hMSCs复合生物衍生骨支架材料的体内外成骨研究
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摘要
为解决临床上由于创伤、感染和肿瘤切除后所造成的大范围骨缺损难以修复的问题,本实验将来源于猪松质骨的生物衍生骨和鼠尾胶原结合作为支架材料,以人骨髓间充质干细胞(human mesenchymal stem cells ,hMSCs)为骨组织工程的种子细胞,并将两者进行体外复合培养和成骨诱导后移植到大鼠的骨缺损模型中进行骨愈合研究。
本实验通过各种理化处置制备了来源于猪松质骨的生物衍生骨支架材料,大大降低了抗原性;并将其与鼠尾胶原复合,制成了生物衍生骨-胶原凝胶复合支架材料,增强了其细胞相容性,有利于细胞的吸附、分化和增殖;经检测复合支架材料具有良好的组织相容性和生物安全性;在体外对hMSCs -支架材料复合物进行成骨诱导后,hMSCs表达成骨特异性标记OPN和OCN,表达强度比单纯hMSCs诱导组强,同时未诱导的复合物中hMSCs也有表达,表明本研究制备的复合支架材料除具有一定的骨诱导作用外,同时也能增强hMSCs的成骨能力;建立了标准化的大鼠骨缺损模型后,将诱导后的hMSCs-支架材料复合物移植到缺损处,证明hMSCs-支架材料复合物具有明显的促进骨缺损修复的作用。
总之,本研究获得的复合支架材料引起的免疫反应较弱,具有良好的生物相容性和细胞相容性,并成功的利用hMSCs与支架材料的共培养来建造组织工程化人工骨、修复骨缺损,为临床大范围骨缺损修复奠定了很好的基础。
The repair of bone defect is always a very important and hot spot for both clinicians and laboratory researchers. As far as concerned, the repair and regeneration of the wide-bound bone defect caused by trauma, infection and tumor excision remain a significant clinical challenge. With the development of histological engineering by the last decade, scientists are trying to create artificial bones by inducing the osteogenic differentiation from potential cells in vitro, promoting amplification of differentiated cells and then seeding them into the degradable scaffold. It has a very promising perspective since it does well for the repair and regeneration of the wide-bound bone defect.
It has been a hot spot of histological engineering to identify promising candidate seed cells. Stem cells possess self-renewal, long-term cell viability, and multilineage differentiation potential, and all the characteristics make them taken as the best seed cells. However, application of multipotentonic embryonic stem cells may be restricted because of ethical issues and immunological complications. To avoid possible ethical problems and immune rejection, it is considered that adult stem cells are a more logical population for engineered tissue, thus being attractive to both clinical and laboratory scientists.
MSCs are multipotential stem cells derived from mesoderm, possessing potent proliferative capacity and self-renewal and multilineage differentiation potential. MSCs can differentiate into osteocytes, chondroncytes, adipocytes, myocytes, hepatocytes, neuron-like cells and neuroglial cells with the controlled conditions and cytokines. It is of advantage to use the MSCs as the seed cells and all of the characteristics of MSCs make them taken as the best seed cells: 1) easy procurement and minimal possibility of clinical discomfort and injury; 2) no ethical problems and immune rejection; 3) potent multiple amplification in vitro; and 4) easily induced differentiation and inserting and expression of exogenous genes easily.
A range of synthetic materials including polylactic acid and hydroxyapatite ceramic have been investigated for bone tissue engineering applications. However, no single material has been able to satisfy all the requirements for creating optimal scaffolding. They have several drawbacks: confused three-dimensional space structure, no secretion potential of osteoinductive factor and the low cell compatibility.
So it is very necessary and urgent to identify other candidate scaffolds to avoid those problems. We create a kind of bio-derived scaffold from the spongy bone of the pig after deproteiniztion, decalcification, defatting and elimination of cells and antigens. It expresses not only osteoinductive factors and possesses low immunogenicity, high cell compatibility and no obvious architectural alteration of the main framework since the main content is collagen.
The capacity of the pure scaffold only to induce osteogenic differentiation is limited and as the development of the histological engineering, complex materials have been developed to enhance the osteogenic differentiation potential. The fact that hMSCs poorly adhere to the pure bio-derived scaffold only limits its application. The collagen lattice from rat tail has been often used as the extracellular matrix in in vitro cell culture. It shows good cytocompatibility and three dimensional ultrastructural features. The three dimensional structure of the collagen has strength and the pores, which keeps the biological osmolarity. This property anchor and support the cell being adherent to the scaffold and thus provide a proper microenvironment for proliferation and differentiation of hMSCs.
