THE CELLULAR COMPLEMENT OF THE SKELETAL SYSTEM STUDIED AUTORADIOGRAPHICALLY WITH TRITIATED THYMIDINE (H3TDR) DURING GROWTH AND AGING

An autoradiographic study has been made of mouse femora from birth to one year of age. Tritiated thymidine was used to study the proliferative rate of various skeletal tissues. The labeling index of the periosteum was found to be highest at birth (0.085). At 8 weeks of age the index fell sharply to 0.007 and remained low throughout the entire period of study. Chondro-osteogenic cells of the periosteum, especially those at the perichondrial zone, were most frequently labeled initially. The labeling index of the epiphyseal disk remained high (0.054 to 0.048) until about the 6th week of age. Following this age the index fell sharply to 0.018 at 8 weeks and 0.002 at 26 weeks of age. Autoradiographs showed that the cells of the articulating surface of the epiphysis and disk are derived at least in part from migrating chondro-osteogenic cells of the periosteum residing at the perichondrial region, and that these cells serve as the progenitor pool necessary for both circumferential growth and expansion of the epiphyseal disk.     

骨膜游离移植修复犬股骨颈骨折

目的　探讨犬自体髂骨骨膜游离移植治疗股骨颈骨折的效果。方法　选用毕格犬 7只 ,共 14个髋关节 ,制成股骨颈骨折模型 ,骨折经螺钉固定后 ,取髂骨骨膜移植于骨折处。于术后 1个月和 3个月X线拍片并取髋关节标本观察。结果　术后 1个月 :X线见骨折线模糊 ;肉眼观察 :移植的骨膜与股骨颈生长在一起 ;镜下观察 :骨膜内毛细血管大量增生 ,大量类骨质及软骨细胞生成。术后 3个月 :X线见骨折愈合 ;肉眼观察 :骨膜移植处有大量骨组织生长 ,填满了骨折端 ;镜下 :骨膜内血管网非常丰富 ,大量骨细胞生成 ,新生骨小梁深入到股骨颈原有骨小梁中并与之融合。结论　犬自体髂骨骨膜游离移植可以成活和成骨 ,能重建股骨颈血运 ,促进骨折愈合。     

Effects of platelet-derived growth factor on bone formation in vitro

Platelet-derived growth factor (PDGF) is a polypeptide found in a variety of tissues, including bone, where it could act as an autologous regulator of skeletal remodeling. Therefore, a recombinant B chain homodimer of human PDGF was studied for its effects on bone formation in cultured rat calvariae. PDGF at 10-100 ng/ml stimulated [3H]thymidine incorporation into DNA by up to sixfold and increased the DNA content and the number of colcemid-induced metaphase arrested cells. This effect was observed in the fibroblast and precursor cell-rich periosteum. As a result of its mitogenic actions, PDGF enhanced [3H]proline incorporation into collagen, an effect that was observed primarily in the osteoblast-rich central bone. The effect of PDGF was not specific for collagen since it also increased noncollagen protein synthesis. In addition, PDGF increased bone collagen degradation. PDGF and insulin-like growth factor (IGF) I had additive effects on calvarial DNA synthesis, but PDGF opposed the stimulatory effect of IGF I on collagen synthesis and IGF I prevented the PDGF effect on collagen degradation. In conclusion, PDGF stimulates calvarial DNA synthesis which causes an increased number of collagen-synthesizing cells, but PDGF also enhances bone collagen degradation.     

天然虫草对小鼠脾脏影响的组织化学和免疫细胞化学的观察

本文用３０只成年雄性小鼠,分为实验和对照两组。实验组每日灌服虫草液一次,共７日。对照组每日灌服生理盐水一次。按时将动物处死,取其脾脏进行组织化学和免疫细胞化学染色。结果显示：（１）虫草组小鼠脾脏白髓成份增加,生发中心明显,嗜派罗宁细胞增多。显微分光光度计检测结果显示虫草组脾脏内ＤＮＡ含量高于对照组,表明虫草能促进小鼠脾脏ＲＮＡ和ＤＮＡ的合成。（２）虫草组脾内ＩｇＧ＋和ＩｇＭ＋浆细胞明显增加,边缘区变宽,可能虫草有促进抗体形成的作用。此外本实验还证明,两组小鼠脾内巨噬细胞数量无明显差别     

