人脂肪干细胞体内外构建组织工程软骨的实验研究
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摘要
目的:探讨在体内外环境下,种子细胞、支架材料与环境因素之间的相互作用;明确组织工程软骨对关节软骨损伤模型的治疗作用。
方法:从人脂肪抽吸物中分离、纯化间充质干细胞,进行体外培养、鉴定;对细胞进行成软骨诱导分化,与海藻酸钠钙凝胶复合,在体外及裸鼠皮下和关节内构建组织工程软骨,观察组织工程软骨对损伤软骨的修复作用及种子细胞、支架材料、环境因素三者之间的相互作用。
结果:
1.从人脂肪组织中可以得到大量间充质干细胞。
2.ADMSCs可成软骨诱导,与海藻酸钠钙凝胶复合后,在微球内分布均匀,细胞呈球形,大部分细胞存活。
3.组织工程软骨促进裸鼠关节软骨损伤修复,新生软骨与正常软骨的细胞形态及基质成分接近;可见软骨下骨结构形成。
结论:
ADMSCs是良好的软骨组织工程种子细胞来源。ADMSCs与海藻酸钠钙凝胶具有良好的组织相容性。经体外成软骨诱导的ADMSCs复合海藻酸钠后,在裸鼠皮下和受损关节内可以保持软骨细胞表型;可以促进其关节软骨损伤的修复。
The defects of articular cartilage induced by injuries or diseases are commonly encountered in hand surgery. Due to its intrinsic properties that lack of vascular supply and the low metabolic activity of chondrocytes, articular cartilage exhibits a limited capacity for intrinsic repair. Thus, caitilage defects, without surgical intervention will either remain injured or progressively worsen with time. Pieces of articular cartilage may become elevated flaps and irritate the synovial lining of the arthrosis causing swelling (effusion) and mechanical symptoms of catching. If the flap of cartilage becomes detached, it can become a loose body, causing locking of the arthrosis so that it does not bend or straighten fully. Surgical techniques and options have focused on the healing of articular cartilage defects. Many of these strategies appear to satisfactory in some cases and can decrease pain and morbidity in the short term, but not effectively sa a long term. Therefore defects of articular cartilage continues to present amajor problem in clinical medicine.
The term“tissue engineering”was officially put forward in 1987 . and has been developed very quickly in recent years. Tissue engineering merges the fields of cell biology, engineering, materials science and surgery to fabricate new functional tissue using living cells and a matrix or scaffold . It is concerned with the creation of biological substitutes designed to maintain, restore or improve the function of damaged tissues and organs.
Mesenchymal stem cells (MSCs) have been isolated not only from bone marrow, but also from many other tissues such as adipose tissue, skeletal muscle, liver, brain and pancreas. Because MSC were found to have the ability to differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons, hepatocytes and insulin-producing cells, MSCs have generated a great deal of interest for their potential use in regenerative medicine and tissue engineering. In 2001, Zuk et al. showed that a population of adipose derived cells similar to the SVF fraction contained cells with multilineage potential. In their work, cells isolated from human lipoaspirates,termed PLA cells,underwent adipogenesis, osteogenesis, chondrogenesis, and myogenesis in vitro, suggesting that the SVF fraction of adipose may be composed not just of lineage-limited preadipocytes but of multipotent stem cells. Obtained through cosmetic procedures, such as lipoaspirates, adipose-derived mesenchymal stem cells (ADMSCs) can be harvested in large quantities, with minimal pain and morbidity incurred by the patient. This simple fact may make substantial quantities of adult stem cells available for either clinical or tissue engineering applications or for molecular characterization.
