Alultrastructural Features And Study On Molecular Mechanism Of Multiple Osteochondroma

Multiple osteochondroma(MO)is a kind of skeletal dysplasia,which can form different size of bone uplift.It is an autosomal dominant genetic disease and most of the patients are teenagers who have a history of family.MO can interfere with the growth of normal osteoepiphysis,leading to bone deformities.Most of the patients present short stature,body force line abnormality and dysfunction.Moreover,this tumor is characterized by a layer of cartilage that is shaped like a hat named cartilage cap on the top of the tumor.The cartilage cap can promote tumor growth by the chondrification.The tumor growth contributes to local pain,bone deformities,activity obstacle,or oppression towards adjacent neurovascular coursing corresponding symptoms.The sudden growth of tumor can lead to malignant transformation.To date,the drug for multiple osteochondroma is still in the stage of laboratory and the main treatment is surgery,including tumor resection and orthopaedic surgery to improve the exterior deformation and limb function.Recently,with the advancement of new methods of molecular biology,the study of MO gradually transforms from macro to micro,and people pay more attention to the study of pathogenesis.Pathological anatomy shows the cauliflower shape of MO which is constituted by perichondrium,cartilage cap and osseous tumors.Perichondrium is a key component of cartilage,which plays in buffering and lubrication role in physiologicalactivity and alters in MO patients.However,to date,little is known about perichondrium in MO.Molecular biology research shows that a variety of cytokines play an important role in the process of bone formation.Reports reveal that the expression of Sex Determining Region Y Box 9(Sox9)is associated with the development of osteochondroma,and Sox9 expression can induce the formation of chondrosarcoma cells.Besides,the Osterix gene is also found to be closely tied to osteoblast differentiation and bone formation,and it can regulate expression of many osteogenesis factors.In the present study,we observed the ultrastructures of cartilage cap and perichondrium in multiple osteochondroma by using scanning electron microscopy and transmission electron microscope,which increase our knowledge about ultrastructural structure of MO.These findings explore the pathogenesis of this disease and provide reliable basis for ultrastructural diagnosis of MO.We further investigated the expression levels of Sox9 and Osterix in osteochondromas tissues and peripheral blood and carried out the correlation analysis between Sox9 and Osterix to explore the possible etiology and pathogenesis of MO.This research was divided into three parts:1.Alultrastructural features of cartilage cap in multiple osteochondromas;2.Alultrastructural features of perichondrium in multiple osteochondromas;3.Study on molecular mechanism of multiple osteochondromas.Part 1 Alultrastructural Features of Cartilage Cap in Multiple OsteochondromasMethods1.Sample selection.MO group: 30 osteochondroma tissue samples were obtained from patients who were diagnosed with MO and had undergone routine tumor resection.Control group: 30 cartilage tissue samples of iliac wing bones were obtained from patients who undergone osteotomy of hip dislocation.2.Scanning electron microscopy of cartilage in normal cartilage tissue and cartilage cap in MO patients.3.Transmission electron microscopy of cartilage in normal cartilage tissue cartilage cap in MO patients.Results1.Results from scanning electron microscopy of cartilage in normal cartilage tissue and cartilage cap in MO patients.Control group(normal cartilage tissue of iliac wing bones): A small amount of chondrocytes are observed in the tissue,and chondrocytes exists in the pit of cartilage;The shapes of cartilage cell are circular or irregular,and bits of bumps are found on the cell surface;A large number of scattered and sparse collagen fibers exist around the pit of cartilage.MO group(cartilage cap of osteochondromas in MO patients): A large number of irregular cartilage lacuna are observed in cartilage tissue and mounts of cartilage cells exist in cartilage lacuna;The volume of chondrocyte increased significantly;cell shapes are irregular;The surface of cells are rich in protuberance;The dense structure of the collagen fibers are observed around the cartilage lacuna of cartilage tissue surface.2.Results from transmission electron microscopy of cartilage in normal cartilage tissue and cartilage cap in MO patients.Control group(normal cartilage tissue of iliac wing bones): A small number of chondrocytes are found;The majority of cell shapes are oval;A small amount of short microvilli are observed on cell surface;The nucleus are irregular in shape and nuclear chromatin are condensed;Few organelles are observed in cytoplasm;The rough endoplasmic reticulum exhibit a funicular distribution;The ribosome particles are observed on part of rough endoplasmic reticulum surfaces;The mitochondria are circular in the cytoplasm;The volume of mitochondria is decreased;Glycogen granules are distributed fascicularly on the mitochondria;MO group(cartilage cap of osteochondromas in MO patients): A large number of chondrocytes are found;cartilage cell volume was increased significantly;The nuclei are round with an increased volume;The nucleus is rich in chromatin and the nucleolus is obvious;The nucleo-cytoplasmic ratio is high;A small number of mitochondria are observed and they were round or oval;The number of rough endoplasmic reticulums are increased with expansion,and low electron density substances deposit on rough endoplasmic reticulum pool and a small number of lysosomes are found;Free ribosomes are observed in cells;Some protein-like substances deposit in intercellular space.Part 2 Alultrastructural Features of Perichondrium in Multiple OsteochondromasMethods1.Sample selection.MO group: 30 osteochondroma tissue samples were obtained from patients who were diagnosed with MO and had undergone routine tumor resection.Control group: 30 cartilage samples of iliac wing bones were obtained from patients who undergone osteotomy of hip dislocation.2.Scanning electron microscopy of