneration experiments prove that the collagen scaffold provided by the invention has a good bone regeneration promoting effect under ultrasonic stimulation.

Description

Technical Field

[0001] The present invention relates to the field of bone tissue engineering and biomedical materials, and in particular to the application of an ultrasonically responsive piezoelectric collagen scaffold. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Bone defects caused by various diseases such as tumors, osteoporosis and infection are one of the main causes of disability, seriously affecting the quality of life of patients and bringing huge socioeconomic burden. Traditional methods for treating bone defects include autologous or allogeneic bone transplantation and growth factor loading, the disadvantages of which include limited sources, secondary injury and infection risks, and immune rejection. Material-mediated physical signals have been widely used in tissue engineering. They have the advantages of high safety, convenient application and low cost, and are therefore regarded as substitutes for growth factors. Among them, piezoelectric signals have shown great potential in regulating cell differentiation and tissue regeneration. For example, piezoelectric bone cement made of polymethyl methacrylate and BaTiO3 was used to promote osteogenic differentiation of stem cells, while hydroxyapatite (HAp) / polyvinylidene fluoride-trifluoroethylene copolymer P(VDF-TrFE) composite scaffolds promoted bone regeneration through the piezoelectric effect of P(VDF-TrFE) and the inherent osteogenic ability of HAp. Despite the potential for bone regeneration, the low biodegradability and biosafety limit the in vivo application of these materials. On the other hand, degradable piezoelectric polymers represented by poly-L-lactic acid (PLLA) can also be used for tissue regeneration. However, the degradation product of PLLA, lactic acid, is a relatively strong acid that may cause chronic inflammatory responses, thereby delaying tissue repair. In addition, processing cost and time are also important considerations in practical applications. Therefore, it remains a challenge to construct natural piezoelectric materials with good biocompatibility, biodegradability, no secondary inflammation, and high cost-effectiveness to enhance bone regeneration.

[0004] Collagen is the most abundant protein in the human body. Due to its good biocompatibility, it has become one of the FDA-approved natural biomaterials that can be used for tissue regeneration. Natural collagen has a hierarchical self-assembly structure. This hierarchical structure includes primary structure (polypeptide chain), secondary structure (α-helical chain) and tertiary structure (three-dimensional helical fiber). The complete tertiary structure gives collagen a good piezoelectric effect. However, during the preparation process, natural collagen scaffolds usually lose their inherent piezoelectric effect due to the destruction of the tertiary structure. Collagen scaffolds that have lost their piezoelectric effect cannot promote osteogenic differentiation, so growth factors are needed to assist in accelerating bone regeneration. At present, there is no research that directly uses the intrinsic piezoelectric effect of natural collagen for bone repair. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides an application of an ultrasonically responsive piezoelectric collagen scaffold.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] An application of an ultrasound-responsive piezoelectric collagen scaffold directly or indirectly used as a scaffold material for bone defect repair or bone tissue engineering;

[0008] The ultrasonically responsive piezoelectric collagen scaffold is made of full-thickness animal connective tissue as raw material, and is a membrane scaffold obtained after hair and subcutaneous fat tissue are removed and decellularized.

[0009] In one or more embodiments, the power of ultrasound is 2 to 3 W / cm 2 , preferably 2.15W / cm 2 .

[0010] In one or more embodiments, the animal is selected from any one of pigs, cattle, sheep, horses or deer, preferably pigs.

[0011] In one or more embodiments, the connective tissue is skin.

[0012] In one or more embodiments, a method for preparing an ultrasound-responsive piezoelectric collagen scaffold comprises:

[0013] (1) Using full-thickness animal connective tissue as raw material, removing hair and subcutaneous fat tissue, and obtaining a first intermediate;

[0014] (2) decellularizing the first intermediate product using sodium hydroxide and sodium dodecyl sulfate (SDS) to obtain a second intermediate product;

[0015] (3) decellularizing the second intermediate product using sodium dodecyl sulfate (SDS) to obtain a third intermediate product;

[0016] (4) treating the third intermediate product with pancreatic enzyme to obtain a fourth intermediate product;

[0017] (5) decellularizing the fourth intermediate product again using sodium dodecyl sulfate (SDS) to obtain a fifth intermediate product;

[0018] (6) Treating the fifth intermediate product with sodium chloride to obtain the ultrasonically responsive piezoelectric collagen scaffold.

[0019] Preferably, in step (2), the concentration of sodium hydroxide in sodium hydroxide and sodium dodecyl sulfate is 1.8 to 2.2 g / L, preferably 2.0 g / L;

[0020] The concentration of sodium dodecyl sulfate is 0.75 to 0.85 g / L, preferably 0.8 g / L.

[0021] Preferably, in step (2), the decellularization treatment time is 11.5 to 12.5 hours, preferably 12 hours.

[0022] Preferably, in step (4), the concentration of pancreatic enzyme is 2.8-3.2 g / L, preferably 3 g / L.

[0023] Preferably, in step (4), the pancreatin treatment time is 11.5 to 12.5 hours, preferably 12 hours.

[0024] Preferably, in step (5), the concentration of sodium dodecyl sulfate is 2.3-2.5 g / L, preferably 2.4 g / L.

[0025] Preferably, in step (5), the decellularization treatment time is 1.5 to 2.5 hours, preferably 2 hours.

[0026] Preferably, in step (6), the mass fraction of sodium chloride is 5.5-7%, preferably 6%.

[0027] Preferably, in step (6), the time for treatment with sodium chloride is 5.5 to 7 hours, preferably 6 hours.

[0028] The beneficial effects of the present invention are:

[0029] (1) In the present invention, full-thickness pig skin is used as raw material, and the hair and subcutaneous fat tissue are removed to obtain a membrane scaffold after decellularization. The membrane scaffold retains a complete tertiary structure, which is the source of its piezoelectric effect; at the same time, the piezoelectric force microscope (PFM) results show that the membrane scaffold has a periodic piezoelectric signal, which further proves that the collagen scaffold provided by the present invention has a piezoelectric effect. The experimental results show that bone marrow mesenchymal stem cells can adhere to the surface of the collagen scaffold and promote the repair and regeneration of bone tissue. Furthermore, the subcutaneous ectopic bone regeneration experiment in rats proves that the collagen scaffold provided by the present invention has a good effect of promoting bone regeneration under ultrasonic stimulation.

[0030] (2) The collagen scaffold provided by the present invention is composed of collagen. When complete bone tissue is obtained during the bone repair process, the collagen scaffold will slowly degrade and be excreted from the body through human absorption, metabolism and other pathways, so that the repaired bone defect site is completely formed by the growth of autologous bone tissue, without any other foreign matter remaining in the human body, and will not cause any adverse effects on the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0032] Figure 1 These are scanning electron microscope images of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1; wherein A is a scanning electron microscope image of the collagen scaffold PiezoCol, and B is a scanning electron microscope image of the collagen scaffold NCol;

[0033] Figure 2 Characterization of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1, wherein A is an XRD graph; B is an FTIR graph; C and E are amplitude images of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1, respectively; D and F are ferroelectric amplitude curves and phase curves of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1;

[0034] Figure 3are the results of biocompatibility testing, wherein A is the cell proliferation of bone marrow mesenchymal stem cells (BMSCs) inoculated on the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1 for 24 hours; B is the cell proliferation of bone marrow mesenchymal stem cells (BMSCs) inoculated on the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1 for 48 hours; C and D are the morphology and adhesion of cells on the collagen scaffold PiezoCol and the collagen scaffold NCol detected by immunofluorescence, respectively; E is a SEM image;

[0035] Figure 4 To detect the intracellular calcium concentration of cells grown on the scaffold by fluorescence;

[0036] Figure 5 This is an in vivo experiment to detect the effect on osteogenic differentiation. A is the Micro-CT result, B is the volume of ectopic new bone, and C is the surface area of ​​ectopic new bone. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0038] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0040] Example 1

[0041] After removing hair and subcutaneous fat tissue from fresh full-thickness pig skin, in order to obtain a decellularized and complete tertiary structure collagen scaffold PiezoCol, a membrane scaffold is obtained after decellularization. Specifically, the pig skin is processed by the following steps:

[0042] (1) Treatment with a mixed solution of 2.0 g / L sodium hydroxide and 0.8 g / L sodium dodecyl sulfate (SDS) for 12 hours;

[0043] (2) Rinse with deionized water for 15 minutes, repeat twice;

[0044] (3) 2.4 g / L SDS treatment for 2 h;

[0045] (4) Rinse with deionized water for 15 minutes, repeat twice;

[0046] (5) 3.0 g / L pancreatin treatment for 12 h;

[0047] (6) Rinse with deionized water for 15 minutes, repeat twice;

[0048] (7) 2.4 g / L SDS treatment for 2 hours;

[0049] (8) Rinse with deionized water for 15 minutes, repeat twice;

[0050] (9) 6% sodium chloride treatment for 6 hours;

[0051] (10) Rinse with deionized water for 20 minutes and repeat three times.

[0052] After freeze-drying, the piezoelectric collagen scaffold PiezoCol was cut into a square membrane scaffold with a side length of about 1 cm and a thickness of about 1 mm.

[0053] Comparative Example 1

[0054] After removing hair and subcutaneous fat tissue from fresh full-thickness pig skin, in order to obtain a decellularized collagen scaffold NCol that does not retain a complete tertiary structure, a membrane scaffold is obtained after decellularization. Specifically, the pig skin is processed by the following steps:

[0055] (1) Treatment with a mixed solution of 2.0 g / L sodium hydroxide and 0.8 g / L sodium dodecyl sulfate (SDS) for 12 hours;

[0056] (2) Rinse with deionized water for 15 minutes, repeat twice;

[0057] (3) 2.4 g / L SDS treatment for 2 h;

[0058] (4) Rinse with deionized water for 15 minutes, repeat twice;

[0059] (5) 3.0 g / L pancreatin treatment for 12 h;

[0060] (6) Rinse with deionized water for 15 minutes, repeat twice;

[0061] (7) 2.4 g / L SDS treatment for 2 hours;

[0062] (8) Rinse with deionized water for 15 minutes, repeat twice;

[0063] (9) 6% sodium chloride treatment for 6 hours;

[0064] (10) Rinse with deionized water for 20 minutes, repeat three times;

[0065] (11) Soak in 1 mol / L sodium hydroxide for 30 minutes.

[0066] After freeze-drying, the non-piezoelectric collagen scaffold NCol was cut into a square membrane scaffold with a side length of about 1 cm and a thickness of about 1 mm.

[0067] Figure 1 The SEM images of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1 are shown in FIG. Figure 1 It can be seen that the collagen fibers on the surface and cross-section of the collagen scaffold PiezoCol show a complete tertiary structure, while the collagen scaffold NCol has no complete tertiary structure.

[0068] Figure 2 A is the XRD pattern of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1. Figure 2 As can be seen in A, the collagen scaffold PiezoCol exhibits the characteristic peaks of collagen fibers.

[0069] Figure 2 Figure B is the FTIR images of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1. The FTIR results indicate that both the collagen scaffold PiezoCol and the collagen scaffold NCol show characteristic peaks of collagen, but the collagen scaffold NCol shows more amino absorption peaks and NH stretching bands, indicating that the collagen scaffold exposed more amino groups after alkali destruction.

[0070] Figure 2 Figures C and E are amplitude images of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1, respectively, which further proves that the collagen scaffold PiezoCol shows a complete tertiary structure, while the collagen scaffold NCol has no complete tertiary structure.

[0071] Figure 2 Figures D and F are the ferroelectric amplitude curves and phase curves of the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1. The results show that the collagen scaffold PiezoCol has a periodic piezoelectric signal, while the collagen scaffold NCol hardly shows any piezoelectric signal. This indicates that the intact tertiary structure is retained, which is the source of the piezoelectric signal of the collagen scaffold.

[0072] Example 2

[0073] In vitro experiments to study the effect of piezoelectric collagen scaffold PiezoCol on the biological activity of BMSCs:

[0074] 2.1 Biocompatibility testing:

[0075] (1) Bone marrow mesenchymal stem cells (BMSCs) were inoculated on the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCoI prepared in Comparative Example 1, and cell proliferation was detected by CCK-8;

[0076] (2) Detect the morphology and growth of cells on the material by immunofluorescence;

[0077] (3) The morphology and adhesion of cells on the material were detected by scanning electron microscopy (SEM).

[0078] The results are as follows Figure 3 As shown, Figure 3 A in the middle shows the cell proliferation of bone marrow mesenchymal stem cells (BMSCs) after 24 hours of inoculation on the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1; Figure 3 Figure B shows the cell proliferation of bone marrow mesenchymal stem cells (BMSCs) after 48 hours of inoculation on the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1. Figure 3 As can be seen from Figures A and B, the collagen scaffold PiezoCol prepared in Example 1 and the collagen scaffold NCol prepared in Comparative Example 1 both have good cell compatibility. Figure 3 C and D are immunofluorescence detection of cell morphology and adhesion on collagen scaffold PiezoCol and collagen scaffold NCol, respectively. Figure 3 As can be seen in C and D, a large number of bone marrow mesenchymal stem cells (BMSCs) adhere to the surface of the collagen scaffold. At the same time, the SEM images suggest that bone marrow mesenchymal stem cells (BMSCs) adhere to the scaffold surface by extending a large number of pseudopodia ( Figure 3 Middle E).

[0079] 2.2 Intracellular calcium ion concentration detection:

[0080] The intracellular calcium concentration of cells grown on the scaffold was detected by fluorescence.

[0081] The results are as follows Figure 4 As shown, from Figure 4 It can be seen that during osteogenic differentiation, piezoelectric stimulation increases intracellular calcium ions (Ca 2+ ) content, and 2.15W / cm 2 The mechanical vibration induced by (1MHz) ultrasound stimulation affects the intracellular calcium ions (Ca 2+) content has almost no effect.

[0082] Example 3

[0083] In vivo assay to detect the effect on osteogenic differentiation:

[0084] The animal experiment was divided into six groups, namely, blank control group (Ctrl), NCol group, PiezoCol group, blank control + ultrasound group (Ctrl / U), NCol + ultrasound group (NCol / U), and PiezoCol + ultrasound group (PiezoCol / U). Collagen scaffolds loaded with bone marrow mesenchymal stem cells (BMSCs) were implanted subcutaneously on the back of 8-week-old male Wistar rats. The ultrasound group was irradiated with 2.15W / cm 2 Ultrasonic massage was performed every two days at a power of 1 MHz for 10 minutes. All animals were killed 4 weeks after surgery and samples were collected. After the rats were killed, micro-computed tomography (Micro-CT) was performed on samples from different groups to observe the effect of collagen scaffolds on subcutaneous ectopic bone regeneration.

[0085] The results are as follows Figure 5 As shown in the figure, Micro-CT results showed that obvious bone-like tissue was generated under the skin in the PiezoCol / U group, and the volume and surface area of ​​the bone-like tissue were significantly increased compared with the non-ultrasound group.

[0086] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of an ultrasonically responsive piezoelectric collagen scaffold directly or indirectly used as a scaffold material for bone defect repair or bone tissue engineering, characterized in that: The ultrasonically responsive piezoelectric collagen scaffold is made of full-thickness animal connective tissue as raw material, and is a membrane scaffold obtained after hair and subcutaneous fat tissue are removed and decellularized.

2. The use according to claim 1, characterized in that The power of ultrasound is 2-3 W / cm 2 , preferably 2.15W / cm 2 .

3. The use according to claim 1, characterized in that The animal is selected from any one of pigs, cattle, sheep, horses or deer, preferably pigs; Or, the connective tissue is the skin.

4. The use according to claim 1, characterized in that A method for preparing an ultrasound-responsive piezoelectric collagen scaffold comprises: (1) Using full-thickness animal connective tissue as raw material, removing hair and subcutaneous fat tissue, and obtaining a first intermediate; (2) decellularizing the first intermediate product using sodium hydroxide and sodium dodecyl sulfate (SDS) to obtain a second intermediate product; (3) decellularizing the second intermediate product using sodium dodecyl sulfate (SDS) to obtain a third intermediate product; (4) treating the third intermediate product with pancreatic enzyme to obtain a fourth intermediate product; (5) decellularizing the fourth intermediate product again using sodium dodecyl sulfate (SDS) to obtain a fifth intermediate product; (6) Treating the fifth intermediate product with sodium chloride to obtain the ultrasonically responsive piezoelectric collagen scaffold.

5. The use according to claim 4, characterized in that In step (2), the concentration of sodium hydroxide in sodium hydroxide and sodium dodecyl sulfate is 1.8 to 2.2 g / L, preferably 2.0 g / L; The concentration of sodium dodecyl sulfate is 0.75 to 0.85 g / L, preferably 0.8 g / L.

6. The use according to claim 4, characterized in that In step (2), the decellularization treatment time is 11.5 to 12.5 hours, preferably 12 hours.

7. The use according to claim 4, characterized in that In step (4), the concentration of pancreatic enzyme is 2.8-3.2 g / L, preferably 3 g / L; Alternatively, in step (4), the pancreatin treatment time is 11.5 to 12.5 hours, preferably 12 hours.

8. The use according to claim 4, characterized in that In step (5), the concentration of sodium dodecyl sulfate is 2.3-2.5 g / L, preferably 2.4 g / L.

9. The use according to claim 8, characterized in that In step (5), the decellularization treatment time is 1.5 to 2.5 hours, preferably 2 hours.

10. The use according to claim 4, characterized in that In step (6), the mass fraction of sodium chloride is 5.5-7%, preferably 6%; Alternatively, in step (6), the time for treatment with sodium chloride is 5.5 to 7 hours, preferably 6 hours.

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