Three-dimensional scaffold structure, which is needed to be degradable and to maintain stable volume and shape. a good scaffold must facilitate cell attachment without provoking an immune response, permit the diffusion of nutrients from the blood, and, at least initially, mimic the mechanical properties of the tissue. Additionally, the scaffold can be constructed with or designed to release factors that can beneficially manipulate the behavior of the cells in the device. Alginate, or alginic acid, is an unbranched binary copolymer of (1-->4)-linked beta-D-mannuronic acid and alpha-L-guluronic acid. Alginate readily forms binding interactions with a variety of divalent metal ions, such as Ca2+ and Ba2+. This binding has been used to cross-link bulk alginates for a wide variety of applications, particularly in areas of tissue engineering, medical devices, and wound-healing dressings.
Regeneration of damaged cartilage in different pathological situations is a major goal which could be achieved through physiologic regeneration. The results of this study suggested that MSCs seem to be the best candidates for cell therapy for regeneration of injured tissue. They are easily isolated and can be rapidly amplified. When these cells are implanted into cartilage defects, they appear to be able to proliferate and differentiate into chondrocytes. This procedure is of considerable relevance for the regeneration of large areas of cartilage lesions. However, complete healing is difficult to obtain and requires integration of newly regenerated tissue with the surrounding host tissue and true differentiation through pathways involved in embryonic development. This goal may be achieved through the combination of MSC mediated therapy and gene transfer of selective differentiating cytokines. Objective:
To investigate the biological features of human adipose-derived mesenchymal stem cells (ADMSCs ) to isolate and culture, then differentiate into adipocyte osteoblast and chondrocyte in vitro, and discuss the feasibility of ADMSCs as seed cells in cartilage tissue engineering. To study the feasibility of applying ADMSCs as seed cells and alginate as the scafold in constructing tissue engineering cartilage in vitro. To study the relation of seed cells, scaffold structure and microenvironment; investigate the results of the articular cartilage repaired by implantation ADMSCs suspended in calcium alginate. Methods:
1. Human adipose was obtained from the healthy womanhood during the abdominal liposuction procedure and digested with collagenaseⅠto isolate ADMSCs , which were incubated in high-glucose DMEM with 0.10 volume fraction fetal bovine serum (FBS) for primary culture. Growth curve of the third-pas sage ADMSCs was drawn by cytometry; the express ions of CD13 and CD34 were assayed by flow cytometry and immunofluorescence technique. The third-passage ADMSCs were cultured in high-glucose DMEM supplemented with selective differentiating cytokines for adipogenic and osteogenesis induction. The appearance and generation of ADMSCs were observed everyday under inverted microscope, and the differentiations to adipocyte and osteoblast were verified by specific staining.
2. Human adipose was obtained from the healthy womanhood during the abdominal liposuction procedure and digested with collagenaseⅠto isolate ADMSCs , which were incubated in high-glucose DMEM with 0.10 volume fraction fetal bovine serum (FBS) for primary culture. The third-passage ADMSCs were cultured in high-glucose DMEM supplemented with selective differentiating cytokines for chondrogenic induction. The appearance and generation of ADMSCs were observed everyday under inverted microscope, and the differentiations to chondrocyte were verified by AB-PSA staining and immunocytochemical staining.
3. Applying ADMSCs as seed cells and alginate as the scafold in constructing tissue engineering cartilage in vitro. Tissue engineering cartilage were impianted subcutaneously and articular in athymic mice after cultured for 1 week in vitro. New cartilage formation was assessed though gross observation and histological examination.
Results:
1. ADMSCs are easily isolated and can be rapidly amplified. ADMSCs cultured in vitro were in flat fusiform shape and stably passaged, during which ADMSCs kept the same shape. The expression of stromal cell-associated marker CD13 and stem cell-associated marker CD34 was positive. The characteristics of adipocyte and osteoblast were shown after the ADMSCs had exposed to the defined medium.