perichondrium in normal cartilage tissue and perichondrium in MO patients.3.Transmission electron microscopy of perichondrium in normal tissue and perichondrium in MO patients.Results1.Results from scanning electron microscopy of perichondrium in normal tissue and perichondrium in MO patients.Control group(normal perichondrium of iliac wing bones): The dense structure and beam pattern of the collagen fibers are observed in normal perichondrium;Collagen fibers in normal perichondrium array orderly and loosely.MO group(perichondrium of osteochondromas in MO patients): Ollagen fibers in cartilage perichondrium of MO patients are compact and desultory compared with control group;Collagen fibers in perichondrium of MO patients are obviously compact and arrayed like cluster compared with control group.2.Results from transmission electron microscopy of cartilage in normal cartilage tissue and erichondrium in MO patients.Control group(normal perichondrium of iliac wing bones): The nuclei in fibroblast cells is circular;Nucleolus volume is small and irregularly shaped;The cell surface appear no small bumps;Chromatin aggregation exists in nuclear;Collagen fibers in normal perichondrium array orderly and loosely;There is not collagen fiber and capillaries around the perichondrium;Chromatin aggregation exists in nuclear of fibroblast cells;Rough endoplasmic reticulum and mitochondrial are abundant;No secretion is observed in fibroblast cells;Collagen fibers array orderly and loosely;Mitochondria are thick and abundant rough endoplasmic reticulum are observed in fibroblast cells;The secretion of collagen fiber and capillary formation are not observed around fibroblasts.MO group(perichondrium of osteochondromas in MO patients): Fibroblasts secrete lots of filaments collagen fiber with local intensive distribution,and fibroblast cell nucleus is irregular;Low electron density substances deposit around the nucleus and the secretion of collagen fiber observed around the nucleus;Lots of homogeneous collagen are observed in cytoplasm;Nucleus volume is increased;Nucleolus is observed obviously;Euchromatin is rich in nucleolus;rough endoplasmic reticulum is abundant with expansion;Mitochondrial mild enlargement are observed in cytoplasm;collagen fibers array disorderly with local denseness;new capillary blood vessels or atresia are observed locally;Vessel lomen is not observed;Lots of homogeneous collagen are observed in cytoplasm with low electron density;Rough endoplasmic reticulum is abundant with expansion;Mitochondrial mild enlargement are observed in cytoplasm.Part 3 Study on Molecular Mechanism of Multiple OsteochondromasMethods1.Histochemical staining was used to detect the expression levels of Sox9 and Osterix in normal cartilage tissue and osteochondromas tissues of MO patients.2.Correlation analysis between Sox9 and Osterix in osteochondromas tissues of MO patients.3.ELISA was used to detect expression levels of Sox9 and Osterix in peripheral serum of control group and MO patients.4.Correlation analysis between Sox9 and Osterix in peripheral serum of MO patients.5.q RT-PCR was used to detect the expression levels of Sox9 and Osterix m RNAs in normal cartilage tissue and osteochondromas tissues of MO patients.6.Correlation analysis between Sox9 and Osterix m RNAs in osteochondromas tissues of MO patients.7.q RT-PCR was used to detect the expression levels of Sox9 and Osterix in normal cartilage tissue and osteochondromas tissues of MO patients.8.Relationship analysis between the morphologies of collagenous fiber and bloodcapillary and the expression of Sox9 and Osterix in normal cartilage tissue and osteochondromas tissues of MO patients.Results1.Results from histochemical staining show that Sox9 and Osterix express positively in normal cartilage tissue and osteochondromas tissues of MO patients.The protein levels of Sox9 and Osterix in osteochondromas tissues are higher than that in control groups.2.Correlation analysis indicates that the expression levels of Sox9 and Osterix correlate positively in osteochondromas tissues of MO patients.3.Results from ELISA show that the expression levels of Sox9 and Osterix in p peripheral serum of MO patients are higher than that in control groups.4.Correlation analysis presents that the expression levels of Sox9 and Osterix correlate positively in peripheral serum of MO patients.5.Results from q RT-PCR show that the m RNA levels of Sox9 and Osterix in osteochondromas tissues of MO patients are higher than that in control groups.6.Correlation analysis indicates that the expression levels of Sox9 and Osterix m RNAs correlate positively in osteochondromas tissues of MO patients.7.Results from western blot show that the protein levels of Sox9 and Osterix in osteochondromas tissues of MO patients are higher than that in control groups.8.Morphologies of collagen fibers and capillaries may be associated with the expression of Sox9 and Osterix in MO.Conclusion1.Compared with normal cartilage tissue,the alultrastructural features of cartilage cap change significantly.These results indicate the vigorous metabolism of cartilage tissue in cartilage cap of MO patients.The data also suggest the activation of proliferation and protein synthesis in cartilage cell,as well as the density and calcification of collagen fiber in part of the osteochondromas cartilage cap.All the alterations in cartilage tissue pay the way for the development,progression and malignant transformation of MO.2.Compared with normal cartilage tissue,the alultrastructural features of perichondrium alter obviously.These results suggest the vigorous metabolism of cartilage tissue in perichondrium of MO patients.The results also indicate the activation of growth and protein synthesis of cartilage cell,as well as the density rich collagen fiber in perichondrium,which support the development and malignant transformation of MO.3.m RNA and protein levels of Sox9 and Osterix in osteochondromas tissues and peripheral serum are higher than that in control groups.Correlation analysis indicates that the expression levels of Sox9 and Osterix m RNAs correlate positively.4.The expression of Sox9 and Osterix may promote the formation of collagen fibers and capillaries in MO.