The Human Placenta Derived Mesenchymal Stem Cells Seeded On A Silk Fibroin/Hydroxyapatite Scaffold Enhance Bone Repair In A Rabbit Radius Segmental Bone Defect Model

The principle of tissue engineering is to use the engineering methods and tools to build and regenerate various tissues and organs to replace the lesions organization, which means to implant the seed cells with the specific function or differentiation potential and the biodegradable materials (scaffolds) to form a specific tissue or organ with normal structure and function at last, as the seed cells proliferating, secreting extracellular matrix, and the biological material gradual degradated and absorbed. Therefore, the seed cells and the scaffold were main research contents in the Tissue Engineering.The important problems in bone tissue engineering are the source, isolation, and expansion of seed cells. Mesenchymal stem cells (MSCs) are derived from the mesoderm at the early development stage. It can be adopted as an ideal source of seed cells which have the potential to be induced into osteogenic, chondrogenic, and adipogenic cells or even tendon and adipose tissues. MSCs are easy to be obtained, cultured, expanded in vitro as well as to be easily induced into designed tissues. Currently, the bone marrow-derived MSCs (BMSCs) are widely used. However, the amount of MSCs in bone marrow is extremely low and accounts for about 0.01~0.001% of the bone marrow derived cells. Increasing evidence suggests MSCs with osteogenic potential have been isolated from a diverse range of tissues including adipose tissue and perinatal tissues such as umbilical cord, placenta, and umbilical cord blood. Previous findings from our group indicate that MSCs from the placenta (PMSCs) could be obtained and expanded in vitro. In vitro cultured PMSCs still well maintain the same biological characteristics as the BMSCs. In addtion, the cell bank of PMSCs could be set up in advance for the clinical trials, suggesting that PMSCs have a wide application prospect.The biomaterial scaffolds used in bone tissue engineering should be biocompatible and osteoconductive. In addition, they should have proper porosity and pore size for cell attachment and flow transport of nutrients and metabolic waste, as well as proper mechanical properties to match bone tissues. Hydroxyapatite (HA) is the main mineral component of bones. HA has been attracted much attention for its excellent biocompatibility and bioactivity. However, the biodegradation rate of sintered HA ceramics is very slow and the porous HA ceramics is fragile.Silk fibroin (SF), especially, produced by the Bombyx mori silkworms is one of the most abundant natural proteins and can be obtained easily and inexpensively. Previous studies indicated that silk fibroin matrices could support human bone marrow-derived MSCs (BMSCs) attachment and proliferation, the calcium deposition on them and the development of bone-like trabeculae with cuboid cells. However, because of the low hardness and easy deformation under stress, pure silk fibroin materials are difficult to adapt to environmental tissue. Thus, it is necessary to fabricate new silk materials which have highly hardness and toughness and suitable mechanical properties to fit hard tissue and be applied as bone tissue engineering or bone tissue inducing matrices. The low degradation rate, high brittleness of HA, and poor mechanical strength of SF can be well solved through combining HA and SF to be SF/HA scaffold with an appropriate degradation rate and favorable biocompatibility.In Our study we isolated and cultured PMSCs in vitro, explored its immunological characteristics, then produced the bio-composite materials with cultured PMSCs and SF/HA porous materials to repair the rabbit bone defect. Through this study, we want to evaluate the value and potentiality in application of both PMSCs and SF/HA scaffold in bone tissue engineering.PartⅠObject:To investigate the distribution and isolation of MSCs in human placenta and to establish the stable expanding culture system for PMSCs in vitro.Method: To take three tissue samples from the term (38–40 weeks'gestation) placentas of healthy donor mothers, sample one (A) is from the central part of the placenta where the umbilical cord attached, sample two (C) is the fringe part of placenta, and the sample three (B) is the part between part A and part C, to do the full-thickness serial sections, separately, then, immunofluorescence and immunohistochemical staining were adopted to observe the distribution of CD166,CD90 and CD29 positive cells, finally, the HPIAS-1000 color pathological image analysis system was used for the image analysis. Besides, the overall perfusion method was adopted to isolate PMSCs and the cell surface markers and multi-differentiation potential of the cultured PMSCs was identified.Result: Comparing with other area (part B, and part C), CD166, CD90 and CD29 positive cells were found mainly distributed in part A , the OD value in this group was statistically significant (P<0.05), and all these CD166, CD90 and CD29 positive cells were found gathered around the blood vessels. The cultured PMSCs obtained by overall perfusion method were CD73,CD90,CD166 and CD105 positive, while CD34,CD45,CD14 and CD106 were negative. Exposure to osteogenic inductive medium resulted in secretion of extracellular calcium crystals, identified by von Kossa staining, indicating