We obtain high homogenous hMSCs by the density gradient separation method, monocloning screen method and adherent culture system. We create the complex bio-derived bone scaffold and the collagen from mouse tail and compare it’s cytocompatibility with other scaffolds by in vitro co-culture of the relative scaffold with hMSCs. We then create the complex scaffold by mixing together of bio-derived bone scaffold with collagen from mouse tail. Then in vivo transplantation of the complex scaffold, lymphocyte transformation test, using concanavlinA as a stimulant, and complement dependent cytotoxity test to detect the histocompatibility of the bio-derived scaffold are performed. In vitro induction of osteogenic differentiation of hMSCs-scaffold complex has been investigated. We also has established the rat bone fracture model and also transplant the hMSCs-scaffold complex after undergoing induction of osteogenic differentiation between the defect area of the cut ends of bone to detect the bone repair.
Results:
One:
A standardized platform has been established for isolation, cultivation, purification, identification and amplification of hMSCs with homogeneity of higher than 98%. The hMSCs under in vitro culture maintain stable biological characteristics at even passage 12 and stay at the state of undifferentiation, providing an excellent cell source for extended experiments
Two:
Dexamethasone, sodium glycerophosphate and vitamin C are added to the control medium to induce the osteogenic differentiation of hMSCs. The induced cells gather together like nodisity and deposition of calium salts is observed. Also after induction, cells positively express OPN and OCN which are specific to osteoblasts.
Three:
We compared cytocompatiblity of the complex scaffold by mixing together of bio-derived bone scaffold with the collagen from mouse tail with that of nanometer material PLGA on the adherence capacity and the time needed by hMSCs to be adherent to the surface of the scaffold and the proliferation rate. It shows that the collagen from mouse tail has a better cytocompatibility. The bio-derived scaffold has the natural structure of cancellous bone, thus functioning well as the three dimensional scaffolds for the engineered bone. We eventually choose the complex scaffold.
Four:
We perform in vivo transplantation of the complex scaffold, lymphocyte transformation test, using concanavlinA as a stimulant, and the complement dependent cytotoxity test to detect the histocompatibility of the bio-derived scaffold. The immunoreactivity of bio-derived scaffold is low, without obvious tendency to induce inflammation. It appeared to have good biocompatibility and high biological safety.
Five:
We create the mixture suspension of hMSCs and collagen from mouse tail and then incubate the suspension for two minutes. Then the suspension is added to the bio-derived bone scaffold when it reaches the semisolid state, and cultivate in the incubator for 2 to 5 minutes with a condition of 5% carbon dioxide, temperature 37. The complex scaffold has been established when the collagen becomes hard. The three dimensional structure of the collagen has strength and pores, which keeps the biological osmolarity. This property anchor and support the cell being adherent to the scaffold and make cells grow well in pores of the scaffold, and provide a large space for cell.
Scanning electron microscope and light microscope images show that the cells grow in a pattern of multilayer. And some of the cells are well adherent to the surface of the scaffold and highly connected to each other like a net. The deposition of calium salts is around the cells and lines the surface of the scaffold and gradually fills the pores of the scaffold. MTT analysis shows that the viability and the proliferation state have the characteristic proliferation pattern of stem cells.
Six:
After induction of osteogenic differentiation, the OPN and OCN which are specific to osteoblast are positively expressed by hMSCs. The analysis of activity for ALP shows that the ALP activity of induced cells is strikingly higher than that of control group, and the hMSCs have completely differentiated to the osteoblast at day 20.
Seven:
The bone defect model of rat has been successfully established and provides a good basis for the osteogenic differentiation in vivo.
Eight:
We label the hMSCs with Brdu and then transplant the indeuced hMSCs-scaffold complex into the defect area of the rat. After 4 weeks, X-rays showed callus formation in the experimental group. At week 8, the amount of the callus and its density had increased. The defect was filled with newly formed bone and there was no obvious distinction between the callus and the broken ends of the fractured bone. At week 12, the extra callus was absorbed and the broken ends of fractured bone connected to each other very well. In contrast, there were obvious gap between broken ends of fractured bone in the control group. The fracture is ununion. Hematoxylin and eosin stains of the experimental group at week 4 shows that the bony callus was encapsulated by fibrous tissue located in the defect area. By week 8, fibrous tissue had been replaced by irregularly arranged osseous tissue which contained islands of medullary bone marrow. By week 12, the bony tissue was arranged regularly in the defect area and the callus had transformed into mature lamellar bone. The cortical and medullary bone had recovered their anatomical relationship. In contrast, the defect in the control group is filled with amount of necrotic tissues, inflammatory cells and fibrous tissue. Immunochemistry staining shows that the hMSCs survive and continuingly express OPN and OCN which specific to osteoblasts. It suggests that hMSCs- scaffold complex has the ability to promote the healing of the bone defect.
