THE IN VITRO DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES : V. THE FORMATION OF MACROPHAGE LYSOSOMES

A combined morphological, autoradiographic, and cytochemical study at the electron microscope level has been directed towards the formation of electron-opaque granules of cultured macrophages. Labeling of the membrane-bound vesicular structures of pinocytic origin was accomplished with colloidal gold. The initial uptake of gold occurred within micropinocytic vesicles. These electron-lucent vesicles subsequently fused with and discharged their contents into larger pinocytic vacuoles. Colloidal gold was homogeneously distributed in the large pinosomes. In contrast, gold was initially deposited in the periphery of preformed dense granules indicating that these structures were also in constant interaction with the external environment. Colloidal gold was not observed within the cisternae of the endoplasmic reticulum nor within the saccules or vesicles of the Golgi apparatus. There were, however, many small, gold-free vesicles, indistinguishable from Golgi vesicles, which were preferentially aligned about and appeared to fuse with the large pinosomes. The intracellular flow of leucine-H³-labeled protein was followed by electron microscopic autoradiography. After a 15 min pulse of labeled amino acid there was initial labeling of the rough endoplasmic reticulum. Subsequently, much of the label appeared in the Golgi complex. At still later time periods the cytoplasmic dense granules contained the majority of the isotope. Acid phosphatase activity was localized to the dense granules and in the majority of cells to the Golgi apparatus. It is suggested that hydrolytic enzymes are initially synthesized in the endoplasmic reticulum and are then transferred to the Golgi apparatus. Here they are packaged into small Golgi vesicles which represent the primary lysosome of macrophages. The Golgi vesicles subsequently fuse with pinosomes, thereby discharging their hydrolases and forming digestive granules or secondary lysosomes.     

THE IN VITRO DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES : IV. THE ULTRASTRUCTURE OF MACROPHAGE DIFFERENTIATION IN THE PERITONEAL CAVITY AND IN CULTURE

The structure of unstimulated mouse peritoneal phagocytes has been examined by electron microscopy and compared to cells obtained from the inflamed peritoneum and from cultures maintained in vitro. The unstimulated cell resembles the blood monocyte and contains a moderate amount of rough surfaced endoplasmic reticulum, a small but well defined Golgi apparatus and a few, small, electron-opaque granules in the cytoplasm. During in vitro cultivation there are marked changes in cell ultrastructure. Most prominent is the formation of large electron-opaque granules, some of which have a complex matrix containing both electron-opaque and lucent vesicles. In addition, there is an increase in size of the Golgi apparatus with the appearance of new lamellae and tiny, smooth surfaced vesicles. With continued cultivation, large lipid droplets are found in apposition to the rough endoplasmic reticulum. The formation and size of electron-opaque granules as well as the enlargement of the Golgi region is stimulated by high concentrations of serum in the medium. Cells obtained from the peritoneal cavity of lipopolysaccharide stimulated animals demonstrated changes in ultrastructure similar to those seen in cells cultured in vitro.     

Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence

Rabbit blood monocytes, which contain no cytochemically demonstrable peroxidase, develop peroxidatic activity in the RER and perinuclear cisternae within 2 h after adherence to serum- or fibrin-coated surfaces. A similar reactivity appears in surface-adherent human and rat blood monocytes. In both localization and characteristics, this enzyme reactivity in monocytes resembles that normally seen in the resident peritoneal macrophages of the rabbit, as well as in several types of tissue macrophages in other species. Thus this observation supports the concept, presently based on the kinetic data of other investigators, that blood monocytes are the precursors of such cells. Moreover, the appearance of new enzyme activity after adherence may reflect alterations in cellular metabolism resulting from plasma membrane:surface interactions.     

The appearance of transition forms between monocytes and Kupffer cells in the liver of rats treated with glucan. A cytochemical and ultrastructural study

A massive accumulation of mononuclear phagocytes in the rat liver was found after the injection of glucan, a beta-1,3-polyglucose. Portal vessels and central veins contained large numbers of rounded and elongated cells which were adherent to the endothelium. By scanning electron microscopy most of these cells exhibited prominent lemellopodia, raised ridge-like profiles and blebs, the typical features of mononuclear phagocytes. Peroxidase cytochemistry revealed that whereas most cells in portal vessels were monocytes with peroxidase positive and negative granules, the majority of cells in central veins were macrophages exhibiting peroxidase activity in nuclear envelope (NE) and endoplasmic reticulum (ER). In sinusoids monocytes and macrophages were seen side by side. The major finding of the present study was that some cells, adherent to the endothelium or portal vessels or to the lining of sinusoids, exhibited a peroxidase pattern intermediate between monocytes and Kupffer cells, i.e. strong peroxidase activity in cytoplasmic granules, as well as a weak to moderately positive peroxidase reaction in NE and ER. These intermediate cells also contained peroxidase-negative granules with halo, which are usually seen in monocytes. Furthermore, examination of serial ultrathin sections of typical Kupffer cells revealed numerous peroxidase-positive granules and peroxidase-negative granules with halo in their cytoplasm. Finally, dividing Kupffer cells with positive peroxidase reaction in ER were found. These in vivo observations provide ultrastructural and cytochemical evidence in support of the concept of derivation of Kupffer cells from monocytes, demonstrating in addition that Kupffer cells are capable of self-replication in situ.     

Differentiation of macrophages from normal human bone marrow in liquid culture. Electron microscopy and cytochemistry.

To study the various stages of human mononuclear phagocyte maturation, we cultivated bone marrow in an in vitro diffusion chamber with the cells growing in suspension and upon a dialysis membrane. At 2, 7, and 14 days, the cultured cells were examined by electron microscopy and cytochemical techniques for peroxidase and for more limited analysis of acid phosphatase and arylsulfatase. Peroxidase was being synthesized in promonocytes of 2- and 7-day cultures, as evidenced by reaction product in the rough-surfaced endoplasmic reticulum, Golgi complex, and storage granules. Peroxidase synthesis had ceased in monocytes and the enzyme appeared only in some granules. By 7 days, large macrophages predominated, containing numerous peroxidase-positive storage granules, and heterophagy of dying cells was evident. By 14 days, the most prevalent cell type was the large peroxidase-negative macrophage. Thus, peroxidase is present in high concentrations in immature cells but absent at later stages, presumably a result of degranulation of peroxidase-positive storage granules. Clusters of peroxidase-negative macrophages with indistinct borders (epithelioid cells), as well as obvious multinucleated giant cells, were noted. Frequently, the interdigitating plasma membranes of neighboring macrophages showed a modification resembling a septate junction--to our knowledge, representing the first documentation of this specialized cell contact between normal macrophages. We suggest that such junctions may serve as zones of adhesion between epithelioid cells.     

MORPHOLOGY AND PEROXIDASE CYTOCHEMISTRY OF MOUSE PROMONOCYTES, MONOCYTES, AND MACROPHAGES

Mouse promonocytes have been identified and studied in cultures of bone marrow cells. These cells have a diameter of 14–20 µ, and in stained preparations reveal a large, indented or folded nucleus, and basophilic, finely granular cytoplasm. The living promonocyte viewed by phase contrast shows additional features: nucleoli, small dense bodies, and vesicles in the cytoplasm adjacent to the nuclear hilus, and slight membrane ruffling. Prominent ultrastructural components of promonocytes include a well developed Golgi apparatus, small numbers of centrosomal granules and vacuoles, extensive ribosomal aggregates, and finger-like projections of the cell surface. Promonocytes engage in pinocytosis and phagocytosis, but they are less active in these functions than are peripheral blood monocytes of peritoneal macrophages. Promonocytes are positive for peroxidase, the reaction product being localized to granules most of which are centrally situated in the cell. Monocytes in blood or in inflammatory peritoneal exudates display much smaller numbers of peroxidase-positive granules, and various types of mature mouse macrophages are peroxidase negative.     

In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes

The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co- cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes.     

Control of the acute phase response. Demonstration of C-reactive protein synthesis and secretion by hepatocytes during acute inflammation in the rabbit

To determine the cell of origin of C-reactive protein (CRP) and to cast light on the mechanisms leading to the acute phase response, we used an immunoenzymatic technique to visualize this protein in livers from rabbits at intervals after intramuscular injection of turpentine. CRP was detected only in hepatocytes. 8 h after turpentine injection, CRP was demonstrated in occasional periportal hepatocytes. With time, larger numbers of positive cells were detected successively in perilobular, midlobular, and centrilobular areas. On electron microscopy, CRP was detected in rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and Golgi apparatus (GA). When colchicine was administered to inhibit cellular secretion of CRP, intensity of reaction and number of CRP-containing hepatocytes were substantially greater than without colchicine, but the sequence of intralobular distribution was similar. At peak serum response 38 h after turpentine injection, CRP could be demonstrated in most hepatocytes. Electron microscopic studies showed accumulation of CRP on membranes and lumina of RER, SER, GA, and in cytoplasmic vacuoles. These findings indicate that CRP is produced by progressively increasing numbers of hepatocytes after inflammatory stimulus and suggest that a mediator, acting initially in portal zones, is responsible for recruitment of cells to CRP production.     

Ultrastructural studies of human lymphoid cells. mu and J chain expression as a function of B cell differentiation

J chain expression was examined as a function of the stage in differentiation along the B cell axis in humans. Intracellular distribution of J and mu chains in leukemic HLA-DR+ null and pre-B cells, and in normal B cells stimulated with pokeweed mitogen (PWM) was determined by immunoelectron microscopy and radioimmunoassay (RIA). J chain was detected in leukemic null and pre-B cells on free and membrane-bound ribosomes in the cytoplasm, or on perinuclear cisternae. Mu chain was found on free ribosomes and ribosomal clusters in leukemic pre-B cells but was absent in the leukemic null cells. In pre-B cell lines, mu chain was seen within rough endoplasmic reticulum (RER) and the Golgi apparatus whereas J chain was not detected in these organelles. However, both mu and J chain were detected in RER and the Golgi apparatus of immature and mature plasma cells induced by PWM stimulation of normal peripheral blood lymphocytes. Low levels of J chain were also detected by RIA in lysates of leukemic null and pre-B cells. Most of the intracellular J chain became detectable after reduction and alkylation of cell lysates, and free J chain was not found in the culture supernatants. The amount of intracellular and secreted immunoglobulin-bound J chain increased dramatically after PWM stimulation of peripheral blood lymphocytes. The majority of J chain- positive cells seen over an 8 d culture interval were lymphocytes and lymphoblasts, while mu chain was found primarily in plasma cells. These results suggest that J chain expression precedes mu chain synthesis during B cell differentiation and that a combination of the two chains for secretion is not initiated until the onset of plasma cells maturation.     

Exocrine pancreas of streptozotocin-diabetes rats treated with insulin.

The effect of insulin treatment on the exocrine pancreas of streptozotocin-diabetes rats was investigated by light and electron microscopy. In the diabetic rats treated with daily lente insulin injection for four weeks, the islets became hyperplastic and proliferative, although degenerative and atrophic islets caused by streptozotocin remained if diabetic rats were not treated with insulin. Fibrosis and degeneration of the acinar cells were not found in all the diabetic rats by light microscopic examinations. Electron microscopic examinations showed that acinar cells of the exocrine pancreas of the diabetic rats without insulin treatment were characterized by irregular dilatation and prominent lamellar arrangement of rough endoplasmic reticulum and by nuclear pyknosis. After one hour of a single injection of regular insulin, rough endoplasmic reticulum of the acinar cells of the diabetic rats was rapidly activated and many intracisternal granules appeared. When the daily injection of insulin was continued, the acinar cells became to show regular arrangement of rough endoplasmic reticulum, much less vacuolarizations and less immature zymogen granules in comparison with those of the untreated diabetic rats. Exocrine pancreas of the insulin-treated rats revealed a lot of autophagic vacuoles which were supposed to derive from lysosomes. These results suggested that insulin had a repairing effect on the damaged acinar cells in diabetic state.     

Differences in maturation of tick-borne encephalitis virus in mammalian and tick cell line

OBJECTIVE: The maturation process of tick-borne encephalitis virus (TBEV) in the tick RA-257 and porcine PS cells was studied by transmission electron microscopy and the E and NS1 proteins were localized in the infected cells. METHODS: The porcine PS and tick RA-257 cell lines were infected with TBEV and examined at different time points post infection under an electron microscope. The E and NS1 proteins were localized with monoclonal antibodies on ultrathin cryosections. RESULTS: The first virus particles and virus-induced vesicles appeared inside hypertrophied and dilated rough endoplasmic reticulum (RER) cisternae in PS cells 15 h p.i. In the course of progressing maturation, the virus particles came up inside the Golgi apparatus and then probably left the cell by the exocytic pathway. Free nucleocapsids did not appear. The observed pattern corresponded to a trans-type maturation. The maximum of the infected PS cell survival was about 50 h p.i. Immunolocalization of some viral proteins (the envelope protein E and the nonstructural protein NS1) revealed the proteins in the cytosol and on the membrane of hypertrophied RER cisternae. On the other hand, the maturation process exhibited different features in the case of the tick RA-257 cells. The nucleocapsids appeared in the cytosol 24 h p.i. and enveloped viral particles were observed in the lumen of vacuoles. Infection of RA-257 cells caused only minor ultrastructural changes and resulted in persistent infection. Immunolocalization of viral proteins in the tick cell line also differed. Proteins E and NS1 were localized in the cytosol and on the vacuolar and plasma membranes. CONCLUSION: The TBEV maturation pathway in the mammalian host cell line differs from the pathway that the virus undergoes in the tick vector cell line.     

Stimulation of insulin secretion reveals heterogeneity of pancreatic B cells in vivo.

We examined the immunofluorescence and ultrastructural changes of insulin-producing B cells in the center and at the periphery of islets of Langerhans during in vivo stimulation by glucose and glibenclamide. A decreased insulin immunostaining was detected in islets from the splenic rat pancreas after 1.5 h of glucose stimulation. By contrast, immunofluorescence changes became apparent in islets from the duodenal pancreas only after greater than 3 h of hyperglycemia. In both cases, the immunolabeling of central B cells decreased before that of peripheral B cells. Similar changes were seen following in vivo stimulation of insulin secretion by glibenclamide. At the ultrastructural level, hyperglycemia decreased the volume density of B cell secretory granules and increased that of rough endoplasmic reticulum and Golgi apparatus. These changes were also detected earlier in central than in peripheral B cells and earlier in splenic than in duodenal islets. The data show that B cells form a heterogeneous population in vivo.     

Conversion of monocytes to cells capable of anchorage-independent growth in vitro

We investigated the time-course involved in the conversion of mouse blood monocytes in vitro in cells capable of anchorage-independent growth. Two criteria were used to define when monocytes were fully converted to cells similar to mononuclear phagocytes present in inflammatory exudate, such as thioglycollate medium (TM)-elicited peritoneal exudate. They were the production of high levels of plasminogen activators and an ability to undergo anchorage-independent growth. Resident peritoneal macrophages were used as controls and for comparison. Our studies indicated that monocytes, but not resident peritoneal macrophages, could be converted to cells similar to TM- elicited mononuclear phagocytes after 2 d in culture.     

A review of macromolecular transport and secretion at the cellular level.

This is a review of current information concerning mechanisms involved in transport and secretion of macromolecules in exocrine glands. Emphasis has been placed on information available for pancreatic acinar cells. The review was prompted by the availability of considerable amounts of new information developed during the past several years. Exportable proteins in the pancreatic acinar cells are synthesized on ribosomes attached to the endoplasmic reticulum. Following synthesis, nascent proteins are transported from ribosomes attached to the endoplasmic reticulum into intracisternal spaces bound by the endoplasmic reticulum. The proteins are then carried to the Golgi complex by transitional elements. Zymogen granules are formed in the Golgi complex and migrate to the cell apex. Appropriate stimulation leads to fusion of the zymogen granule membrane and apical plasmalemma followed by a break in the membrane and consequent release of the granule content into the ductules. The extact molecular events involved in the process of secretion are not known. The roles of cAMP and cGMP in pancreatic secretion are supported by indirect evidence only. The role of calcium in secretion is apparent, but further investigation is needed to delineate the exact mechanism of its action. Membrane depolarization and associated ionic fluexes seem to play a significant role.     

Intracellular localization of the radiation enhancer motexafin gadolinium using interferometric Fourier fluorescence microscopy

Motexafin gadolinium (MGd) is a unique therapeutic agent that localizes in cancer cells and increases tumor response to ionizing radiation and certain chemotherapeutics. The in vitro intracellular localization, accumulation, and retention of MGd in murine EMT6 mammary sarcoma and Rif-1 fibrosarcoma cell lines were studied using interferometric Fourier fluorescence microscopy. MGd cellular uptake was semiquantified using its characteristic fluorescence emission band centered at 758 nm. Colocalization studies were performed using mitochondrial, endoplasmic reticulum, Golgi apparatus, nuclear, and lysosomal fluorescent organelle probes, and verified using interferometric Fourier spectroscopy. Cellular uptake was gradual and increased significantly with incubation time. MGd localized primarily within the lysosomes and endoplasmic reticulum, and to a lesser extent within the Golgi apparatus and mitochondria. Mitochondrial staining was increased in media without serum. No nuclear uptake was detected in the Rif-1 cells, but after 48 h nuclear uptake was observed in 15% of EMT6 cells. These results indicated that MGd accumulates within cytoplasmic compartments. The sustained intracellular localization of MGd may, in part, account for its unique radiation and chemotherapy enhancement properties. Interferometric Fourier fluorescence microscopy is a potentially powerful tool in delineating and verifying localization sites of therapeutic agents.     

HEMATOPOIETIC ORIGIN OF MACROPHAGES AS STUDIED BY CHROMOSOME MARKERS IN MICE

The origin of macrophages was studied in mouse radiation chimeras by chromosome marker technique. Macrophage cultures were established from peritoneal exudate, from lung washings, and from organ cultures of bone marrow, spleen, lymph node, thymus, and lung. Cultured macrophages were induced to divide by adding conditioned medium from L cell cultures. In chimeras which were lethally irradiated and given injections of bone marrow or spleen cells, dividing macrophages were of donor type, independent of the source of the macrophages. When chimeras were established by injections of a mixture of bone marrow cells and cells from other hematopoietic tissues of two genetically different donors, the ratio of cells with different genotypes was approximately the same in bone marrow cells and in macrophage cultures. Thymus, lymph node, and peritoneal exudate cells were not found to contain precursor cells for macrophages. Precursor cells for macrophages and for bone marrow cells appeared to be equally sensitive to sublethal irradiation. The results indicate that macrophages from different sources can all be derived from hematopoietic tissues, and suggest that only hematopoietic tissues contain precursor cells for macrophages which are capable of in vitro division. The close relationship between the source of cells in bone marrow and in macrophage cultures suggests that, at the maturation level at which the irradiated host is repopulated, the precursor or stem cells for macrophages may be identical with those for myeloid and erythroid series of cells.     

Maturation and interrelationship of mouse mononuclear phagocytes in bone marrow, peripheral blood and peritoneal cavity in terms of erythrophagocytic activity.

Ingestion by mononuclear cells in bone marrow, peripheral blood and the peritoneal cavity of red cells treated with antibodies and various chemicals was studied in an attempt to characterize these cell lines during development and to clarify the interrelationship among them. Bone marrow mononuclear phagocytes became intensively phagocytic during the second day of culture, indicating that the maturation time from progenitor cells to macrophages was about 48 hr. Blood monocytes exhibited very active erythrophagocytic activity after 24 to 48 hr cell culture. Uptake of sensitized red cells by both the cell lines was markedly inhibited by IgG irrespective of time periods of cell culture. Majority of peritoneal lymphocyte-like cells (LLC) separated from other forms of mononuclear cells were shown to become capable of erythrophagocytosis with maturation time to macrophages between 24 to 48 hrs. Under non-stimulated steady state, uptake of sensitized red cells by peritoneal macrophages was far less sensitive to IgG inhibition than that by bone marrow mononuclear phagocytes and blood monocytes, suggesting that IgG receptor does not play main role for the erythrophagocytosis by peritoneal macrophages. Peritoneal cells collected under inflammatory stimulus exhibited a character similar to bone marrow mononuclear phagocytes and blood monocytes with regard to IgG receptor. It was suggested that under steady state peritoneal macrophages arise and mature from local proliferative pool with some different phagocytic mechanism from that of bone marrow mononuclear phagocytes.     

Novel type of proliferating lymphoplasmacytoid cell with a characteristic spotted immunofluorescence pattern

We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.     

Interleukin 1 beta is localized in the cytoplasmic ground substance but is largely absent from the Golgi apparatus and plasma membranes of stimulated human monocytes

The subcellular location of IL-1 beta was determined using a postsectioning immunoelectron microscopic method on ultrathin frozen sections of human monocytes stimulated with LPS. This methodology permits access of antibody probes to all sectioned intracellular compartments, and their visualization at high resolution. Staining was performed with a rabbit antibody that specifically recognized amino acids 197-215 in the 33-kD IL-1 beta precursor molecule, followed by affinity-purified goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles. Approximately 90% of the IL-1 beta antigens were localized in the ground substance of the cytoplasm at 4 or 20 h after activation, when both intracellular and extracellular accumulation of IL-1 beta was well underway. No significant IL-1 beta staining was observed on the outer cell membrane, nor within the lumens of the endoplasmic reticulum (ER), the Golgi apparatus, or secretory vesicles. In contrast, lysozyme was localized in the ER and dense secretory granules using these methods. Our results suggest that IL-1 beta is not anchored on the plasma membrane, and that its secretion occurs by a novel mechanism that does not use a secretory leader sequence, nor the classical secretory pathway involving the ER and Golgi apparatus.     

大鼠骨髓间充质干细胞成骨诱导分化的超微结构变化

背景：目前对骨髓间充质干细胞成骨诱导分化的超微结构观察的报道甚少。目的：采用全骨髓贴壁法分离培养大鼠骨髓间充质干细胞,成骨诱导并染色鉴定,利用电镜观察诱导前后细胞超微结构变化特点。方法：全骨髓贴壁法体外分离、培养、纯化大鼠骨髓间充质干细胞,流式细胞仪检测细胞表面标记物,成骨染色鉴定向成骨方向诱导分化,扫描电镜及透射电镜观察成骨诱导前后细胞超微结构变化。结果与结论：培养的第3代骨髓间充质干细胞纯度高、活力强,成骨诱导后的碱性磷酸酶活性染色、钙化结节染色均呈阳性扫描电镜及透射电镜观察显示,经向成骨细胞诱导分化后,细胞形态铺展,不规则,其线粒体、粗面内质网、空泡明显增多表明细胞功能活跃。