2. Chondrogenic induction of PLA cells, under micromass conditions, resulted in cell condensation after induction and was followed by ridge and spheroid/nodule formation. ADMSCs cultured in chondrogenic differentiation medium, AB-PSA staining was positive and the typeⅡcollagen immunocytochemical staining appeared positive with brown cytoplasm.The cell morphology of induced ADMSCs was evident that they obtained the differentiation phenotype as chondrogenic lineages. The expression of the typeⅡcollagen and Sox9 is an early and practically unique marker of chondrogenic differentiation. RT-PCR analysis of mRNA encoding typeⅡcollagen and Sox9 showed that high lever expression for 2 weeks.The chondrogenesis of ADMSCs were supported by presence of some other important components of cartilaginous extracellular matrix, such as typeⅨ,Ⅹ,Ⅺcollagen, AGN、COMP and Link Protein. In contrast to this, there was a trend to decrease in expression of the typeⅠcollagen after induction. Shortly after ADMSCs were seeded onto scaffolds, the calcium alginate microsphere presented the translucent globes and smooth surface. ADMSCs presented single cell condition and evenlyspred around in the microsphere. 3. In the impianted subcutaneously groups, the uninduced ADMSCs underwent fibroblast differentiation. In contrast to this, the induced ADMSCs still shown characteristic of chondrogenic. The groups with a single full thickness osteochondral injury underwent a fibrocartilaginous repair that go with serve osteoarthritis. The healing with alginate groups shown minor osteoarthritis. But the damage of articular cartilage did not heal. The healing with ADMSCs groups can significant attempt to recreate the normal articular cartilage.
Conclusions:
1. This study indicates that liposuction aspirates can provide abundant ADMSCs with the potential of multilineage differentiation and proliferation over extended periods. Adipose tissue can be a promising adult stem cell source. ADMSCs can be used as seed cells for cartilage tissue engineering.
2. ADMSCs can be induced to differentiate towards chondrocytes in conditioned medium in vitro. Calcium alginate represent good compatibility and adhesion with ADMSCs without little toxicity. It appears that ADMSCs and calcium alginate microsphere may provide an excellent support for cell transplantation.
3. The induced ADMSCs still shown characteristic of chondrogenic after impianted subcutaneously. The induced ADMSCs still shown characteristic of chondrogenic after impianted in damaged articular cartilage, and significant attempt to recreate the normal articular cartilage.
方法:从人脂肪抽吸物中分离、纯化间充质干细胞,进行体外培养、鉴定;对细胞进行成软骨诱导分化,与海藻酸钠钙凝胶复合,在体外及裸鼠皮下和关节内构建组织工程软骨,观察组织工程软骨对损伤软骨的修复作用及种子细胞、支架材料、环境因素三者之间的相互作用。
结果:
1.从人脂肪组织中可以得到大量间充质干细胞。
2.ADMSCs可成软骨诱导,与海藻酸钠钙凝胶复合后,在微球内分布均匀,细胞呈球形,大部分细胞存活。
3.组织工程软骨促进裸鼠关节软骨损伤修复,新生软骨与正常软骨的细胞形态及基质成分接近;可见软骨下骨结构形成。
结论:
ADMSCs是良好的软骨组织工程种子细胞来源。ADMSCs与海藻酸钠钙凝胶具有良好的组织相容性。经体外成软骨诱导的ADMSCs复合海藻酸钠后,在裸鼠皮下和受损关节内可以保持软骨细胞表型;可以促进其关节软骨损伤的修复。
The defects of articular cartilage induced by injuries or diseases are commonly encountered in hand surgery. Due to its intrinsic properties that lack of vascular supply and the low metabolic activity of chondrocytes, articular cartilage exhibits a limited capacity for intrinsic repair. Thus, caitilage defects, without surgical intervention will either remain injured or progressively worsen with time. Pieces of articular cartilage may become elevated flaps and irritate the synovial lining of the arthrosis causing swelling (effusion) and mechanical symptoms of catching. If the flap of cartilage becomes detached, it can become a loose body, causing locking of the arthrosis so that it does not bend or straighten fully. Surgical techniques and options have focused on the healing of articular cartilage defects. Many of these strategies appear to satisfactory in some cases and can decrease pain and morbidity in the short term, but not effectively sa a long term. Therefore defects of articular cartilage continues to present amajor problem in clinical medicine.