Collectively, the immunoreactivity of bio-derived scaffold is low, without obvious tendency to induce inflammation. It appeared to have good biocompatibility and high biological safety. The bio-derived scaffold has good compatibility with the mesenchymal stem cells. And the scaffold promotes osteoblast differentiation of the mesenchymal stem cells. It is of importance to provide a basis for the clinical engineered repair of the bone defect.
本实验通过各种理化处置制备了来源于猪松质骨的生物衍生骨支架材料,大大降低了抗原性;并将其与鼠尾胶原复合,制成了生物衍生骨-胶原凝胶复合支架材料,增强了其细胞相容性,有利于细胞的吸附、分化和增殖;经检测复合支架材料具有良好的组织相容性和生物安全性;在体外对hMSCs -支架材料复合物进行成骨诱导后,hMSCs表达成骨特异性标记OPN和OCN,表达强度比单纯hMSCs诱导组强,同时未诱导的复合物中hMSCs也有表达,表明本研究制备的复合支架材料除具有一定的骨诱导作用外,同时也能增强hMSCs的成骨能力;建立了标准化的大鼠骨缺损模型后,将诱导后的hMSCs-支架材料复合物移植到缺损处,证明hMSCs-支架材料复合物具有明显的促进骨缺损修复的作用。
总之,本研究获得的复合支架材料引起的免疫反应较弱,具有良好的生物相容性和细胞相容性,并成功的利用hMSCs与支架材料的共培养来建造组织工程化人工骨、修复骨缺损,为临床大范围骨缺损修复奠定了很好的基础。
The repair of bone defect is always a very important and hot spot for both clinicians and laboratory researchers. As far as concerned, the repair and regeneration of the wide-bound bone defect caused by trauma, infection and tumor excision remain a significant clinical challenge. With the development of histological engineering by the last decade, scientists are trying to create artificial bones by inducing the osteogenic differentiation from potential cells in vitro, promoting amplification of differentiated cells and then seeding them into the degradable scaffold. It has a very promising perspective since it does well for the repair and regeneration of the wide-bound bone defect.
It has been a hot spot of histological engineering to identify promising candidate seed cells. Stem cells possess self-renewal, long-term cell viability, and multilineage differentiation potential, and all the characteristics make them taken as the best seed cells. However, application of multipotentonic embryonic stem cells may be restricted because of ethical issues and immunological complications. To avoid possible ethical problems and immune rejection, it is considered that adult stem cells are a more logical population for engineered tissue, thus being attractive to both clinical and laboratory scientists.
MSCs are multipotential stem cells derived from mesoderm, possessing potent proliferative capacity and self-renewal and multilineage differentiation potential. MSCs can differentiate into osteocytes, chondroncytes, adipocytes, myocytes, hepatocytes, neuron-like cells and neuroglial cells with the controlled conditions and cytokines. It is of advantage to use the MSCs as the seed cells and all of the characteristics of MSCs make them taken as the best seed cells: 1) easy procurement and minimal possibility of clinical discomfort and injury; 2) no ethical problems and immune rejection; 3) potent multiple amplification in vitro; and 4) easily induced differentiation and inserting and expression of exogenous genes easily.
A range of synthetic materials including polylactic acid and hydroxyapatite ceramic have been investigated for bone tissue engineering applications. However, no single material has been able to satisfy all the requirements for creating optimal scaffolding. They have several drawbacks: confused three-dimensional space structure, no secretion potential of osteoinductive factor and the low cell compatibility.
So it is very necessary and urgent to identify other candidate scaffolds to avoid those problems. We create a kind of bio-derived scaffold from the spongy bone of the pig after deproteiniztion, decalcification, defatting and elimination of cells and antigens. It expresses not only osteoinductive factors and possesses low immunogenicity, high cell compatibility and no obvious architectural alteration of the main framework since the main content is collagen.
The capacity of the pure scaffold only to induce osteogenic differentiation is limited and as the development of the histological engineering, complex materials have been developed to enhance the osteogenic differentiation potential. The fact that hMSCs poorly adhere to the pure bio-derived scaffold only limits its application. The collagen lattice from rat tail has been often used as the extracellular matrix in in vitro cell culture. It shows good cytocompatibility and three dimensional ultrastructural features. The three dimensional structure of the collagen has strength and the pores, which keeps the biological osmolarity. This property anchor and support the cell being adherent to the scaffold and thus provide a proper microenvironment for proliferation and differentiation of hMSCs.
We obtain high homogenous hMSCs by the density gradient separation method, monocloning screen method and adherent culture system. We create the complex bio-derived bone scaffold and the collagen from mouse tail and compare it’s cytocompatibility with other scaffolds by in vitro co-culture of the relative scaffold with hMSCs. We then create the complex scaffold by mixing together of bio-derived bone scaffold with collagen from mouse tail. Then in vivo transplantation of the complex scaffold, lymphocyte transformation test, using concanavlinA as a stimulant, and complement dependent cytotoxity test to detect the histocompatibility of the bio-derived scaffold are performed. In vitro induction of osteogenic differentiation of hMSCs-scaffold complex has been investigated. We also has established the rat bone fracture model and also transplant the hMSCs-scaffold complex after undergoing induction of osteogenic differentiation between the defect area of the cut ends of bone to detect the bone repair.
Results:
One:
A standardized platform has been established for isolation, cultivation, purification, identification and amplification of hMSCs with homogeneity of higher than 98%. The hMSCs under in vitro culture maintain stable biological characteristics at even passage 12 and stay at the state of undifferentiation, providing an excellent cell source for extended experiments
Two:
Dexamethasone, sodium glycerophosphate and vitamin C are added to the control medium to induce the osteogenic differentiation of hMSCs. The induced cells gather together like nodisity and deposition of calium salts is observed. Also after induction, cells positively express OPN and OCN which are specific to osteoblasts.
Three:
We compared cytocompatiblity of the complex scaffold by mixing together of bio-derived bone scaffold with the collagen from mouse tail with that of nanometer material PLGA on the adherence capacity and the time needed by hMSCs to be adherent to the surface of the scaffold and the proliferation rate. It shows that the collagen from mouse tail has a better cytocompatibility. The bio-derived scaffold has the natural structure of cancellous bone, thus functioning well as the three dimensional scaffolds for the engineered bone. We eventually choose the complex scaffold.
Four:
We perform in vivo transplantation of the complex scaffold, lymphocyte transformation test, using concanavlinA as a stimulant, and the complement dependent cytotoxity test to detect the histocompatibility of the bio-derived scaffold. The immunoreactivity of bio-derived scaffold is low, without obvious tendency to induce inflammation. It appeared to have good biocompatibility and high biological safety.
Five:
We create the mixture suspension of hMSCs and collagen from mouse tail and then incubate the suspension for two minutes. Then the suspension is added to the bio-derived bone scaffold when it reaches the semisolid state, and cultivate in the incubator for 2 to 5 minutes with a condition of 5% carbon dioxide, temperature 37. The complex scaffold has been established when the collagen becomes hard. The three dimensional structure of the collagen has strength and pores, which keeps the biological osmolarity. This property anchor and support the cell being adherent to the scaffold and make cells grow well in pores of the scaffold, and provide a large space for cell.
Scanning electron microscope and light microscope images show that the cells grow in a pattern of multilayer. And some of the cells are well adherent to the surface of the scaffold and highly connected to each other like a net. The deposition of calium salts is around the cells and lines the surface of the scaffold and gradually fills the pores of the scaffold. MTT analysis shows that the viability and the proliferation state have the characteristic proliferation pattern of stem cells.
Six:
After induction of osteogenic differentiation, the OPN and OCN which are specific to osteoblast are positively expressed by hMSCs. The analysis of activity for ALP shows that the ALP activity of induced cells is strikingly higher than that of control group, and the hMSCs have completely differentiated to the osteoblast at day 20.
Seven:
The bone defect model of rat has been successfully established and provides a good basis for the osteogenic differentiation in vivo.
Eight:
We label the hMSCs with Brdu and then transplant the indeuced hMSCs-scaffold complex into the defect area of the rat. After 4 weeks, X-rays showed callus formation in the experimental group. At week 8, the amount of the callus and its density had increased. The defect was filled with newly formed bone and there was no obvious distinction between the callus and the broken ends of the fractured bone. At week 12, the extra callus was absorbed and the broken ends of fractured bone connected to each other very well. In contrast, there were obvious gap between broken ends of fractured bone in the control group. The fracture is ununion. Hematoxylin and eosin stains of the experimental group at week 4 shows that the bony callus was encapsulated by fibrous tissue located in the defect area. By week 8, fibrous tissue had been replaced by irregularly arranged osseous tissue which contained islands of medullary bone marrow. By week 12, the bony tissue was arranged regularly in the defect area and the callus had transformed into mature lamellar bone. The cortical and medullary bone had recovered their anatomical relationship. In contrast, the defect in the control group is filled with amount of necrotic tissues, inflammatory cells and fibrous tissue. Immunochemistry staining shows that the hMSCs survive and continuingly express OPN and OCN which specific to osteoblasts. It suggests that hMSCs- scaffold complex has the ability to promote the healing of the bone defect.
Collectively, the immunoreactivity of bio-derived scaffold is low, without obvious tendency to induce inflammation. It appeared to have good biocompatibility and high biological safety. The bio-derived scaffold has good compatibility with the mesenchymal stem cells. And the scaffold promotes osteoblast differentiation of the mesenchymal stem cells. It is of importance to provide a basis for the clinical engineered repair of the bone defect.
引文
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