Bioelectromagnetics 36:35^44 (2015)
Pulsed Electromagnetic Field May Accelerate in vitro Endochondral Ossification JueWang,1 NaTang,2 Qiang Xiao,1 Li Zhang,1 Yu Li,1 Juan Li,1 JunWang,1 Zhihe Zhao,1 and LijunTan1* 1
State Key Laboratory of Oral Diseases,West China Hospital of Stomatology, Sichuan University, Chengdu, China 2 Stomatology Department of Sichuan Medical Science Academy, Sichuan Provincial People’s Hospital, Chengdu, China
Recapitulation of embryonic endochondral bone formation is a promising alternative approach to bone tissue engineering. However, the time-consuming process is one of the reasons the approach is unpractical. Here, we aimed at accelerating the in vitro endochondral ossification process of tissue engineering by using a pulsed electromagnetic field (PEMF). The rat bone marrow-derived stem cells were chondrogenic or hypertrophic differentiated in a three-dimensional pellet culture system, and treated with different intensities of PEMF (1, 2, and 5 mT with modulation frequency 750 Hz, carrier frequency 75 Hz and a duty ratio of 0.8, 3 h/day for 4 weeks). The effects of PEMF on hypertrophy and endochondral ossification were assessed by safranin O staining, immunohistochemistry, and quantitative real-time polymerase chain reaction. The results suggest that PEMF at 1, 2, and 5 mT may inhibit the maintenance of the cartilaginous phenotype and increase cartilage-specific extracellular matrix degradation in the late stage of chondrogenic differentiation. In addition, among the three different intensities, only PEMF at 1 mT directed the differentiation of chondrogenicinduced stem cell pellets to the hypertrophic stage and promoted osteogenic differentiation. Our findings provide the feasibility to optimize the process of in vitro endochondral ossification with PEMF stimulation. Bioelectromagnetics 36:35–44, 2015. © 2014 Wiley Periodicals, Inc. Key words: bone repair; bone marrow-derived stem cells; tissue engineering; cartilage; physical stimulation
INTRODUCTION Attempts to engineer bone tissue generally focus on intramembranous bone formation. However, bone tissue engineering through intramembranous bone formation for clinical application is not successful, partly because of the lack of sufficient vasculature during initial stage [Meijer et al., 2007]. Therefore, the concept of recapitulation of endochondral bone formation has been investigated as a feasible approach [Sasaki et al., 2010; Scotti et al., 2010, 2013]. It is a more physiological process by which the embryonic intermediate cartilaginous template is gradually replaced by bone tissue. Because cartilage by nature is equipped to survive in hypoxic conditions, repair of bone defects over cartilaginous templates may help overcome the lack of vascularization [Coyle et al., 2009]. This will be of benefit especially for larger tissue engineering constructs. However, this approach for bone tissue engineering has been largely overlooked, mostly because of the long process length time involved [Tortelli et al., 2010]. Thus, to achieve functional and timely mechanical and morphological development, it is important to find strategies to 2014 Wiley Periodicals, Inc.
accelerate the hypertrophic and endochondral ossification process. Pulsed electromagnetic field (PEMF) is a safe, non-invasive, clinically beneficial physical stimulus, which has been used clinically to promote the healing of fracture non-union and osteoarthritis [De Mattei et al., 2007; Griffin et al., 2011; Ryang et al., 2013]. Previous studies have revealed that PEMF can enhance the activity of osteoblasts and promote osteogenic Grant sponsor: National Natural Science Foundation of China; grant numbers: 30900287, 1030034, 30900286. Conflicts of interest: None. *Correspondence to: Lijun Tan, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, 14#, 3rd section, Renmin South Road, Chengdu 610041, China. E-mail: [email protected] Received for review 18 March 2014; Accepted 13 August 2014 DOI: 10.1002/bem.21882 Published online 30 October 2014 in Wiley Online Library (wileyonlinelibrary.com).
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differentiation of mesenchymal stem cells (MSCs), and can stimulate chondrocyte proliferation and extracellular matrix (ECM) synthesis; therefore, PEMF can be used to control construct differentiation in tissue engineering [Sun et al., 2010; Mayer-Wagner et al., 2011; Zhou et al., 2011; Ongaro et al., 2012; Fini et al., 2013]. Indeed, PEMF is regarded as a convenient and effective way to alter the physical microenvironment of bone tissue through electromagnetic effect rather than mechanical energy [Trock, 2000]. To assess the effects of PEMF on hypertrophy and endochondral ossification process, collagen type I, collagen type II, collagen type X, Sox9, bone sialoprotein (BSP), transforming growth factor-b3 (TGF-b3), osterix (Osx), and runt-related transcription factor 2 (Runx2) were used as the differentiation markers in this study. During embryonic endochondral bone formation, MSCs condense and differentiate into immature chondrocytes. The chondrocytes then synthesize a matrix that mainly consists of collagen II and aggrecan. TGF-b3 stimulates production of ECM components and is involved in the formation of new cartilage [Blaney Davidson et al., 2006]. Subsequently, the chondrocytes further differentiate and enter the prehypertrophic stage, producing Runx2 [Komori, 2010]. Expression of Runx2 stimulates the upregulation of hypertrophic markers collagen X. Once collagen X is synthesized, the cells are differentiated into the hypertrophic stage. During this final stage of differentiation, the expression of chondrogenic genes, such as Sox9 and TGF-b3, is downregulated in hypertrophic chondrocytes as a necessary step to initiate cartilage-bone transition [Hattori et al., 2010]. The hypertrophic cells are now characterized by expression of the osteogenic genes: collagen I, osterix, and BSP [Pelttari et al., 2006; Gawlitta et al., 2010]. In vitro chondrogenic potential of rat bone marrow-derived stem cells (BMSC) has been documented in monolayer cultures [Worster et al., 2006], three-dimensional scaffold cultures [Coleman et al., 2007], and pellet cultures [Muraglia et al., 2003]. In the present study, we constructed the in vitro chondrogenic and hypertrophic differentiation model with rat BMSC pellets and optimized PEMF intensity to accelerate the in vitro endochondral ossification process and propose that the procedure is a potential strategy for bone formation and repair. MATERIALS AND METHODS Cell Culture BMSC were harvested from the posterior tibia and femur bone marrow of 2-week-old male SpragueBioelectromagnetics
Dawley rats (Experimental Animal Center of Sichuan University, Chengdu, China), as reported previously [Qi et al., 2009]. Passage 3 cells were used for subsequent experiments. MSCs were identified as CD34 (), CD45 (), CD29 (þ), and CD44 (þ). Chondrogenic and Hypertrophic Induction of Rat BMSC Pellets Culture Passage 3 cells were dissociated, centrifuged, and resuspended to a concentration of 5 105 cells/ml in chondrogenic medium (RASMX-90042; Cyagen Biosciences, Guangzhou, China) containing 100 ml/L dexamethasone (RASMX-90042; Cyagen Biosciences), 3 ml/L ascorbate (RASMX-90042; Cyagen Biosciences), 10 ml/L Insulin-Transferrin-Selenium (ITS, RASMX90042; Cyagen Biosciences), 1 ml/L sodium pyruvate (RASMX-90042; Cyagen Biosciences), 1 ml/L proline (RASMX-90042; Cyagen Biosciences), and 10 ml/L TGF-b3 (RASMX-90042; Cyagen Biosciences). Cells were then centrifuged at 150g for 5 min and maintained at 37 8C in a humidified atmosphere containing 5% CO2. After 24 h, cells were aggregated into pellets, and cell pellets were carefully transferred to 24-well plates, four pellets per well with 1 ml chondrogenic medium (RASMX-90042; Cyagen Biosciences). The medium was changed every 2–3 days, and cells were cultured for 4 weeks. In order to induce a hypertrophic phenotype, BMSC pellets were cultured for an additional week in hypertrophic medium lacking TGF-b3, but supplemented with 50 nM thyroxine (T1775; Sigma-Aldrich, St. Louis, MO), 7.0 103 M b-glycerophosphate (RASMX-90021; Cyagen Biosciences), 108 M dexamethasone (RASMX-90042; Cyagen Biosciences), and 2.5 104 M ascorbic acid (RASMX-90042; Cyagen Biosciences) after 3 weeks of chondrogenic induction [Mueller and Tuan, 2008; Scotti et al., 2010]. PEMF Stimulation The intensity of the PEMF stimulus and the culture conditions in each seven experimental groups of rat BMSC pellets are shown in Table 1. KDSC4000 PEMF (Chengdu Miracle Chemical, Chengdu, China) stimulation was applied for 3 h/day for 4 weeks with modulation frequency of 750 Hz, carrier frequency of 75 Hz, and three different intensities of 1, 2, and 5 mT (Fig. 1). The specific PEMF signal (75 Hz/750 Hz) was chosen based on previous studies [Fini et al., 2005]. The carrier frequency 75 Hz and pulse duration 1.3 ms were commonly used in previous PEMF studies. The modulation frequency was calculated and set to 750 Hz to meet the requirement. The strength of the magnetic field (1, 2, or 5 mT) was determined by the magnetic field computing software (Chengdu Miracle Chemical) associated with the PEMF device.