The term“tissue engineering”was officially put forward in 1987 . and has been developed very quickly in recent years. Tissue engineering merges the fields of cell biology, engineering, materials science and surgery to fabricate new functional tissue using living cells and a matrix or scaffold . It is concerned with the creation of biological substitutes designed to maintain, restore or improve the function of damaged tissues and organs.
Mesenchymal stem cells (MSCs) have been isolated not only from bone marrow, but also from many other tissues such as adipose tissue, skeletal muscle, liver, brain and pancreas. Because MSC were found to have the ability to differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons, hepatocytes and insulin-producing cells, MSCs have generated a great deal of interest for their potential use in regenerative medicine and tissue engineering. In 2001, Zuk et al. showed that a population of adipose derived cells similar to the SVF fraction contained cells with multilineage potential. In their work, cells isolated from human lipoaspirates,termed PLA cells,underwent adipogenesis, osteogenesis, chondrogenesis, and myogenesis in vitro, suggesting that the SVF fraction of adipose may be composed not just of lineage-limited preadipocytes but of multipotent stem cells. Obtained through cosmetic procedures, such as lipoaspirates, adipose-derived mesenchymal stem cells (ADMSCs) can be harvested in large quantities, with minimal pain and morbidity incurred by the patient. This simple fact may make substantial quantities of adult stem cells available for either clinical or tissue engineering applications or for molecular characterization.
Three-dimensional scaffold structure, which is needed to be degradable and to maintain stable volume and shape. a good scaffold must facilitate cell attachment without provoking an immune response, permit the diffusion of nutrients from the blood, and, at least initially, mimic the mechanical properties of the tissue. Additionally, the scaffold can be constructed with or designed to release factors that can beneficially manipulate the behavior of the cells in the device. Alginate, or alginic acid, is an unbranched binary copolymer of (1-->4)-linked beta-D-mannuronic acid and alpha-L-guluronic acid. Alginate readily forms binding interactions with a variety of divalent metal ions, such as Ca2+ and Ba2+. This binding has been used to cross-link bulk alginates for a wide variety of applications, particularly in areas of tissue engineering, medical devices, and wound-healing dressings.
Regeneration of damaged cartilage in different pathological situations is a major goal which could be achieved through physiologic regeneration. The results of this study suggested that MSCs seem to be the best candidates for cell therapy for regeneration of injured tissue. They are easily isolated and can be rapidly amplified. When these cells are implanted into cartilage defects, they appear to be able to proliferate and differentiate into chondrocytes. This procedure is of considerable relevance for the regeneration of large areas of cartilage lesions. However, complete healing is difficult to obtain and requires integration of newly regenerated tissue with the surrounding host tissue and true differentiation through pathways involved in embryonic development. This goal may be achieved through the combination of MSC mediated therapy and gene transfer of selective differentiating cytokines. Objective:
To investigate the biological features of human adipose-derived mesenchymal stem cells (ADMSCs ) to isolate and culture, then differentiate into adipocyte osteoblast and chondrocyte in vitro, and discuss the feasibility of ADMSCs as seed cells in cartilage tissue engineering. To study the feasibility of applying ADMSCs as seed cells and alginate as the scafold in constructing tissue engineering cartilage in vitro. To study the relation of seed cells, scaffold structure and microenvironment; investigate the results of the articular cartilage repaired by implantation ADMSCs suspended in calcium alginate. Methods:
1. Human adipose was obtained from the healthy womanhood during the abdominal liposuction procedure and digested with collagenaseⅠto isolate ADMSCs , which were incubated in high-glucose DMEM with 0.10 volume fraction fetal bovine serum (FBS) for primary culture. Growth curve of the third-pas sage ADMSCs was drawn by cytometry; the express ions of CD13 and CD34 were assayed by flow cytometry and immunofluorescence technique. The third-passage ADMSCs were cultured in high-glucose DMEM supplemented with selective differentiating cytokines for adipogenic and osteogenesis induction. The appearance and generation of ADMSCs were observed everyday under inverted microscope, and the differentiations to adipocyte and osteoblast were verified by specific staining.
2. Human adipose was obtained from the healthy womanhood during the abdominal liposuction procedure and digested with collagenaseⅠto isolate ADMSCs , which were incubated in high-glucose DMEM with 0.10 volume fraction fetal bovine serum (FBS) for primary culture. The third-passage ADMSCs were cultured in high-glucose DMEM supplemented with selective differentiating cytokines for chondrogenic induction. The appearance and generation of ADMSCs were observed everyday under inverted microscope, and the differentiations to chondrocyte were verified by AB-PSA staining and immunocytochemical staining.
3. Applying ADMSCs as seed cells and alginate as the scafold in constructing tissue engineering cartilage in vitro. Tissue engineering cartilage were impianted subcutaneously and articular in athymic mice after cultured for 1 week in vitro. New cartilage formation was assessed though gross observation and histological examination.
Results:
1. ADMSCs are easily isolated and can be rapidly amplified. ADMSCs cultured in vitro were in flat fusiform shape and stably passaged, during which ADMSCs kept the same shape. The expression of stromal cell-associated marker CD13 and stem cell-associated marker CD34 was positive. The characteristics of adipocyte and osteoblast were shown after the ADMSCs had exposed to the defined medium.
2. Chondrogenic induction of PLA cells, under micromass conditions, resulted in cell condensation after induction and was followed by ridge and spheroid/nodule formation. ADMSCs cultured in chondrogenic differentiation medium, AB-PSA staining was positive and the typeⅡcollagen immunocytochemical staining appeared positive with brown cytoplasm.The cell morphology of induced ADMSCs was evident that they obtained the differentiation phenotype as chondrogenic lineages. The expression of the typeⅡcollagen and Sox9 is an early and practically unique marker of chondrogenic differentiation. RT-PCR analysis of mRNA encoding typeⅡcollagen and Sox9 showed that high lever expression for 2 weeks.The chondrogenesis of ADMSCs were supported by presence of some other important components of cartilaginous extracellular matrix, such as typeⅨ,Ⅹ,Ⅺcollagen, AGN、COMP and Link Protein. In contrast to this, there was a trend to decrease in expression of the typeⅠcollagen after induction. Shortly after ADMSCs were seeded onto scaffolds, the calcium alginate microsphere presented the translucent globes and smooth surface. ADMSCs presented single cell condition and evenlyspred around in the microsphere. 3. In the impianted subcutaneously groups, the uninduced ADMSCs underwent fibroblast differentiation. In contrast to this, the induced ADMSCs still shown characteristic of chondrogenic. The groups with a single full thickness osteochondral injury underwent a fibrocartilaginous repair that go with serve osteoarthritis. The healing with alginate groups shown minor osteoarthritis. But the damage of articular cartilage did not heal. The healing with ADMSCs groups can significant attempt to recreate the normal articular cartilage.
Conclusions:
1. This study indicates that liposuction aspirates can provide abundant ADMSCs with the potential of multilineage differentiation and proliferation over extended periods. Adipose tissue can be a promising adult stem cell source. ADMSCs can be used as seed cells for cartilage tissue engineering.
2. ADMSCs can be induced to differentiate towards chondrocytes in conditioned medium in vitro. Calcium alginate represent good compatibility and adhesion with ADMSCs without little toxicity. It appears that ADMSCs and calcium alginate microsphere may provide an excellent support for cell transplantation.
3. The induced ADMSCs still shown characteristic of chondrogenic after impianted subcutaneously. The induced ADMSCs still shown characteristic of chondrogenic after impianted in damaged articular cartilage, and significant attempt to recreate the normal articular cartilage.
引文
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