Pulsed electromagnetic fields alter phenotypic expression in chondroblasts in tissue culture

We hypothesize that pulsed electromagnetic fields (PEMF) alter phenotypic expression of chondroblasts by promoting the production of alkaline phosphatase (AP) and altering the structure of proteoglycans. Chondroblasts from the hypertrophic zone of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from 16 day chick embryos. Cultures were randomly designated control (C) or experimental (E). E received PEMF for 24 h in a 6 h on, 6 h rest sequence. The controls were in the same incubator shielded by Mu metal. Assays for AP activity were performed and normalized to protein content. Proteoglycan synthesis assay involved labeling with 35S fractionating in a 5% to 20% surcrose gradient determining total protein and chondroitin sulfate content. PEMF showed no change of AP activity on F. A high AP basal activity was found in HC, but was not increased above the control. PEMF increased AP in the SC samples (E/C ratio). The sucrose gradient data showed a shift in peaks for SC only altering the ratio of carbohydrate to protein for the SC. Analysis of carbohydrate and protein indicated that the effect was decreased synthesis or degradation of protein. We conclude that PEMF alters the phenotypic expression of sternal chondroblasts in our in vitro system.     

Calcium binding by chondroitin sulfate

The binding of calcium by chondroitin sulfate C was studied by employing biological and physicochemical methods for the quantitative determination of ionic calcium. Mean values for logK _j, the logarithm of the uncorrected formation constant for a calcium-chondroitin sulfate complex, were 1.64 with the frog heart technique, 1.55 with the murexide method, 1.39 with ultrafiltration and 1.04 with the calcium selective electrode. The magnitude of these values suggests that chondroitin sulfate has a relatively high binding capacity for calcium, but calculated values for an exchange parameterK _p indicate that calcium has a low affinity for the polysaccharide.

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Zusammenfassung Die Bindung von Calcium mit Chondroitinsulfat C wurde untersucht mittels biologischer und physikalisch-chemischer Methoden für die quantitative Bestimmung des ionischen Calciums. Im Durchschnitt ergab logK _j, der Logarithmus der unkorrigierten Formationskonstante für einen Calciumchondroitinsulfatkomplex, 1,64 mit der Technik des Froschherzens, 1,55 mit der Murexidmethode, 1,39 mittels Ultrafiltration und 1,04 mit den Calciumselektiven Elektroden. Die Größe dieser Werte zeigt, daß Chondroitinsulfat eine relativ hohe Bindungskapazität für Calcium hat, die berechneten Werte für einen AustauschparameterK _p jedoch, daß Calcium eine kleine Affinität für Polysaccharide hat.

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Résumé La liaison du calcium par le sulfate de chondroitin C a été étudiée en utilisant des méthodes biologiques et physicochimiques pour la détermination quantitative du calcium ionique. Les valeurs moyennes pour logK _j, le logarithme de la constante de formation non corrigée d'un complexe calcium-sulfate de chondroitin C, sont 1,64 par la technique de cur de grenouille, 1,55 par la méthode de murexide, 1,39 par l'ultrafiltration et 1,04 par l'électrode sélective du calcium. L'ordre de grandeur de ces valeurs suggèrent que le sulfate de chondroitin C a une capacité de liaison pour calcium relativement élevée, mais les valeurs calculées pour un paramètre d'échangeK _p indiquent que le calcium a une affinité faible pour le polysaccharide.

     

In vitro chondrogenic differentiation of human articular cartilage derived chondroprogenitors using pulsed electromagnetic field

BackgroundThe ability to grow new cartilage remains the standard goal of any treatment strategy directed at cartilage repair. Chondroprogenitors have garnered interest due to their applicability in cell therapy. Pulsed electromagnetic field (PEMF) favors chondrogenesis by possible upregulation of genes belonging to TGFβ superfamily. Since TGFβ is implicated in chondrogenic signalling, the aim of the study was to evaluate the ability of PEMF to induce chondrogenesis via endogenous TGFβ production in chondroprogenitors vs differentiation using chondrogenic medium inclusive of TGFβ.MethodsChondroprogenitors were harvested from three non-diseased human knee joints via fibronectin assay. Passage 3 pellets were subjected to four different culture conditions: a) negative control contained chondrogenic medium without TGFβ2, b) positive control contained medium with TGFβ2, c) PEMF 1 contained medium of negative control plus single exposure to PEMF and d) PEMF 2 contained medium of negative control plus multiple exposures to PEMF. Following differentiation (day 21), pellets were assessed for gene expression of ACAN, SOX9, COL2A1, TGFβ1, TGFβ2, and TGFβ3. Alcian blue staining to detect glycosaminoglycan deposition was also performed. Medium supernatant was used to detect endogenous latent TGF-β1 levels using ELISA.ResultsAll study arms exhibited comparable gene expression without any significant difference. Although positive control and PEMF study arms demonstrated notably better staining than negative control, the level of latent TGF-β1 was seen to be significantly high in supernatant from positive control (P < 0.05) when compared to other groups.ConclusionOur results indicate that PEMF induced chondrogenesis might involve other signalling molecules, which require further evaluation.     

Long-term pulsed electromagnetic field (PEMF) results in congenital pseudarthrosis

Summary Ninety-one patients with congenital pseudarthrosis of the tibia have been treated with pulsed electromagnetic fields (PEMFs) since 1973 and all except 4 followed to puberty. Lesions were stratified by roentgenographic appearance. Type I and type II had gaps less than 5 mm in width. Type III were atrophic, spindled, and had gaps in excess of 5 mm. Overall success in type I and II lesions was 43 of 60 (72%). Of those 28 patients seen before operative repair had been attempted, 7 of 8 type I lesions healed (88%), whereas 16 of 20 type II lesions healed (80%) on PEMFs and immobilization alone. Only 19% (6 of 31) type III lesions united, only one of which did not require surgery. Sixteen of 91 limbs (18%) were ultimately amputated, most before treatment principles were fully defined in 1980. Fourteen of these 16 patients (88%) had type III lesions. Refracture occurred in 22 patients, most as the result of significant trauma, in the absence of external brace support. Twelve of the 19 refractures, retreated with PEMFs and casts, healed on this regime. Episodic use of PEMFs proved effective in controlling stress fractures in several patients until they reached puberty. PEMFs, which are associated with no known risk, appear to be an effective, conservative adjunct in the management of this therapeutically challenging, congenital lesions.     

Degradative pathways in cultured synovial fibroblasts: selective effects of pulsed electromagnetic fields

A cell culture model for studying the cytokine-mediated degradation of connective tissue was exposed to clinically applied, low-frequency pulsed electromagnetic fields (PEMF), and levels of collagenolytic activity, two lysosomal hydrolases, and prostaglandin E2 were measured. PEMFs reduced the release of two lysosomal enzymes by cultured rabbit synovial fibroblasts but did not affect their response to mononuclear-cell-conditioned medium. PEMF did not alter levels of cytokine activity produced by a mixed mononuclear cell population, nor did they affect the cytokine-mediated release of collagenase or prostaglandin E2 by synovial fibroblasts. The relevance of these findings to the clinical application of PEMF to soft- and hard-tissue injuries is discussed.     

Supraphysiological concentrations of dexamethasone induce elevation of calcium uptake and depression of [3H]-Thymidine incorporation into DNA in cartilagein vitro

Summary When condylar cartilages of neonatal mice are treatedin vitro with dexamethasone they respond with a significant depression of DNA synthesis. Concomitant with the retardative effect upon the incorporation of [³H]-thymidine, dexamethasone enhances the uptake of⁴⁵Ca by the condylar tissue. The hormone's effect upon calcium uptake is relatively rapid and takes place within 2 hours whereas the effect upon the incorporation of [³H]-thymidine takes place after a lag period of 24 hours. The present findings tend to indicate the casual relationship between the above effects of dexamethasone in fast growing cartilage. Further, there is evidence that both of the above responses are specific and are protein and mRNA synthesis dependent. Control studies using both a calcium inonophore (A23187) and a calcium blocker (D-600) provided data that further substantiate the proposed association among dexamethasone, calcium metabolism, and DNA synthesis in neonatal growth cartilage.     

The effect of pulsed electromagnetic fields on bone cell metabolism and calvaria resorption in vitro,and on calcium metabolism in the live rat

Summary The effects of three pulsed electromagnetic fields were investigated on bone cells and calvaria in culture, and on calcium metabolism in the live rat. No significant effect was seen on: 1) the proliferation of calvaria cells in culture; 2) alkaline phosphatase level, lactic acid release and collagen synthesis by confluent calvaria cells, with the exception of one pulse which produced a small increase in the latter when expressed as DNA; 3) resorption of calvaria in culture; 4) intestinal absorption, urinary excretion and net balance of calcium, bone formation and bone resorption in the live rat.on leave from Department of Pharmacology, School of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan     

Effects of mechanical and osmotic pressure on the rate of glycosaminoglycan synthesis in the human adult femoral head cartilage: an in vitro study

We studied the effects of mechanical and osmotic compression on sulphate incorporation into glycosaminoglycans of human femoral head cartilage. We found that both mechanical and osmotic compression produce the same lowering of sulphate uptake relative to uncompressed controls. It appears that this effect is not associated with changes in solute transport or changes in solute concentration in the matrix, but is due, in part at least, to an increased osmotic pressure acting on the chondrocytes. A second mechanism of action might be involved directly through the increased proteoglycan concentration in the pericellular environment, resulting from a reduction in the water content. We also found that glycosaminoglycan synthesis returned to its control level when the conditions prevailing in the matrix, in the absence of pressure or added solute, were restored.     

Low dose short duration pulsed electromagnetic field effects on cultured human chondrocytes: An experimental study

Background:

Pulsed electromagnetic field (PEMF) is used to treat bone and joint disorders for over 30 years. Recent studies demonstrate a significant effect of PEMF on bone and cartilage proliferation, differentiation, synthesis of extracellular matrix (ECM) and production of growth factors. The aim of this study is to assess if PEMF of low frequency, ultralow field strength and short time exposure have beneficial effects on in-vitro cultured human chondrocytes.

Materials and Methods:

Primary human chondrocytes cultures were established using articular cartilage obtained from knee joint during joint replacement surgery. Post characterization, the cells were exposed to PEMF at frequencies ranging from 0.1 to 10 Hz and field intensities ranging from 0.65 to 1.95 μT for 60 min/day for 3 consecutive days to analyze the viability, ECM component synthesis, proliferation and morphology related changes post exposure. Association between exposure doses and cellular effects were analyzed with paired''t’ test.

Results:

In-vitro PEMF exposure of 0.1 Hz frequency, 1.95 μT and duration of 60 min/day for 3 consecutive days produced the most favorable response on chondrocytes viability (P < 0.001), ECM component production (P < 0.001) and multiplication. Exposure of identical chondrocyte cultures to PEMFs of 0.65 μT field intensity at 1 Hz frequency resulted in less significant response. Exposure to 1.3 μT PEMFs at 10 Hz frequency does not show any significant effects in different analytical parameters.

Conclusions:

Short duration PEMF exposure may represent a new therapy for patients with Osteoarthritis (OA).     

茜素红半定量测定细胞基质钙含量的实验研究

目的使用茜素红检测的方法，对细胞在成骨诱导和未诱导状态下，细胞基质中钙盐沉积情况进行半定量分析，研究细胞基质中钙盐沉积情况变化趋势规律，为临床实践提供理论参考。方法对4例正常人骨髓间充质干细胞（Bone marrow mesenchymal stem cells，BMSCs）及4例正常人脂肪间充质干细胞（Adipose-derived mesenchymal stem cells，ASCs），使用茜素红半定量法检测成骨诱导及未诱导细胞基质中钙盐沉积情况进行半定量分析，研究细胞基质中钙盐沉积情况变化趋势规律。结果对于相同代次的间充质干细胞，未诱导组的钙盐沉积情况，在7d后没有显著的改变：而诱导组钙盐的沉积在10～14d明显增多。实验结果同时显示，同一样本的间充质干细胞，随着代次增加，未诱导组钙盐的沉积时间延后，且沉积量逐渐减少。诱导组钙盐的沉积情况，7d时没有显著的改变；14d时，随代次增加，钙盐沉积量逐渐减少。结论体外成骨诱导的第3～4代的间充质干细胞在第10～14天已经具有较强的成骨活性，可能比较适宜体内回植。本实验为组织T程骨的体外构建时间提供了理论依据。     

Effects of a specially pulsed electric field on an animal model of wound healing

The possible beneficial effects of a specially pulsed electric field (PEF) on wound healing were investigated in this study. We made a pair of triangular, full-thickness, dorsal incisions in the skin of 32 healthy male mice (one control group and three exposure groups). The treatment groups were kept between parallel plates in a partially insulated exposed environment. Group I was exposed to an electric field intensity of 10 kV/m, group II was exposed to 1.9 kV/m, and group III was exposed to 0.9 kV/m. PEFs were applied to the subjects for 20–22 h and 8 consecutive days. We determined the differences in wound recovery between the groups based on the following parameters: collagen fiber density, inflammatory infiltration density, capillary proliferation, and existence of exudates. We found that a 0.9 kV/m–1.9 kV/m chopped direct current (DC) electric field with a 30 μs repetition time favorably affected collagen synthesis and wound recovery. Despite the intensity of 0.9–1.9 kV/m, PEF accelerated healing, but 10 kV/m decelerated this recovery process.     

Calcium oxalate crystallizing properties of polyanions elaborated by cultured renal proximal tubular cells

To study the influence of renal polyanions on crystallization of urinary calcium oxalate, we recovered polyanionic macromolecules from media conditioned by primary cultures of renal proximal tubular epithelial cells of rats in serum-free, hormonally defined medium. Cells cultured on microporous supports showed a higher degree of morphological and functional proximal differentiation into a polarized monolayer than those on plastic impervious substrata. Papainization of the polyanions yielded the glycosaminoglycans chondroitin/dermatan sulphate and heparan sulphate. These accounted respectively for 60% and 80% of the crystal nucleation-promoting activities of polyanions recovered from the apical and basal media conditioned by polarized cultures on microporous supports. Similar relative activities were observed among the urinary glycosaminoglycans and polyanions similarly tested. Primary cultures of polarized proximal tubular epithelial cells are useful then as an in vitro model to study the crystallizing activities of polyanionic macromolecules produced by renal cells.     

软骨寡聚基质蛋白在慢性胰腺炎损伤中退变腺泡细胞内的表达

目的 研究正常胰腺、慢性胰腺炎与胰腺癌组织中软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP)mRNA和蛋白表达水平的差异,揭示COMP在慢性胰腺炎样损伤中的意义。方法 采用Northern印迹法、Western印迹法、原位杂交法与免疫组化方法对14例慢性胰腺炎、14例胰腺癌及15例正常胰腺组织进行分析。结果 在慢性胰腺炎组织中和胰腺癌组织中类似慢性胰腺炎损伤的退变腺泡细胞胞浆内,存在高水平的COMP mRNA信号与免疫反应;而在胰腺癌细胞、正常胰腺组织的导管细胞与胰岛细胞的胞浆内,COMP mRNA信号与免疫反应微弱或缺如。结论 COMP在慢性胰腺炎及胰腺癌中类似慢性胰腺炎损伤的退变腺泡细胞内高表达,可能与慢性胰腺炎中腺泡细胞功能异常有关。     

体外培养组织工程皮肤几种活性肽及细胞外基质的表达

目的观察体外培养组织工程皮肤的生物学活性. 方法将表皮细胞和(/或)成纤维细胞种植于脱细胞真皮表面,经培养后形成各种皮肤替代物,分别于接种后3、7 d时收集培养上清,采用酶联免疫吸附法(ELISA)测定白细胞介素(IL)-6、IL-8和转化型生长因子β1(TGF-β1)的含量,采用放射免疫法测定层粘连蛋白、透明质酸、Ⅰ型和Ⅲ型胶原的含量. 结果各皮肤替代物上清中均可检测到一定量的细胞因子、生长因子和细胞外基质成分,培养7 d与3 d时相比,除TGF-β1外,其余各指标的含量均明显上升(P＜0.01).含表皮细胞和成纤维细胞的皮肤替代物与单纯含成纤维细胞的皮肤替代物相比,培养上清中Ⅰ、Ⅲ型胶原含量明显减少(P＜0.01). 结论体外培养的皮肤替代物具有较强的生物学活性,种植表皮细胞对成纤维细胞的功能具有一定的调节作用.     

Influence of different types of electromagnetic fields on skin reparatory processes in experimental animals

Wound healing is a very complex process, some phases of which have only recently been explained. Magnetic and electromagnetic fields can modulate this process in a non-thermal way. The aim of this research was to compare the influence of constant and pulsed electromagnetic fields and low-level laser therapy (LLLT) on wound healing in experimental animals. The experiment was conducted on 120 laboratory rats divided into four groups of 30 animals each (constant electromagnetic field, pulsed electromagnetic field, LLLT and control group). It lasted for 21 days. Under the influence of the constant electromagnetic field the healing of the skin defect was accelerated in comparison with the control group. The difference was statistically significant in all the weeks of the experiment at the P < 0.01 level. Accelerated healing was also observed under the influence of the pulsed electromagnetic field (P < 0.05). In the group of animals exposed to LLLT, the healing of the skin defect was faster than in the control group. The statistical significance was at the P < 0.05 level. Different types of electromagnetic fields have a promoting effect on the wound healing process.     

软骨细胞老化过程中胞外基质表达水平的改变

目的研究猪耳软骨细胞在体外培养传代过程中,细胞老化相关基因和胞外基质相关基因表达水平的改变。方法取体外培养猪耳软骨细胞,通过光镜,对各代细胞的形态学变化进行观测。RT-PCR检测各代细胞中bcl-2、端粒酶、Ⅰ型胶原和Ⅱ型胶原在mRNA表达水平上的改变。结果体外培养软骨细胞随着老化的进程,逐渐显现衰老和凋亡的特征。凋亡抑制基因bcl-2和控制细胞分裂的端粒酶的表达显著下降(P<0.01)。软骨细胞的特征性基质分子Ⅱ型胶原在第3代时表达水平有显著下降(P<0.01),降至原代细胞的5.47%±1.04%,在第4代几乎不再表达。而Ⅰ型胶原在第5代时,有显著上升(P<0.01);在第9代时又显著下降(P<0.01)。结论猪软骨细胞经体外培养,由第4代起逐渐发生去分化,丧失软骨细胞的表型。同时,细胞的增殖分裂能力亦逐步减退,细胞凋亡现象明显增加。     

Suppression of Parathyroid Hormone and Bone Resorption by Calcium Carbonate and Calcium Citrate in Postmenopausal Women

This study was conducted to compare the suppressive effects of calcium carbonate and calcium citrate on bone resorption in early postmenopause. Calcium citrate is thought to be better absorbed. We therefore tested the hypothesis that calcium as citrate is more effective than calcium as carbonate in suppressing parathyroid hormone (PTH) and C-terminal telopeptide. Twenty-five healthy postmenopausal women were recruited in this double blind crossover study. The subjects were randomly allocated to receive either 1,000 mg of elemental calcium as carbonate or 500 mg of calcium as citrate. They were given the alternate calcium dose 1 week later. Serum measurements of total and ionized calcium, phosphate, PTH, and CrossLaps were repeated 12 hours after each dose. Analysis of variance found no significant difference between measures for the two salts. Tests for equivalence indicated that 500 mg of calcium citrate may be superior to 1,000 mg of calcium carbonate in raising serum total and ionized calcium (P = 0.04 and 0.05, respectively). For all parameters measured, 500 mg of calcium citrate was at least as beneficial as 1,000 mg of calcium carbonate. Calcium citrate is at least as effective as calcium carbonate in suppressing PTH and C-terminal telopeptide cross-links, at half the dose. This may be because calcium as citrate is better absorbed than calcium as carbonate. If calcium citrate can be used in lower doses, it may be better tolerated than calcium carbonate.     

Calcium phosphate stones produced by Madin-Darby canine kidney (MDCK) cells inoculated in nude mice

The canine renal distal tubular cell line Madin-Darby canine kidney (MDCK) forms calcium phosphate microliths during a long-term culture in vitro. We identified osteopontin (OPN) and calprotectin (CPT) from a urinary stone matrix. We recently also detected the expression of OPN and CPT in MDCK cells. The relationship between the mechanism of the stone formation and these stone matrix proteins is not yet known. Here, MDCK cells were cultured and inoculated in the subcutis of nude mice. After 4, 8 and 12 weeks, the inoculated tissues were resected, fixed and immunostained with polyclonal anti-human OPN and polyclonal anti-human CPT antibodies. Some serial specimens were stained with von Kossa's procedure. MDCK cells formed some follicular formations in the subcutis of nude mice at least at 12 weeks after transplantation. At 8 weeks after the inoculation, we detected small calcium phosphate stones with MDCK cells trapped in the follicles. The cells forming the stones also expressed both OPN and CPT. The CPT expression sites coincided with the stone formation sites. We confirmed that MDCK cells inoculated in nude mice had stone-forming potential, and we speculate that OPN and CPT play important roles in stone formation by MDCK cells. Received: 17 November 1997 / Accepted 28 October 1998