PEMF Stimulation of Endochondral Ossification TABLE 1. Experimental Groups, the Intensity of the PEMF Stimulus and the Culture Conditions in Each Seven Experimental Groups of Rat BMSC Pellets Group Group Group Group Group Group Group Group
Procedure C N Nh PS1 PS2 PS3 PSh
No induction þ No PEMF stimulation CI for 4wk þ No PEMF stimulation CI for 3wk þ HI for 1wk þ No PEMF stimulation CI for 4wk þ 1 mT PS for 4wk CI for 4wk þ 2 mT PS for 4wk CI for 4wk þ 5 mT PS for 4wk CI for 3wk þ HI for 1wk þ 1 mT PS for 4wk
Groups C, N, and Nh were control groups. CI, chondrogenic induction; HI, hypertrophic induction; PS, PEMF stimulation.
By entering the operating current and the sample position, the software was able to calculate the maximum field strength of certain position, and the amplitude represents a peak-to-peak value. Chondrogenically differentiated rat BMSC pellets were divided into three different groups based on the intensity: group PS1, 1 mT; group PS2, 2 mT; and group PS3, 5 mT. For group PS1, PS2, and PS3, the cultures were simultaneously treated with PEMF and chondrogenic medium over the 4-week period. Group PSh was cultured in hypertrophic medium for 1 week
Fig. 1. Schematic representation of PFMF pulse protocol and PEMF generating device. A: The PEMF output waveform consisted of a pulsed burst repeated at 75 Hz (burst duration,13 ms; pulse duration,1.3 ms). B: The PEMF device was comprised of a control box and a magnetic field unit.The magnetic field unit was placed in an incubator (37 8C, 5% CO 2). BMSC pellets cultured in 24-well plates were placed between the top and bottom coils of the devicefor thepulsedelectromagnetic field stimulationfor 3 h/ day.
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with PEMF stimulation of 1 mT, after 3 weeks of chondrogenic induction. Group PS1 pellets displayed the most osteogenic effect by 1 mT PEMF stimulation among groups PS1, PS2, and PS3 in the first part of our study. Therefore 1 mT intensity was adopted in Group PSh. Group C was the negative control consisting of uninduced pellets. Group N was the chondrogenic positive control consisting of pellets that were chondrogenic-induced for 4 weeks with no PEMF stimulation. Group Nh was the hypertrophic positive control consisting of pellets that were cultured in hypertrophic medium for 1 week without PEMF stimulation, after 3 weeks of chondrogenic induction. All seven experimental groups were cultured in one incubator. PEMF stimulation was applied in another incubator (no current to the coils for the control groups). MTT Assay for Cell Proliferation Activity of Rat BMSC Cell proliferation activity was determined using the 3-(4, 5-dimethylthiazol-2)-2, 5-diphenyltetrazolium bromide (MTT) assay kit according to the manufacturer’s protocol (Amresco, Solon, OH). BMSC were seeded at 2 104 cells/well in 200 ml a-minimum essential medium (a-MEM, A1049001; Gibco, Carlsbad, CA)/10% fetal bovine serum (FBS, F2442; Sigma– Aldrich, Grand Island, NY) and exposed to different intensities of PEMF stimulation (1 mT, 2 mT, 5 mT for 3 h/day). A no-stimulation group was used as control. Samples (n ¼ 10) were collected at the same time on days 1, 2, 3, 4, and 5 of cell culture for the MTT colorimetric test [Wang et al., 2013]. Immunohistochemical and Histochemical Staining The cell pellets were fixed in 4% paraformaldehyde for 48 h, rinsed with distilled water, dehydrated in a graded series of ethanols, embedded in paraffin, and cut into 5 mm thick sections that were collected on slides. The sections were subjected to hematoxylin eosin and safranin O-fast green staining. Expression of collagen type I, collagen type II, collagen type X, and BSP were detected by immunohistochemical staining method using specific primary antibodies as follows: collagen type I (08A001277; MP Biomedicals, Solon, OH), collagen type II (08A001280; MP Biomedicals), collagen type X (ab7046; AbCam, Cambridge, UK), BSP (A4232.1/A4232.2; Immundiagnostik, Bensheim, Germany), as well as biotinylated secondary antibodies (Dako, Carpinteria, CA).The immunobinding was detected using the appropriate avidin-biotin complex (ABC PK-4000; Vector Laboratories, Peterborough, UK). Image-Pro Plus 6.0 software (Media Cybernetics, Bioelectromagnetics
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Silver Spring, MD) was used for semi-quantitative analysis of immunohistochemical images using the following scores: area, integrated optical density (IOD) and mean optical density (MOD ¼ IOD SUM/area SUM). Real-Time Polymerase Chain Reaction Analysis Twelve pellets per condition were homogenized in 1 ml Trizol reagent (Invitrogen, Carlsbad, CA). Equal amounts of RNA samples were reverse-transcribed using the SYBR PrimeScrip RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s protocol. Each real-time polymerase chain reaction (PCR) was carried out in triplicate in an ABI PRISM 7300 Fast Real-time System (Applied Biosystems, Foster City, CA) using SYBR Premix Ex Taq II (TaKaRa) with primers listed in Table 2. The PCR amplification reaction was carried out for 40 cycles by denaturing at 95 8C for 5 s, and annealing at 60 8C for 30 s. b-actin was used for normalization. Value of the negative control group (group C) was designated as 1. Statistical Analysis All experiments were performed a minimum of three times. All values are expressed as mean standard deviation of three biological repeats. Statistical comparisons were made using one-way analysis of variance, followed by multiple comparisons using Student–Newman–Keuls test (SNK test). A value of P < 0.05 was considered statistically significant. RESULTS Effect of PEMF on Cell Proliferation The MTT assay showed that PEMF stimulation (1 mT, 2 mT, 5 mT) did not cause significant changes
Fig. 2. Growth curves of rat BMSC from controland experimental groups.1, 2, and 5 mTrepresent various doses of PEMF intervention.