osteogenic differentiation. When cells were cultured in adipogenic inductive medium, intracytoplasmic lipid vacuoles were observed, and confirmed by oil red O staining. After been cultured in chondrogenic inductive medium, chondrogenic differentiation was identified demonstrated by positive type II collagen staining. After been exposed to the neural inducer, most PMSCs showed the typical morphology of neurons with many projections, and the induced cells were NSE,GFAP, neural cell specific markers, positive by immunofluorescence staining, while if change the inductive medium to the endothelial one, 7 days later, cell body was found gradually retracted, three-dimensional sense enhanced, and the endothelial cell-specific markers KDR and v-WF were expressed on the induced cells by immunofluorescence staining.Conclusion: CD166, CD90 and CD29 positive cells were found in placenta, moreover there was a certain correlation between the distribution of these cells and blood vessels in the placenta. The region where the umbilical cord attached was proved to be the MSCs-rich region. Besides, overall perfusion method could be adopted to culture the PMSCs in vitro, and the cultured cells were proved having the ability to differentiate into bone, cartilage, fat, nerves and endothelial cell.PartⅡObject:To study the immunological characteristics of PMSCs and its compatibility with silk fibroin (SF).Method: The two-way mixed lymphocyte reaction (MLR) system both for human and the rabbits were established. After adding PMSCs which was treated by the mitomycin in the MLR system, the lymphocyte proliferation rate were measured by 3H incorporation, and the content of IL-2 and IFN-γin the supernatant was detected by ELISA to observe the effects of human PMSCs on lymphocyte proliferation both from human and the rabbits. Meanwhile, PMSCs were cultured in the SF coating flasks, then its phenotype was analysised by flow cytometry, its adipogenic, chondrogenic, and osteogenic differentiation potential were determined by specific staining, and its immune regulation ability was detected in the MLR system, finally its growth condition was observered under the electric microscope.Result: The proliferation of T lymphocytes could be inhibited to different extents by adding different ratios of PMSCs to human or rabbit PBMCs, and the inhibition rate increased as the ratio of PMSCs to PBMCs increased. Meanwhile, PMSCs could reduce cytokines IL-2, IFN-γsecretion in the mixed lymphocyte reaction system. After being cultured on the SF film, the growth characteristics, surface markers, multi-differentiation capacity of PMSCs were not changed; the electric microscope observation results showed that PMSCs proliferated in the SF film materials after been co-cultured for 8 days, secreted large amounts of granular, mesh matrix substances well filled in the material clearance.Conclusion: The cultured PMSCs had the immune regulation ability and inhibited xenogeneic immune cells as well. PMSCs also held the cross-mesoderm differentiation ability, and could be induced to differentiate to neural cells and endothelial cells. SF had good biocompatibility and had no effects on the PMSCs growth characteristics, surface markers and multi-differentiation capacity.PartⅢObject:To explore the silk fibroin/hydroxyapatite (SF/HA) porous materials serving as a delivery vehicle for human PMSCs in a rabbit bone defect model so as to evaluate its value and mechanism of application in bone tissue engineering.Method: The BrDU labeling PMSCs were cultured in SF/HA porous materials, then transplanted them to the radial defect animal model. A total of 24 healthy adult New Zealand rabbits were randomly divided into experimental group, control group and the blank control group, and a 15-mm length of radius defect model was established. The gross observation result, histological result and the radiographic examination result were analyzed on 2nd, 4th, 8th, and 12th week after transplantation to evaluate fracture healing.Result: Radiological and histological examination results of 2, 4, 8, 12 weeks after implantation showed that new bone formation in the experimental group were better than the control group, while in the blank control group, no new bone formation was detected at each time point. Immunofluorescence staining showed BrDU-positive cells were still visible exists 8 weeks after transplantation in the experimental group. Moreover, results from radiographic examination showed significant differences in the radiographic score between experimental groups and control groups at the 8th week and the12th week(P<0.05).Conclusion: PMSCs/SF/HA composites materials could be adopted to construct tissue-engineered bone which can repair the bone defect. And it proved that SF/HA porous materials could be applied as a scaffold for bone tissue engineering, and PMSCs were a new source of seed cells.Summary: CD166, CD90 and CD29 positive cells were found in placenta, moreover there was a certain correlation between the distribution of these cells and blood vessels in the placenta. Besides, overall perfusion method could be adopted to isolate the PMSCs in placenta, and the cultured cells held the cross-mesoderm differentiation ability and the immune regulation ability. SF had good biocompatibility and SF/HA porous materials could be applied as a scaffold for bone tissue engineering, and PMSCs were a source of seed cells.