FACTORS INFLUENCING THE FREQUENCY OF MESOSOMES OBSERVED IN FIXED AND UNFIXED CELLS OF STREPTOCOCCUS FAECALIS

Mesosomes of Streptococcus faecalis (American Type Culture Collection 9790) were seen about 92% less frequently in freeze fractures of unfixed cells than in freeze fractures and sections of fixed cells. This difference in frequency was not related to any period of unbalanced macromolecular synthesis induced by chemical fixation. All measured synthetic processes (DNA, RNA, and protein synthesis, and glycerol incorporation) were halted with either osmium tetroxide (OS) or glutaraldehyde fixation. That fewer mesosomes were seen in freeze fractures of unfixed cells was probably due to the difficulty of observing cross-fractured mesosomes in this organism in the unfixed state. Unfortunately, mesosomes probably preferentially cross fracture in the unfixed state and therefore are usually only observed, infrequently, in those cases where the freeze fracture follows the surface layer of a mesosomal membrane. However, the addition of glycerol to unfixed cells, especially in the chilled state, greatly increased the frequency of observation of cytoplasmic mesosomes in freeze fractures. It is thought that glycerol, like chemical fixation, increases the number of surface-fractured mesosomes, which in turn increases the frequency of mesosome observation. It was also observed that cellular autolysis occurring during OS fixation seemingly reduced the number of mesosomes observed in thin sections and freeze fractures of OS-fixed cells.     

RATE AND TIME OF DNA SYNTHESIS OF INDIVIDUAL CHINESE HAMSTER CELLS

The duration of DNA synthesis of a diploid cell line of Chinese hamster fibroblasts was determined in a comparative study by the FLM technique, and also by a new technique for measuring the rate of DNA synthesis of individual cells. These methods produced comparable results when applied during exponential growth of the cells. The rate of DNA synthesis was measured by means of quantitative autoradiography following a short-term incubation of the cells with 5 × 10^-6 M FUdR and 10^-5 M ¹⁴C-TdR. The choice of the medium for this purpose did not seem to be critical. The autoradiographic silver grains over cells and ¹⁴C-standard sources are counted by microphotometry using incident light bright-field. The direct measurements of DNA synthesis rate are ‘compartment’ statistics which have been converted into ‘flux’ parameters for comparison with the FLM method and applicability in cell-kinetic calculations. Frequency distributions of the rate of DNA synthesis of individual cells thus obtained may resemble normal distributions quite closely. They result from several factors: differences in the rate of synthesis in different parts of the S-phase, the density distribution of cells within the S-phase, the variation in the time of DNA synthesis among individual cells, and the experimental error. In the case of a pronounced partial synchronization as probably has been present in one experiment performed in the lag phase, an incorrect time of DNA synthesis may result from the rate values. Due to the variation in DNA synthesis rate in different parts of the S-phase it is not possible to determine the duration of DNA synthesis of an individual cell. However, the mean values of DNA synthesis time are reliable. The new method will be preferentially applied for determining the duration of DNA synthesis of human cells in as far as difficulties are encountered with the classical methods. In addition, it may be used to advantage for studying cells which make up low percentages in mixed populations. It finally permits a safer morphological classification of the cells under study than is possible with the classical methods.     

Targeted activation of androgen receptor signaling in the periosteum improves bone fracture repair

Low testosterone level is an independent predictor of osteoporotic fracture in elderly men as well as increased fracture risk in men undergoing androgen deprivation. Androgens and androgen receptor (AR) actions are essential for bone development and homeostasis but their linkage to fracture repair remains unclear. Here we found that AR is highly expressed in the periosteum cells and is co-localized with a mesenchymal progenitor cell marker, paired-related homeobox protein 1 (Prrx1), during bone fracture repair. Mice lacking the AR gene in the periosteum expressing Prrx1-cre (AR^-/Y;Prrx1::Cre) but not in the chondrocytes (AR^-/Y;Col-2::Cre) exhibits reduced callus size and new bone volume. Gene expression data analysis revealed that the expression of several collagens, integrins and cell adhesion molecules were downregulated in periosteum-derived progenitor cells (PDCs) from AR^-/Y;Prrx1::Cre mice. Mechanistically, androgens-AR signaling activates the AR/ARA55/FAK complex and induces the collagen-integrin α2β1 gene expression that is required for promoting the AR-mediated PDCs migration. Using mouse cortical-defect and femoral graft transplantation models, we proved that elimination of AR in periosteum of host mice impairs fracture healing, regardless of AR existence of transplanted donor graft. While testosterone implanted scaffolds failed to complete callus bridging across the fracture gap in AR^-/Y;Prrx1::Cre mice, cell-based transplantation using DPCs re-expressing AR could lead to rescue bone repair. In conclusion, targeting androgen/AR axis in the periosteum may provide a novel therapy approach to improve fracture healing.Subject terms: Bone development, Metabolic bone disease     

Capacitively coupled electric fields accelerate proliferation of osteoblast-like primary cells and increase bone extracellular matrix formation in vitro

Over the last few years, electric and electromagnetic fields have gained more and more significance in the therapy of bone fracture healing and bone disease. Yet, the underlying mechanisms on a cellular and molecular level are not completely understood. In the present study we have investigated the effects of capacitively coupled, pulsed electric fields on cellular proliferation, alkaline phosphatase activity, and matrix protein synthesis of osteoblast-like primary cells in vitro. Cells were derived from bovine periosteum and electrically stimulated by saw-tooth pulses of 100 V external voltage and 16 Hz frequency. This corresponds to an electric field of 6 kV/m across the cell membranes as could be shown by computer simulation. Field application caused acceleration of cell culture development. A significant increase of proliferation concurrent with an enhancement of alkaline phosphatase activity was observed in sub-confluent cultures. Exposure of confluent osteoblast-like primary cells to electric fields resulted in enhanced synthesis and secretion of extracellular matrix-related proteins. These findings suggest that capacitively coupled electric fields accelerate bone cell proliferation and differentiation in vitro and enhance the synthesis of cells leading to promoted matrix formation and maturation.     

SOME EFFECTS OF BLEOMYCIN ON THE PROLIFERATION, MATURATION TIME and PROTEIN SYNTHESIS OF HAIRLESS MOUSE EPIDERMIS

Hairless mice were given 2 mg Bleomycin i.p. in 1-0 ml saline on two successive days. By a stathmokinetic method, by micro-flow fluorometry and by autoradiography certain kinetic parameters were measured during 10 days after the last injection. Cell counts were made and the turnover time of the differentiating cells estimated. Protein synthesis was estimated by the uptake of radioactive histidine, and dry cell mass measured by weighing. Bleomycin affected cell proliferation in the epidermis by depressing biphasically both the number of cells in, and the passage of cells through, the cell cycle phases: S, G₂ and M, most probably by directly affecting late Gj cells and cells in mitosis. The time between the two minima of depressed DNA synthesis corresponded to the mean generation time of the basal cells. Histidine uptake and dry cell mass were slightly affected, but the turnover time of the differentiating cells was prolonged. Bleomycin thus had a strong long-lasting inhibitory effect on epidermal cell proliferation and a marked inhibitory effect on epidermal cell maturation in mice.     

Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblastic cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.     

人股骨抽提物的骨诱导活性分析

骨诱导因子在骨的发生及损伤修复过程中起重要作用,因而它们在治疗骨缺损、骨质疏松等病症方面有广阔的应用前景。本实验测定了人股骨内活性因子的骨诱导活性。结果表明,人胎骨抽提物能在小鼠体内诱导未分化的间充质细胞向骨细胞转化,对培养人骨细胞的ＤＮＡ合成与碱性磷酸酶活性均有刺激作用。抽提物经初步纯化后,活性有所增强。     

Muscle-bone interactions during fracture healing

Although it is generally accepted that the rate and strength of fracture healing is intimately linked to the integrity of surrounding soft tissues, the contribution of muscle has largely been viewed as a vascular supply for oxygen and nutrient exchange. However, more is becoming known about the cellular and paracrine contributions of muscle to the fracture healing process. Research has shown that muscle is capable of supplying osteoprogenitor cells in cases where the periosteum is insufficient, and the muscular osteoprogenitors possess similar osteogenic potential to those derived from the periosteum. Muscle’s secrotome includes proteins capable of inhibiting or enhancing osteogenesis and myogenesis following musculoskeletal injury and can be garnered for therapeutic use in patients with traumatic musculoskeletal injuries. In this review, we will highlight the current knowledge on muscle-bone interaction in the context of fracture healing as well as concisely present the current models to study such interactions.     

Modification of radiation-induced genetic damage in Drosophila melanogaster male germ cells with nucleic acid precursors. 3. Effects on the premeiotic cells

Using a 2-day brood pattern, the effect of 5-bromodeoxyuridine (BUdR) or 5-bromodeoxycytidine (BCdR) pre-treatment on the radiation-induced yield of sex-linked recessive lethals and translocations was studied in the spermatocytes and late gonial cells (p.i. DNA synthesis cells) of D. melanogaster. The p.i. DNA synthesis cells were irradiated (I.2 kR γ-radiation) in the pre-meiotic or post-meiotic stage. Irradiation of p.i. DNA synthesis cells in the pre-meiotic stage resulted in enhanced lethal frequency with BUdR (3.0%) and BCdR (2.9%) over the other pre-treatment conditions: saline (S), thymidine (TdR) and deoxycitydine (CdR) in the spermatocytes but not in the late gonial cells. The radiosensitizing property was evident with BCdR even when the p.i. DNA synthesis cells were irradiated in the post-meiotic stage; but not with BUdR pre-treatment. Probable reasons for the contradicting results reported in the literature were discussed.     

CELL ARREST IN Gl AND G2 OF THE MITOTIC CYCLE OF VICIA FABA ROOT MERISTEMS

Experiments were performed with cultured excised primary root tips of Vicia faba ‘Longpod’ to determine: (1) the proportion of meristematic cells arrested in Gl and in G2 during carbohydrate starvation, and to determine if the proportion is fixed or can be varied experimentally; (2) the effect of increased starvation on the ability of arrested cells in Gl and G2 to initiate DNA synthesis and mitosis, respectively, when exogenous sucrose was supplied; and (3) whether puromycin, cycloheximide, or actinomycin D prevented the initiation of DNA synthesis and the onset of mitosis. Microspectrophotometry of nuclear DNA and autoradiographic measurements of incorporated ³H-thymidine showed that 72 hr of starvation immediately after excision produced tissue with more than 70 % of the cells arrested in G2 and less than 30 % in Gl. If cultured for three days and then starved for 72 hr, the tissue had nearly equal numbers of cells arrested in Gl and G2. As the duration of starvation increased, the time required to initiate DNA synthesis and to divide when carbohydrate was replenished also increased. Inhibition of protein synthesis by puromycin and cycloheximide prevented the initiation of DNA synthesis and mitosis, but actinomycin D, an inhibitor of RNA synthesis, did not prevent division of cells from G2 nor DNA synthesis by cells from Gl. The experiments demonstrated that the mitotic cycle of Vicia has two major controls, one in Gl and another in G2, and that other factors determine how many cells are affected by either of these cycle controls.     

Gradients in plastid and mitochondrial DNA synthesis inFunaria protonemata: Visualization by bromodeoxyuridine immunohistochemistry

Summary DNA synthesis was investigated by visualizing sites of bromodeoxyuridine incorporation with antibodies in protonemata of the mossFunaria hygrometrica. In apically elongating tip cells a pronounced gradient of organelle DNA synthesis from tip to base was visible, reflecting the distribution of proliferating organelles within the tip cell. Side branch development coincided with reinitiation of replication of plastid and mitochondrial DNA.Abbreviations BrdU 5-bromo-2-deoxyuridine - DABCO diazabicyclol (2,2,2) octan - FITC fluorescein-5-isothiocyanat - HSA human serum albumine - IgG immunoglobulin G - PBS saline phosphate buffer     

Stimulation of bone formation by prostaglandin E2

We examined the effect of prostaglandin E2 (PGE2), in the presence or absence of cortisol, on bone formation in 21-day fetal rat calvaria maintained in organ culture for 24 to 96 h. [3H]Thymidine and [3H] proline incorporation were used to assess DNA and collagen synthesis, respectively. Changes in dry weight and DNA content were assessed after 96 h. PGE2 (10(-7) M) stimulated both DNA and collagen synthesis in calvaria. The effect on DNA synthesis was early (24 h), transient and limited to the periosteum. Collagen synthesis was stimulated at a later time (96 h), predominantly in the central bone. Cortisol (10(-7) M) inhibited DNA and collagen synthesis. The addition of PGE2 reversed the inhibitory effects of cortisol on DNA synthesis and content and increased collagen synthesis in central bone to levels above control untreated cultures. We conclude that PGE2 has stimulatory effects on bone formation and can reverse the inhibitory effects of cortisol. Hence the effects of cortisol may be mediated in part by their ability to reduce the endogenous production of prostaglandins.     

CELL SPECIFICITY OF CHALONE-TYPE INHIBITORS OF DNA SYNTHESIS RELEASED BY BLOOD LEUCOCYTES AND ERYTHROCYTES

Inhibitors of DNA synthesis released into balanced salt solution by rat erythrocytes and by rat leucocytes have been found to possess target-cell-specific properties which would be expected of chalones. When assayed in short-term in vitro cultures the erythrocyte product reduced DNA synthesis (as measured autoradiographically) in erythroblasts present in populations of bone-marrow cells but did not affect the DNA synthesis in myeloid or lymphoid cells. The leucocyte product, under the same culture conditions, reduced DNA synthesis in leucocyte precursor cells. The grain counts over nuclei of different cell types were recorded as well as the DNA labelling index. Results so far obtained cannot ascribe the erythrocyte-chalone-produced reduction in labelling index to a blockage of entry into S phase. This cell-specific inhibitor may reduce continuing DNA synthesis in S phase cells to undetectable levels, compared with synthesis in control media. The leucocyte product, however, most probably prevents entry of leucocyte precursor cells into S phase. Possible relevance of these inhibitors as components of physiological control mechanisms or as therapeutic agents is discussed.     

U46619双向调节系膜细胞DNA合成的研究

Mene用血清或佛波醇肉豆寇乙酸(PMA)预失活化系膜细胞的PKC,可抑制U46619所致的IP合成及细胞内Ca~(2 )的升高,提示PKC预先活化后,可对再次活化PLC-PKC的物质起负反馈抑制作用。血清中含有血液凝固时血小板释放的血小板源性生长因子(PDGF),可活化PLC及PKC,可能对U46619的作用起负反馈抑制,使U46619促增殖作用丧失。第三,TXA_2可能直接或间接刺激前列腺素(PG)E_2或PGI_2的合成,而后两者有抑制细胞增殖的作用。不论何种机制参与,U46619这种抑制作用在病理上可能有积极意义:在正常状态下,体内肾小球系膜细胞为静息状态,肾小球合成的PG及TX极少,当肾小球产生炎症时,肾小球白细胞及血小板浸润增多,合成的PG、TX及PDGF增多,这时TX的升高可能有抑制PDGF所致细胞增殖的作用。 摘要 本实验用[~3H]TdR掺入法测定TXA_2类似物U46619对大鼠系膜细胞DNA合成的调节作用,测定系膜细胞合成的DAG及1,4,5-IP_3量,用PKC抑制剂Calphostin C预处理系膜细胞,观察其对U46619促增殖作用的影响。结果表明,U46619促进生长停滞的系膜细胞的DNA合成及IP_3的生成。本文首次证实U46619也促进DAG的生成并发现PKC抑制剂可抑制U46619的促增殖作用,提示U46619使生长停滞的系膜细胞的PLC活化,促进IP及DAG生成,进而激活PKC,促进系膜细胞DNA合成     

Anatomical factors relative to the racial selectivity of femoral neck fracture

A comparative racial study of 200 femora from 50 American White and 50 American Negro female skeletons was carried out to determine whether any anatomical differences in femoral from exist between these groups which might account for the racial selectivity of hip fracture, Significant racial differences were found in neck-shaft angle, angle of inclination and oblique length. Negro females have longer femora, larger neck-shaft angles and a smaller angle of inclination than have White females. These differences in femoral morphology may, in some measure, contribute to the greater incidence of hip fracture in female Whites.