in rat BMSC proliferation in two-dimensional culture system (Fig. 2). Due to the failure of control pellets to form a sufficiently robust ECM in stromal medium (group C), cell proliferation analysis was conducted with monolayer cultured stem cells, and the data of group C were thus not included in the remaining results. Histological Characteristics of PEMF-Stimulated Stem Cell Pellets In group N (4 weeks chondrogenic differentiation), regions with cartilaginous morphology were present, accompanied by central necrosis and an eosinophilic matrix, which accumulated mainly in the external zone. In groups PS1 (PEMF at 1 mT) and PS2 (PEMF at 2 mT), chondrogenic pellets displayed cartilaginous features, including cells in lacunae embedded in an abundant matrix. Interestingly, in group PS3, 5 mT PEMF induced notable differences in morphology, with prominent central necrosis and large
TABLE 2. Primer Sequences Used for Real-Time RT-PCR Analysis Target gene b-Actin Sox9 Col10a1 Col1a Runx2 Osx TGF-b3
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Primer sequence Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
50 -ACGGTCAGGTCATCACTATCG-30 50 -GGCATAGAGGTCTTTACGGATG-30 50 -TGCTCGGAACTGTCTGGAAACT-30 50 -GAGGAGGAGGGAGGGAAAACA-30 50 -GATGCCTCTTGTCAGTGCTAACC-30 50 -GATCTTGGGTCATAGTGCTGCTG-30 50 -GGCAAGACAGTCATCGAATACA-30 50 -GATGGAGGGAGTTTACACGAAG-30 50 -GAAATGCCTCTGCTGTTATGAA-30 50 -AAAGTGAAACTCTTGCCTCGTC-30 50 -AAGTTCACCTGTCTGCTCTGCTC-30 50 -GGCTGATTGGCTTCTTCTTCC-30 50 -AGGTTTTCCGTTTCAATGTGTC-30 50 -TTGGCTATGTGTTCATCAGGTC-30
Accession number NM_031144 XM_003750950 XM_001053056 NM_053304 NM_001278483 NM_001037632 NM_013174
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empty areas in the ECM aligned beneath the periphery of the pellet. In group Nh, the hypertrophic pellets displayed a basophilic matrix and marked necrosis centrally with a ring of peripheral cells. In group PSh, the hypertrophic pellets stimulated with 1 mT PEMF showed a scattered eosinophilic matrix without obvious peripheral cells. In group C, control pellets grown in stromal medium failed to form a sufficiently robust ECM (Fig. 3, left panel). Expression of Chondrogenic Markers Affected by PEMF In group N, strong safranin O staining suggested high glycosaminoglycans (GAG) expression, especially in the region of necrosis. The safranin O staining area and intensity decreased with increasing intensity of PEMF stimulation. In group Nh, as chondrogenesis progressed to hypertrophy, reduced level of GAG was detected. Higher safranin O staining was present in group PSh, the 1 mT hypertrophic pellets (Fig. 3, right panel). Collagen type II was detected in PEMF stimulated pellets as well as in control group N. It was also detected in group Nh between the internal necrosis area and the outer rim of elongate cells, whereas no sign of collagen type II was present in group PSh (Fig. 4). Expression of Hypertrophic and Osteogenic Markers Affected by PEMF Type X collagen, a marker for chondrocyte hypertrophic differentiation, was present throughout group PS1 (1 mT) compared with no expression in group N after 4 weeks’ chondrogenic induction. But it was barely expressed in group PS2 (2 mT) and group PS3 (5 mT). The hypertrophic group (Nh) was uniformly positive for type X collagen, whereas only low and localized levels of type X collagen were detected in hypertrophic pellets exposed to 1 mT PEMF stimulation (group PSh; Fig. 4). In group N, group PS1, group Nh, and group PSh, immunohistochemical analysis showed high levels of homogeneous staining for type I collagen. In groups PS2 (2 mT) and PS3 (5 mT), type I collagen staining intensity decreased significantly (Fig. 4). BSP expression was enhanced in group PS1 (1 mT) compared with group N. It was confined predominantly to the outer rim underneath the superficial layer of the pellets, with notably stronger intensity in the periphery of the lacunae and an overlap with type X collagen. In group PS2 (2 mT), decreased BSP expression was detected compared to group PS1, while BSP production was not detected in group PS3 (5 mT). Group PSh displayed diffused deposition of BSP, while group Nh was negative for BSP (Fig. 4).
Fig. 3. Safranin red O-fast green staining of rat BMSC pellets. PS1, PS2, PS3: chondrogenic groups exposed to PEMF (PS1, 1mT; PS2, 2 mT; PS3, 5 mT; 75 Hz frequency) for 3 h/day for 4 weeks. Group N: chondrogenic positive control consisting of pellets that were chondrogenic-induced for 4 weeks with no PEMF stimulation. PSh: cultured in hypertrophic medium for 1week with1mT PEMF, after 3 weeks of chondrogenic induction. Group Nh: hypertrophic positive control consisting of pellets that were cultured in hypertrophic medium for 1 week without PEMF stimulation, after 3 weeks of chondrogenic induction.The asterisk and the arrowindicate centralnecrosis and eosinophilic matrixthat hadaccumulatedmainlyinthe externalzone, respectively; Scalebar ¼100 mm. Bioelectromagnetics
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Fig. 4. Effect of PEMF treatments on chondrogenic- and osteogenic-related protein expression in rat BMSC pellets. A: immunohistochemistry results of collagen II, collagen I, collagen X, and BSPin BMSCpelletswith PEMF stimulation.Scalebar ¼10 mm.B:Meanopticaldensityof collagen II, collagen I, collagen X, and BSPin immunohistochemical staining of PEMF-stimulated chondrogenic groups. P < 0.05 when compared to group N.C:Mean opticaldensityof collagen II, collagen I, collagen X, and BSP in immunohistochemical staining of PEMF-stimulated hypertrophic group. P < 0.05 whencomparedtogroup Nh.
Effect of PEMF Treatment on Chondrogenicand Osteogenic-Related Gene Expression Profile of Rat BMSC In PS groups, PEMF led to a significant decrease in Sox9 and TGF-b3 mRNA expression compared Bioelectromagnetics
with group N. A similar effect was seen in group PSh compared with group Nh (Figs. 5 and 6). Consistent with the immunohistochemistry observations, PEMF treatment significantly induced more Col10a1 mRNA expression in PS groups compared with group N, but significantly lower expression was
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Fig. 5. Effect of PEMF stimulation on chondrogenic and osteogenic gene expression in chondrogenicrat BMSCpellets.Quantitativereal-time RT-PCRanalysisofchondrogenic- andosteogenicrelated gene expressionlevelsusing total RNAisolated from PEMF-stimulated chondrogenic pellets. P < 0.05 whencomparedtogroup N (Y-axis: 2DDCt).
detected in group PSh compared with group Nh (Figs. 5 and 6). Real-time RT-PCR showed that expression of Runx2, a gene critical for chondrocyte maturation and osteoblast differentiation, was upregulated in PS groups over controls (group N), whereas it was inhibited by 1 mT PEMF treatment in the group PSh compared with group Ph. Gene expression level of osterix, the gene that controls osteoblast lineage commitment and subsequent osteoblast differentiation, was significantly increased by PEMF treatment in both PS groups and group PSh (Figs. 5 and 6). DISCUSSION Defining the molecular mechanism of the effect of PEMF on chondrogenic and hypertrophic differentiation of BMSC has potential value in bone engineering. The MTT assay was used to investigate whether selected intensities of PEMF stimulation affected BMSC proliferation at the early stage of preliminarily expansion. No significant change was detected compared to the controls.
As to the effect of PEMF on chondrogenesis, lower level of GAG (principally a measure of aggrecan) and collagen type II protein expression was detected, as well as a significant decrease in Sox9 and TGF-b3 mRNA expression in PEMF groups (PS groups). This suggests that PEMF may inhibit the maintenance of the cartilaginous phenotype and promote cartilage specific ECM degradation in chondrogenically differentiated rat BMSC pellets [Hattori et al., 2010]. A decreased collagen type II protein expression was also detected in BMSC pellets imposed with PEMF stimulation and hypertrophic medium simultaneously (PSh group). However, increased GAG expression was detected in PSh group compared with Nh group. This indicates that PEMF may inhibit or interfere with aggrecan (GAG) degradation in the presence of hypertrophic medium. As the two major components of cartilage ECM, many proteolytic enzymes are responsible for the degradative events of collagen type II and aggrecan, such as matrix metalloproteinases and the aggrecanases [Ortega et al., 2004; Mackie et al., 2008]. PEMF may have influenced the Bioelectromagnetics
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Fig. 6. Effect of PEMF stimulation on chondrogenic and osteogenic gene expression in hypertrophic differentiatedrat BMSCpellets.Quantitativereal-time RT-PCRanalysisof chondrogenic- and osteogenic-relatedgene expressionlevelsusingtotal RNAisolatedfrom PEMF-stimulatedhypertrophicpellets. P < 0.05 whencomparedtogroup Nh (Y-axis: 2DDCt).
function of some proteases assisting with removal of aggrecan. However, there is no literature on this aspect currently and further investigations are needed. Despite the inhibitory effect on chondrogenic phenotype maintenance, PEMF is crucial in promoting the hypertrophic phase of endochondral ossification. As the only known hypertrophic chondrocyte-specific marker, the expression level of collagen X is increased when the immature chondrocytes differentiate into hypertrophic chondrocytes, and decreased when the hypertrophic chondrocytes differentiate into osteoblasts [Zheng et al., 2003; Gawlitta et al., 2010]. In the present study, in group PS1 (1 mT PEMF), there were higher levels of collagen X gene and protein than group N, indicating that certain intensity PEMF promoted the hypertrophic differentiation of BMSC pellets. Collagen X was strongly expressed in group Nh but weakly in group PSh, implying that PEMF might promote hypertrophic BMSC to differentiate into osteoblasts. PEMF has played a bidirectional regulation role during endochondral ossification on Bioelectromagnetics
collagen type X and accelerated the hypertrophic stage. Promotion of the final stage of endochondral ossification by 1 mT PEMF stimulation was shown by a significant increase in expression of BSP, collagen type I, and osterix in response to both chondrogenic (group PS1) and hypertrophic (group PSh) cues. However, no such effects were detected when cells were exposed to PEMF of 2 mT and 5 mT. Therefore, certain PEMF stimulus may promote the in vitro hypertrophic and endochondral ossification process, and the intensity of 1 mT was optimal in the present study. If wider amplitude range was studied, the optimal amplitude may lay outside the amplitudes tested in the present study. In summary, we investigated the effects of PEMF on hypertrophy and endochondral ossification of rat BMSC pellets in the late chondrogenic and hypertrophic stage. PEMF at 1 mT inhibited the maintenance of the chondrocyte phenotype in chondrogenic differentiated BMSC pellets, caused the earlier onset of
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hypertrophy, and accelerated the endochondral ossification process in hypertrophic chondrocytes. However, the term “endochondral ossification” is usually referred to an in vivo process of bone formation, which consists of certain well-established steps. The present study only mimicked one of these processes. As the bone formation process cannot be accomplished in vitro without the invasion of blood vessels, osteoblasts, and bone marrow cells, further investigation of the application of PEMF is necessary to determine whether similar effects will be achieved in vivo and to determine the underlying mechanisms. REFERENCES Blaney Davidson EN, Vitters EL, van der Kraan PM, van den Berg WB. 2006. Expression of transforming growth factor-beta (TGFbeta) and the TGFbeta signaling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis: Role in cartilage degradation, chondrogenesis and osteophyte formation. Ann Rheum Dis 65:1414–1421. Coleman RM, Case ND, Guldberg RE. 2007. Hydrogel effects on bone marrow stromal cell response to chondrogenic growth factors. Biomaterials 28:2077–2086. Coyle CH, Izzo NJ, Chu CR. 2009. Sustained hypoxia enhances chondrocyte matrix synthesis. J Orthop Res 27:793–799. De Mattei M, Fini M, Setti S, Ongaro A, Gemmati D, Stabellini G, Pellati A, Caruso A. 2007. Proteoglycan synthesis in bovine articular cartilage explants exposed to different low-frequency low-energy pulsed electromagnetic fields. Osteoarthritis Cartilage 15:163–168. Fini M, Giavaresi G, Carpi A, Nicolini A, Setti S, Giardino R. 2005. Effects of pulsed electromagnetic fields on articular hyaline cartilage: Review of experimental and clinical studies. Biomed Pharmacother 59:388–394. Fini M, Pagani S, Giavaresi G, De Mattei M, Ongaro A, Varani K, Vincenzi F, Massari L, Cadossi M. 2013. Functional tissue engineering in articular cartilage repair: Is there a role for electromagnetic biophysical stimulation? Tissue Eng Part B Rev 19:353–367. Gawlitta D, Farrell E, Malda J, Creemers LB, Alblas J, Dhert WJ. 2010. Modulating endochondral ossification of multipotent stromal cells for bone regeneration. Tissue Eng Part B Rev 16:385–395. Griffin XL, Costa ML, Parsons N, Smith N. 2011. Electromagnetic field stimulation for treating delayed union or non-union of long bone fractures in adults. Cochrane Database Syst Rev 4:CD008471. Hattori T, Müller C, Gebhard S, Bauer E, Pausch F, Schlund B, Bösl MR, Hess A, Surmann-Schmitt C, von der Mark H, de Crombrugghe B, von der Mark K. 2010. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development 137:901–911. Komori T. 2010. Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell Tissue Res 339:189– 195. Mackie EJ, Ahmed YA, Tatarczuch L, Chen KS, Mirams M. 2008. Endochondral ossification: How cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol 40:46–62.
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Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B. 2003. Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte– specific expression in vivo. J Cell Biol 162:833– 842. Zhou J, Ming LG, Ge BF, Wang JQ, Zhu RQ, Wei Z, Ma HP, Xian CJ, Chen KM. 2011. Effects of 50 Hz sinusoidal electromagnetic fields of different intensities on proliferation, differentiation and mineralization potentials of rat osteoblasts. Bone 49:753–761.
Pulsed Electromagnetic Field May Accelerate in vitro Endochondral Ossification JueWang,1 NaTang,2 Qiang Xiao,1 Li Zhang,1 Yu Li,1 Juan Li,1 JunWang,1 Zhihe Zhao,1 and LijunTan1* 1
State Key Laboratory of Oral Diseases,West China Hospital of Stomatology, Sichuan University, Chengdu, China 2 Stomatology Department of Sichuan Medical Science Academy, Sichuan Provincial People’s Hospital, Chengdu, China
Recapitulation of embryonic endochondral bone formation is a promising alternative approach to bone tissue engineering. However, the time-consuming process is one of the reasons the approach is unpractical. Here, we aimed at accelerating the in vitro endochondral ossification process of tissue engineering by using a pulsed electromagnetic field (PEMF). The rat bone marrow-derived stem cells were chondrogenic or hypertrophic differentiated in a three-dimensional pellet culture system, and treated with different intensities of PEMF (1, 2, and 5 mT with modulation frequency 750 Hz, carrier frequency 75 Hz and a duty ratio of 0.8, 3 h/day for 4 weeks). The effects of PEMF on hypertrophy and endochondral ossification were assessed by safranin O staining, immunohistochemistry, and quantitative real-time polymerase chain reaction. The results suggest that PEMF at 1, 2, and 5 mT may inhibit the maintenance of the cartilaginous phenotype and increase cartilage-specific extracellular matrix degradation in the late stage of chondrogenic differentiation. In addition, among the three different intensities, only PEMF at 1 mT directed the differentiation of chondrogenicinduced stem cell pellets to the hypertrophic stage and promoted osteogenic differentiation. Our findings provide the feasibility to optimize the process of in vitro endochondral ossification with PEMF stimulation. Bioelectromagnetics 36:35–44, 2015. © 2014 Wiley Periodicals, Inc. Key words: bone repair; bone marrow-derived stem cells; tissue engineering; cartilage; physical stimulation
INTRODUCTION Attempts to engineer bone tissue generally focus on intramembranous bone formation. However, bone tissue engineering through intramembranous bone formation for clinical application is not successful, partly because of the lack of sufficient vasculature during initial stage [Meijer et al., 2007]. Therefore, the concept of recapitulation of endochondral bone formation has been investigated as a feasible approach [Sasaki et al., 2010; Scotti et al., 2010, 2013]. It is a more physiological process by which the embryonic intermediate cartilaginous template is gradually replaced by bone tissue. Because cartilage by nature is equipped to survive in hypoxic conditions, repair of bone defects over cartilaginous templates may help overcome the lack of vascularization [Coyle et al., 2009]. This will be of benefit especially for larger tissue engineering constructs. However, this approach for bone tissue engineering has been largely overlooked, mostly because of the long process length time involved [Tortelli et al., 2010]. Thus, to achieve functional and timely mechanical and morphological development, it is important to find strategies to 2014 Wiley Periodicals, Inc.
accelerate the hypertrophic and endochondral ossification process. Pulsed electromagnetic field (PEMF) is a safe, non-invasive, clinically beneficial physical stimulus, which has been used clinically to promote the healing of fracture non-union and osteoarthritis [De Mattei et al., 2007; Griffin et al., 2011; Ryang et al., 2013]. Previous studies have revealed that PEMF can enhance the activity of osteoblasts and promote osteogenic Grant sponsor: National Natural Science Foundation of China; grant numbers: 30900287, 1030034, 30900286. Conflicts of interest: None. *Correspondence to: Lijun Tan, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, 14#, 3rd section, Renmin South Road, Chengdu 610041, China. E-mail: [email protected] Received for review 18 March 2014; Accepted 13 August 2014 DOI: 10.1002/bem.21882 Published online 30 October 2014 in Wiley Online Library (wileyonlinelibrary.com).
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differentiation of mesenchymal stem cells (MSCs), and can stimulate chondrocyte proliferation and extracellular matrix (ECM) synthesis; therefore, PEMF can be used to control construct differentiation in tissue engineering [Sun et al., 2010; Mayer-Wagner et al., 2011; Zhou et al., 2011; Ongaro et al., 2012; Fini et al., 2013]. Indeed, PEMF is regarded as a convenient and effective way to alter the physical microenvironment of bone tissue through electromagnetic effect rather than mechanical energy [Trock, 2000]. To assess the effects of PEMF on hypertrophy and endochondral ossification process, collagen type I, collagen type II, collagen type X, Sox9, bone sialoprotein (BSP), transforming growth factor-b3 (TGF-b3), osterix (Osx), and runt-related transcription factor 2 (Runx2) were used as the differentiation markers in this study. During embryonic endochondral bone formation, MSCs condense and differentiate into immature chondrocytes. The chondrocytes then synthesize a matrix that mainly consists of collagen II and aggrecan. TGF-b3 stimulates production of ECM components and is involved in the formation of new cartilage [Blaney Davidson et al., 2006]. Subsequently, the chondrocytes further differentiate and enter the prehypertrophic stage, producing Runx2 [Komori, 2010]. Expression of Runx2 stimulates the upregulation of hypertrophic markers collagen X. Once collagen X is synthesized, the cells are differentiated into the hypertrophic stage. During this final stage of differentiation, the expression of chondrogenic genes, such as Sox9 and TGF-b3, is downregulated in hypertrophic chondrocytes as a necessary step to initiate cartilage-bone transition [Hattori et al., 2010]. The hypertrophic cells are now characterized by expression of the osteogenic genes: collagen I, osterix, and BSP [Pelttari et al., 2006; Gawlitta et al., 2010]. In vitro chondrogenic potential of rat bone marrow-derived stem cells (BMSC) has been documented in monolayer cultures [Worster et al., 2006], three-dimensional scaffold cultures [Coleman et al., 2007], and pellet cultures [Muraglia et al., 2003]. In the present study, we constructed the in vitro chondrogenic and hypertrophic differentiation model with rat BMSC pellets and optimized PEMF intensity to accelerate the in vitro endochondral ossification process and propose that the procedure is a potential strategy for bone formation and repair. MATERIALS AND METHODS Cell Culture BMSC were harvested from the posterior tibia and femur bone marrow of 2-week-old male SpragueBioelectromagnetics
Dawley rats (Experimental Animal Center of Sichuan University, Chengdu, China), as reported previously [Qi et al., 2009]. Passage 3 cells were used for subsequent experiments. MSCs were identified as CD34 (), CD45 (), CD29 (þ), and CD44 (þ). Chondrogenic and Hypertrophic Induction of Rat BMSC Pellets Culture Passage 3 cells were dissociated, centrifuged, and resuspended to a concentration of 5 105 cells/ml in chondrogenic medium (RASMX-90042; Cyagen Biosciences, Guangzhou, China) containing 100 ml/L dexamethasone (RASMX-90042; Cyagen Biosciences), 3 ml/L ascorbate (RASMX-90042; Cyagen Biosciences), 10 ml/L Insulin-Transferrin-Selenium (ITS, RASMX90042; Cyagen Biosciences), 1 ml/L sodium pyruvate (RASMX-90042; Cyagen Biosciences), 1 ml/L proline (RASMX-90042; Cyagen Biosciences), and 10 ml/L TGF-b3 (RASMX-90042; Cyagen Biosciences). Cells were then centrifuged at 150g for 5 min and maintained at 37 8C in a humidified atmosphere containing 5% CO2. After 24 h, cells were aggregated into pellets, and cell pellets were carefully transferred to 24-well plates, four pellets per well with 1 ml chondrogenic medium (RASMX-90042; Cyagen Biosciences). The medium was changed every 2–3 days, and cells were cultured for 4 weeks. In order to induce a hypertrophic phenotype, BMSC pellets were cultured for an additional week in hypertrophic medium lacking TGF-b3, but supplemented with 50 nM thyroxine (T1775; Sigma-Aldrich, St. Louis, MO), 7.0 103 M b-glycerophosphate (RASMX-90021; Cyagen Biosciences), 108 M dexamethasone (RASMX-90042; Cyagen Biosciences), and 2.5 104 M ascorbic acid (RASMX-90042; Cyagen Biosciences) after 3 weeks of chondrogenic induction [Mueller and Tuan, 2008; Scotti et al., 2010]. PEMF Stimulation The intensity of the PEMF stimulus and the culture conditions in each seven experimental groups of rat BMSC pellets are shown in Table 1. KDSC4000 PEMF (Chengdu Miracle Chemical, Chengdu, China) stimulation was applied for 3 h/day for 4 weeks with modulation frequency of 750 Hz, carrier frequency of 75 Hz, and three different intensities of 1, 2, and 5 mT (Fig. 1). The specific PEMF signal (75 Hz/750 Hz) was chosen based on previous studies [Fini et al., 2005]. The carrier frequency 75 Hz and pulse duration 1.3 ms were commonly used in previous PEMF studies. The modulation frequency was calculated and set to 750 Hz to meet the requirement. The strength of the magnetic field (1, 2, or 5 mT) was determined by the magnetic field computing software (Chengdu Miracle Chemical) associated with the PEMF device.
PEMF Stimulation of Endochondral Ossification TABLE 1. Experimental Groups, the Intensity of the PEMF Stimulus and the Culture Conditions in Each Seven Experimental Groups of Rat BMSC Pellets Group Group Group Group Group Group Group Group
Procedure C N Nh PS1 PS2 PS3 PSh
No induction þ No PEMF stimulation CI for 4wk þ No PEMF stimulation CI for 3wk þ HI for 1wk þ No PEMF stimulation CI for 4wk þ 1 mT PS for 4wk CI for 4wk þ 2 mT PS for 4wk CI for 4wk þ 5 mT PS for 4wk CI for 3wk þ HI for 1wk þ 1 mT PS for 4wk
Groups C, N, and Nh were control groups. CI, chondrogenic induction; HI, hypertrophic induction; PS, PEMF stimulation.
By entering the operating current and the sample position, the software was able to calculate the maximum field strength of certain position, and the amplitude represents a peak-to-peak value. Chondrogenically differentiated rat BMSC pellets were divided into three different groups based on the intensity: group PS1, 1 mT; group PS2, 2 mT; and group PS3, 5 mT. For group PS1, PS2, and PS3, the cultures were simultaneously treated with PEMF and chondrogenic medium over the 4-week period. Group PSh was cultured in hypertrophic medium for 1 week
Fig. 1. Schematic representation of PFMF pulse protocol and PEMF generating device. A: The PEMF output waveform consisted of a pulsed burst repeated at 75 Hz (burst duration,13 ms; pulse duration,1.3 ms). B: The PEMF device was comprised of a control box and a magnetic field unit.The magnetic field unit was placed in an incubator (37 8C, 5% CO 2). BMSC pellets cultured in 24-well plates were placed between the top and bottom coils of the devicefor thepulsedelectromagnetic field stimulationfor 3 h/ day.
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with PEMF stimulation of 1 mT, after 3 weeks of chondrogenic induction. Group PS1 pellets displayed the most osteogenic effect by 1 mT PEMF stimulation among groups PS1, PS2, and PS3 in the first part of our study. Therefore 1 mT intensity was adopted in Group PSh. Group C was the negative control consisting of uninduced pellets. Group N was the chondrogenic positive control consisting of pellets that were chondrogenic-induced for 4 weeks with no PEMF stimulation. Group Nh was the hypertrophic positive control consisting of pellets that were cultured in hypertrophic medium for 1 week without PEMF stimulation, after 3 weeks of chondrogenic induction. All seven experimental groups were cultured in one incubator. PEMF stimulation was applied in another incubator (no current to the coils for the control groups). MTT Assay for Cell Proliferation Activity of Rat BMSC Cell proliferation activity was determined using the 3-(4, 5-dimethylthiazol-2)-2, 5-diphenyltetrazolium bromide (MTT) assay kit according to the manufacturer’s protocol (Amresco, Solon, OH). BMSC were seeded at 2 104 cells/well in 200 ml a-minimum essential medium (a-MEM, A1049001; Gibco, Carlsbad, CA)/10% fetal bovine serum (FBS, F2442; Sigma– Aldrich, Grand Island, NY) and exposed to different intensities of PEMF stimulation (1 mT, 2 mT, 5 mT for 3 h/day). A no-stimulation group was used as control. Samples (n ¼ 10) were collected at the same time on days 1, 2, 3, 4, and 5 of cell culture for the MTT colorimetric test [Wang et al., 2013]. Immunohistochemical and Histochemical Staining The cell pellets were fixed in 4% paraformaldehyde for 48 h, rinsed with distilled water, dehydrated in a graded series of ethanols, embedded in paraffin, and cut into 5 mm thick sections that were collected on slides. The sections were subjected to hematoxylin eosin and safranin O-fast green staining. Expression of collagen type I, collagen type II, collagen type X, and BSP were detected by immunohistochemical staining method using specific primary antibodies as follows: collagen type I (08A001277; MP Biomedicals, Solon, OH), collagen type II (08A001280; MP Biomedicals), collagen type X (ab7046; AbCam, Cambridge, UK), BSP (A4232.1/A4232.2; Immundiagnostik, Bensheim, Germany), as well as biotinylated secondary antibodies (Dako, Carpinteria, CA).The immunobinding was detected using the appropriate avidin-biotin complex (ABC PK-4000; Vector Laboratories, Peterborough, UK). Image-Pro Plus 6.0 software (Media Cybernetics, Bioelectromagnetics
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Silver Spring, MD) was used for semi-quantitative analysis of immunohistochemical images using the following scores: area, integrated optical density (IOD) and mean optical density (MOD ¼ IOD SUM/area SUM). Real-Time Polymerase Chain Reaction Analysis Twelve pellets per condition were homogenized in 1 ml Trizol reagent (Invitrogen, Carlsbad, CA). Equal amounts of RNA samples were reverse-transcribed using the SYBR PrimeScrip RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s protocol. Each real-time polymerase chain reaction (PCR) was carried out in triplicate in an ABI PRISM 7300 Fast Real-time System (Applied Biosystems, Foster City, CA) using SYBR Premix Ex Taq II (TaKaRa) with primers listed in Table 2. The PCR amplification reaction was carried out for 40 cycles by denaturing at 95 8C for 5 s, and annealing at 60 8C for 30 s. b-actin was used for normalization. Value of the negative control group (group C) was designated as 1. Statistical Analysis All experiments were performed a minimum of three times. All values are expressed as mean standard deviation of three biological repeats. Statistical comparisons were made using one-way analysis of variance, followed by multiple comparisons using Student–Newman–Keuls test (SNK test). A value of P < 0.05 was considered statistically significant. RESULTS Effect of PEMF on Cell Proliferation The MTT assay showed that PEMF stimulation (1 mT, 2 mT, 5 mT) did not cause significant changes
Fig. 2. Growth curves of rat BMSC from controland experimental groups.1, 2, and 5 mTrepresent various doses of PEMF intervention.
in rat BMSC proliferation in two-dimensional culture system (Fig. 2). Due to the failure of control pellets to form a sufficiently robust ECM in stromal medium (group C), cell proliferation analysis was conducted with monolayer cultured stem cells, and the data of group C were thus not included in the remaining results. Histological Characteristics of PEMF-Stimulated Stem Cell Pellets In group N (4 weeks chondrogenic differentiation), regions with cartilaginous morphology were present, accompanied by central necrosis and an eosinophilic matrix, which accumulated mainly in the external zone. In groups PS1 (PEMF at 1 mT) and PS2 (PEMF at 2 mT), chondrogenic pellets displayed cartilaginous features, including cells in lacunae embedded in an abundant matrix. Interestingly, in group PS3, 5 mT PEMF induced notable differences in morphology, with prominent central necrosis and large
TABLE 2. Primer Sequences Used for Real-Time RT-PCR Analysis Target gene b-Actin Sox9 Col10a1 Col1a Runx2 Osx TGF-b3
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Primer sequence Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
50 -ACGGTCAGGTCATCACTATCG-30 50 -GGCATAGAGGTCTTTACGGATG-30 50 -TGCTCGGAACTGTCTGGAAACT-30 50 -GAGGAGGAGGGAGGGAAAACA-30 50 -GATGCCTCTTGTCAGTGCTAACC-30 50 -GATCTTGGGTCATAGTGCTGCTG-30 50 -GGCAAGACAGTCATCGAATACA-30 50 -GATGGAGGGAGTTTACACGAAG-30 50 -GAAATGCCTCTGCTGTTATGAA-30 50 -AAAGTGAAACTCTTGCCTCGTC-30 50 -AAGTTCACCTGTCTGCTCTGCTC-30 50 -GGCTGATTGGCTTCTTCTTCC-30 50 -AGGTTTTCCGTTTCAATGTGTC-30 50 -TTGGCTATGTGTTCATCAGGTC-30
Accession number NM_031144 XM_003750950 XM_001053056 NM_053304 NM_001278483 NM_001037632 NM_013174
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empty areas in the ECM aligned beneath the periphery of the pellet. In group Nh, the hypertrophic pellets displayed a basophilic matrix and marked necrosis centrally with a ring of peripheral cells. In group PSh, the hypertrophic pellets stimulated with 1 mT PEMF showed a scattered eosinophilic matrix without obvious peripheral cells. In group C, control pellets grown in stromal medium failed to form a sufficiently robust ECM (Fig. 3, left panel). Expression of Chondrogenic Markers Affected by PEMF In group N, strong safranin O staining suggested high glycosaminoglycans (GAG) expression, especially in the region of necrosis. The safranin O staining area and intensity decreased with increasing intensity of PEMF stimulation. In group Nh, as chondrogenesis progressed to hypertrophy, reduced level of GAG was detected. Higher safranin O staining was present in group PSh, the 1 mT hypertrophic pellets (Fig. 3, right panel). Collagen type II was detected in PEMF stimulated pellets as well as in control group N. It was also detected in group Nh between the internal necrosis area and the outer rim of elongate cells, whereas no sign of collagen type II was present in group PSh (Fig. 4). Expression of Hypertrophic and Osteogenic Markers Affected by PEMF Type X collagen, a marker for chondrocyte hypertrophic differentiation, was present throughout group PS1 (1 mT) compared with no expression in group N after 4 weeks’ chondrogenic induction. But it was barely expressed in group PS2 (2 mT) and group PS3 (5 mT). The hypertrophic group (Nh) was uniformly positive for type X collagen, whereas only low and localized levels of type X collagen were detected in hypertrophic pellets exposed to 1 mT PEMF stimulation (group PSh; Fig. 4). In group N, group PS1, group Nh, and group PSh, immunohistochemical analysis showed high levels of homogeneous staining for type I collagen. In groups PS2 (2 mT) and PS3 (5 mT), type I collagen staining intensity decreased significantly (Fig. 4). BSP expression was enhanced in group PS1 (1 mT) compared with group N. It was confined predominantly to the outer rim underneath the superficial layer of the pellets, with notably stronger intensity in the periphery of the lacunae and an overlap with type X collagen. In group PS2 (2 mT), decreased BSP expression was detected compared to group PS1, while BSP production was not detected in group PS3 (5 mT). Group PSh displayed diffused deposition of BSP, while group Nh was negative for BSP (Fig. 4).
Fig. 3. Safranin red O-fast green staining of rat BMSC pellets. PS1, PS2, PS3: chondrogenic groups exposed to PEMF (PS1, 1mT; PS2, 2 mT; PS3, 5 mT; 75 Hz frequency) for 3 h/day for 4 weeks. Group N: chondrogenic positive control consisting of pellets that were chondrogenic-induced for 4 weeks with no PEMF stimulation. PSh: cultured in hypertrophic medium for 1week with1mT PEMF, after 3 weeks of chondrogenic induction. Group Nh: hypertrophic positive control consisting of pellets that were cultured in hypertrophic medium for 1 week without PEMF stimulation, after 3 weeks of chondrogenic induction.The asterisk and the arrowindicate centralnecrosis and eosinophilic matrixthat hadaccumulatedmainlyinthe externalzone, respectively; Scalebar ¼100 mm. Bioelectromagnetics
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Fig. 4. Effect of PEMF treatments on chondrogenic- and osteogenic-related protein expression in rat BMSC pellets. A: immunohistochemistry results of collagen II, collagen I, collagen X, and BSPin BMSCpelletswith PEMF stimulation.Scalebar ¼10 mm.B:Meanopticaldensityof collagen II, collagen I, collagen X, and BSPin immunohistochemical staining of PEMF-stimulated chondrogenic groups. P < 0.05 when compared to group N.C:Mean opticaldensityof collagen II, collagen I, collagen X, and BSP in immunohistochemical staining of PEMF-stimulated hypertrophic group. P < 0.05 whencomparedtogroup Nh.
Effect of PEMF Treatment on Chondrogenicand Osteogenic-Related Gene Expression Profile of Rat BMSC In PS groups, PEMF led to a significant decrease in Sox9 and TGF-b3 mRNA expression compared Bioelectromagnetics
with group N. A similar effect was seen in group PSh compared with group Nh (Figs. 5 and 6). Consistent with the immunohistochemistry observations, PEMF treatment significantly induced more Col10a1 mRNA expression in PS groups compared with group N, but significantly lower expression was
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Fig. 5. Effect of PEMF stimulation on chondrogenic and osteogenic gene expression in chondrogenicrat BMSCpellets.Quantitativereal-time RT-PCRanalysisofchondrogenic- andosteogenicrelated gene expressionlevelsusing total RNAisolated from PEMF-stimulated chondrogenic pellets. P < 0.05 whencomparedtogroup N (Y-axis: 2DDCt).
detected in group PSh compared with group Nh (Figs. 5 and 6). Real-time RT-PCR showed that expression of Runx2, a gene critical for chondrocyte maturation and osteoblast differentiation, was upregulated in PS groups over controls (group N), whereas it was inhibited by 1 mT PEMF treatment in the group PSh compared with group Ph. Gene expression level of osterix, the gene that controls osteoblast lineage commitment and subsequent osteoblast differentiation, was significantly increased by PEMF treatment in both PS groups and group PSh (Figs. 5 and 6). DISCUSSION Defining the molecular mechanism of the effect of PEMF on chondrogenic and hypertrophic differentiation of BMSC has potential value in bone engineering. The MTT assay was used to investigate whether selected intensities of PEMF stimulation affected BMSC proliferation at the early stage of preliminarily expansion. No significant change was detected compared to the controls.
As to the effect of PEMF on chondrogenesis, lower level of GAG (principally a measure of aggrecan) and collagen type II protein expression was detected, as well as a significant decrease in Sox9 and TGF-b3 mRNA expression in PEMF groups (PS groups). This suggests that PEMF may inhibit the maintenance of the cartilaginous phenotype and promote cartilage specific ECM degradation in chondrogenically differentiated rat BMSC pellets [Hattori et al., 2010]. A decreased collagen type II protein expression was also detected in BMSC pellets imposed with PEMF stimulation and hypertrophic medium simultaneously (PSh group). However, increased GAG expression was detected in PSh group compared with Nh group. This indicates that PEMF may inhibit or interfere with aggrecan (GAG) degradation in the presence of hypertrophic medium. As the two major components of cartilage ECM, many proteolytic enzymes are responsible for the degradative events of collagen type II and aggrecan, such as matrix metalloproteinases and the aggrecanases [Ortega et al., 2004; Mackie et al., 2008]. PEMF may have influenced the Bioelectromagnetics
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Fig. 6. Effect of PEMF stimulation on chondrogenic and osteogenic gene expression in hypertrophic differentiatedrat BMSCpellets.Quantitativereal-time RT-PCRanalysisof chondrogenic- and osteogenic-relatedgene expressionlevelsusingtotal RNAisolatedfrom PEMF-stimulatedhypertrophicpellets. P < 0.05 whencomparedtogroup Nh (Y-axis: 2DDCt).
function of some proteases assisting with removal of aggrecan. However, there is no literature on this aspect currently and further investigations are needed. Despite the inhibitory effect on chondrogenic phenotype maintenance, PEMF is crucial in promoting the hypertrophic phase of endochondral ossification. As the only known hypertrophic chondrocyte-specific marker, the expression level of collagen X is increased when the immature chondrocytes differentiate into hypertrophic chondrocytes, and decreased when the hypertrophic chondrocytes differentiate into osteoblasts [Zheng et al., 2003; Gawlitta et al., 2010]. In the present study, in group PS1 (1 mT PEMF), there were higher levels of collagen X gene and protein than group N, indicating that certain intensity PEMF promoted the hypertrophic differentiation of BMSC pellets. Collagen X was strongly expressed in group Nh but weakly in group PSh, implying that PEMF might promote hypertrophic BMSC to differentiate into osteoblasts. PEMF has played a bidirectional regulation role during endochondral ossification on Bioelectromagnetics
collagen type X and accelerated the hypertrophic stage. Promotion of the final stage of endochondral ossification by 1 mT PEMF stimulation was shown by a significant increase in expression of BSP, collagen type I, and osterix in response to both chondrogenic (group PS1) and hypertrophic (group PSh) cues. However, no such effects were detected when cells were exposed to PEMF of 2 mT and 5 mT. Therefore, certain PEMF stimulus may promote the in vitro hypertrophic and endochondral ossification process, and the intensity of 1 mT was optimal in the present study. If wider amplitude range was studied, the optimal amplitude may lay outside the amplitudes tested in the present study. In summary, we investigated the effects of PEMF on hypertrophy and endochondral ossification of rat BMSC pellets in the late chondrogenic and hypertrophic stage. PEMF at 1 mT inhibited the maintenance of the chondrocyte phenotype in chondrogenic differentiated BMSC pellets, caused the earlier onset of
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hypertrophy, and accelerated the endochondral ossification process in hypertrophic chondrocytes. However, the term “endochondral ossification” is usually referred to an in vivo process of bone formation, which consists of certain well-established steps. The present study only mimicked one of these processes. As the bone formation process cannot be accomplished in vitro without the invasion of blood vessels, osteoblasts, and bone marrow cells, further investigation of the application of PEMF is necessary to determine whether similar effects will be achieved in vivo and to determine the underlying mechanisms. REFERENCES Blaney Davidson EN, Vitters EL, van der Kraan PM, van den Berg WB. 2006. Expression of transforming growth factor-beta (TGFbeta) and the TGFbeta signaling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis: Role in cartilage degradation, chondrogenesis and osteophyte formation. Ann Rheum Dis 65:1414–1421. Coleman RM, Case ND, Guldberg RE. 2007. Hydrogel effects on bone marrow stromal cell response to chondrogenic growth factors. Biomaterials 28:2077–2086. Coyle CH, Izzo NJ, Chu CR. 2009. Sustained hypoxia enhances chondrocyte matrix synthesis. J Orthop Res 27:793–799. De Mattei M, Fini M, Setti S, Ongaro A, Gemmati D, Stabellini G, Pellati A, Caruso A. 2007. Proteoglycan synthesis in bovine articular cartilage explants exposed to different low-frequency low-energy pulsed electromagnetic fields. Osteoarthritis Cartilage 15:163–168. Fini M, Giavaresi G, Carpi A, Nicolini A, Setti S, Giardino R. 2005. Effects of pulsed electromagnetic fields on articular hyaline cartilage: Review of experimental and clinical studies. Biomed Pharmacother 59:388–394. Fini M, Pagani S, Giavaresi G, De Mattei M, Ongaro A, Varani K, Vincenzi F, Massari L, Cadossi M. 2013. Functional tissue engineering in articular cartilage repair: Is there a role for electromagnetic biophysical stimulation? Tissue Eng Part B Rev 19:353–367. Gawlitta D, Farrell E, Malda J, Creemers LB, Alblas J, Dhert WJ. 2010. Modulating endochondral ossification of multipotent stromal cells for bone regeneration. Tissue Eng Part B Rev 16:385–395. Griffin XL, Costa ML, Parsons N, Smith N. 2011. