阅读说明：本技术 人羊膜间充质干细胞在制备骨再生产品中的用途 (Application of human amniotic mesenchymal stem cells in preparation of bone regeneration products ) 是由 蒋欣泉 江飞 张文杰 于 2020-05-21 设计创作，主要内容包括：本发明提供人羊膜间充质干细胞用于制备骨再生产品的用途。hAMSCs作为一种成体干细胞,具有来源广、低免疫原性、增殖快等特点,具有临床推广应用的优势,本发明基于对hAMSCs作用机制的新发现,开拓了hAMSCs的新用途。在骨缺损处应用hAMSCs,可以利用其免疫调节作用优化骨缺损微环境,刺激内源性骨再生,弥补单纯使用支架材料缺乏骨诱导性的缺陷,也避免使用单一蛋白类活性因子刺激局部免疫反应而影响骨再生效果。动物颅骨缺损模型中未见明显炎症反应,同时促血管化骨再生的M2巨噬细胞聚集,分泌促血管新生和成骨活性因子。本发明不需专门、复杂和昂贵的设备,操作流程简单,有利于推广使用。(The invention provides application of human amniotic mesenchymal stem cells in preparation of bone regeneration products. As an adult stem cell, the hAMSCs have the characteristics of wide source, low immunogenicity, rapid proliferation and the like, and have the advantages of clinical popularization and application. The hAMSCs are applied to the bone defect part, so that the bone defect microenvironment can be optimized by utilizing the immunoregulation effect of the hAMSCs, endogenous bone regeneration is stimulated, the defect that the pure use of a bracket material lacks osteoinductivity is overcome, and the influence on the bone regeneration effect caused by the stimulation of local immunoreaction by a single protein active factor is avoided. No obvious inflammatory reaction is seen in the animal skull defect model, and M2 macrophages promoting vascularization bone regeneration are gathered and secrete factors promoting angiogenesis and osteogenesis. The invention does not need special, complex and expensive equipment, has simple operation flow and is beneficial to popularization and use.)

1. The application of the human amniotic mesenchymal stem cells in preparing bone regeneration products.

2. The use of claim 1, wherein the human amniotic mesenchymal stem cells are the sole active ingredient or one of the active ingredients of the bone regeneration product.

3. The use of claim 1, wherein the bone regeneration product has at least one of the following functions:

1) inducing monocyte polarization to M2 macrophage;

2) regulating and controlling a bone regeneration microenvironment;

3) promoting bone regeneration.

4. Use according to claim 1, wherein the bone regeneration product is selected from bone regeneration biomaterial composites.

5. The use of claim 4, wherein the density of the human amniotic mesenchymal stem cells loaded in the bone regeneration product is 5 × 10⁶-10⁷/ml。

6. The use of claim 4, wherein the bone regeneration product further comprises a scaffold material.

7. A bone regeneration product comprises an effective amount of human amniotic mesenchymal stem cells.

8. The bone regeneration product of claim 7 wherein the bone regeneration product is a bone regeneration biomaterial composite.

9. The bone regeneration product of claim 8, wherein the density of the human amniotic mesenchymal stem cells loaded in the bone regeneration product is 5 × 10⁶-10⁷/ml。

10. The bone regeneration product of claim 7 further comprising a scaffold material.

11. The method for preparing a bone regeneration biomaterial composite as claimed in claim 10, comprising at least the steps of:

(1) mixing the human amniotic mesenchymal stem cell suspension with a degradable hydrogel material.

(2) And (2) mixing the mixture obtained in the step (1) with a scaffold material to obtain the bone regeneration biomaterial compound.

Technical Field

The invention relates to the technical field of biology, in particular to application of human amniotic mesenchymal stem cells in preparation of a bone regeneration product.

Background

At present, the clinical research on bone defect repair tends to use inorganic or organic scaffold materials to replace autologous bone transplantation or protein active factors to stimulate the regeneration of bones of organisms. Although the common scaffold material has certain bone guiding property, the common scaffold material lacks the function of bone immune regulation and control; protein active factors often stimulate immune responses to affect bone regeneration.

The placenta is usually abandoned as medical waste after the delivery of the pregnant women, has sufficient sources and easy acquisition, and the acquisition does not violate ethical principles. Because of its critical role in maternal/fetal immune tolerance, placental tissue contains amniotic mesenchymal stem cells (hAMSCs). At present, no report of applying hAMSCs to vascularized bone regeneration is found.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide the application of the human amniotic mesenchymal stem cells in preparing bone regeneration products.

The invention is realized by the following technical scheme:

in a first aspect of the invention, the invention provides the use of human amniotic mesenchymal stem cells for preparing bone regeneration products.

In a second aspect of the invention, there is provided a bone regeneration product comprising an effective amount of human amniotic mesenchymal stem cells.

In a third aspect of the present invention, there is provided a method for preparing the bone regeneration biomaterial composite, comprising at least the following steps:

(1) mixing the human amniotic mesenchymal stem cell suspension with a degradable hydrogel material.

(2) And (2) mixing the mixture obtained in the step (1) with a scaffold material to obtain the bone regeneration biomaterial compound.

Compared with the prior art, the invention has the beneficial effects that:

as an adult stem cell, the hAMSCs have the characteristics of wide source, low immunogenicity, rapid proliferation and the like, and have the advantages of clinical popularization and application. The hAMSCs are applied to the bone defect part, so that the bone defect microenvironment can be optimized by utilizing the immunoregulation effect of the hAMSCs, endogenous bone regeneration is stimulated, the defect that the pure use of a bracket material lacks osteoinductivity is overcome, and the influence on the bone regeneration effect caused by the stimulation of local immunoreaction by a single protein active factor is avoided. No obvious inflammatory reaction is seen in the animal skull defect model, and M2 macrophages promoting vascularization bone regeneration are gathered and secrete factors promoting angiogenesis and osteogenesis. The invention does not need special, complex and expensive equipment, has simple operation flow and is beneficial to popularization and use.

Drawings

FIG. 1: schematic illustration of microenvironment regulation of hAMSCs bone defect.

FIG. 2: induction of RAW264.7 by hAMSCs. (a1-a4) M0 macrophage surface iNOS (M1 macrophage surface marker) and MMR (M2 macrophage surface marker) staining negative; (B1-B4) iNOS was positive on the surface of M1 macrophage and MMR staining was negative; (C1-C4) iNOS was negative on the surface of M2 macrophage and positive in MMR staining; (D1-D4) iNOS was negative and MMR stained positive after 72 hours of co-culture of hAMSCs with RAW 264.7.

FIG. 3: and specific staining results of iNOS and MMR of 2w specimen after the hAMSCs are transplanted in the rabbit skull defect model. (A) A control group; (B) a group hAMSC; (C) group Bio-Os; (D) group hAMSC/Bio-Os; (E) counting the iNOS positive area; (F) and (5) carrying out statistics on MMR positive areas. (. p < 0.05;. p <0.01)

FIG. 4: and (3) carrying out specific staining on VEGF and BMP2 of 2w samples after the hAMSCs are transplanted in a rabbit skull defect model. (A) A control group; (B) a group hAMSC; (C) group Bio-Os; (D) group hAMSC/Bio-Os; (E) counting the positive area of VEGF; (F) statistical analysis of positive area of BMP 2. (. p < 0.05;. p <0.01)

FIG. 5: specific staining results of CD31 and ALP of 2w specimen after transplantation of hAMSCs in rabbit skull defect model. (A) A control group; (B) a group hAMSC; (C) group Bio-Os; (D) group hAMSC/Bio-Os; (E) counting the positive area of ALP; (F) and (5) counting the positive area of the CD 31. (. p < 0.05;. p <0.01)

FIG. 6: and (5) bone formation results in 12w samples after hAMSCs are transplanted in the rabbit skull defect model. (A) A control group; (B) a group hAMSC; (C) group Bio-Os; (D) group hAMSC/Bio-Os; (E) and (5) counting the area of the relative new bone. (. p < 0.05;. p <0.01)

Detailed Description

The amnion mesenchymal stem cells (hAMSCs) derived from the placenta tissues have better immunoregulation function. hAMSCs can secrete a variety of cytokines with immune regulation, such as: macrophage Inhibitory Factor (MIF), interleukin 8(IL-8), interleukin 6(IL-6), C-reactive protein (CRP), and the like. These proteins have inhibitory effects on macrophages, neutrophils and natural killer cells (NK cells). Therefore, the hAMSCs are applied to the bone defect part, so that the bone defect microenvironment can be optimized by utilizing the immunoregulation effect of the hAMSCs, endogenous bone regeneration is stimulated, the defect that the pure use of a bracket material lacks bone inductivity is overcome, and the influence on the bone regeneration effect caused by the stimulation of local immunoreaction by a single protein active factor is avoided.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

One embodiment of the invention is application of human amniotic mesenchymal stem cells in preparation of bone regeneration products.

Furthermore, the human amniotic mesenchymal stem cells are the only effective component or one of the effective components of the bone regeneration product.

The bone regeneration product is mainly aimed at mammals, and the mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates and the like. The primate is preferably a monkey, ape or human.

The subject may be an individual with a bone defect.

The bone regeneration product has at least one of the following functions:

1) inducing monocyte polarization to M2 macrophage;

2) regulating and controlling a bone regeneration microenvironment;

3) promoting bone regeneration.

In one embodiment, the bone regeneration product is a bone regeneration biomaterial composite.

The density of the human amniotic mesenchymal stem cells loaded in the bone regeneration product is 5 × 10⁶-10⁷/ml。

Further, the bone regeneration product also comprises a bracket material.

In one embodiment, the scaffold material is selected from bone filler materials. Such as Bio-Os.

In one embodiment, the bone filler material has a particle size of 0.25 to 1 mm.

An embodiment of the invention provides a bone regeneration product, which comprises an effective dose of human amniotic mesenchymal stem cells.

Optionally, the bone regeneration product is selected from a bone regeneration biomaterial composite.

The density of the human amniotic mesenchymal stem cells loaded in the bone regeneration product is 5 × 10⁶-10⁷/ml。

Further, the bone regeneration product also comprises a bracket material.

In one embodiment, the scaffold material is selected from bone filler materials. Such as Bio-Os.

The preparation method of the bone regeneration biomaterial compound at least comprises the following steps:

(1) mixing the human amniotic mesenchymal stem cell suspension with a degradable hydrogel material.

(2) And (2) mixing the mixture obtained in the step (1) with a scaffold material to obtain the bone regeneration biomaterial compound.

The density of the human amniotic mesenchymal stem cells loaded in the bone regeneration product is 5 × 10⁶-10⁷/ml。

In one embodiment, the scaffold material is selected from bone filler materials. Such as Bio-Os.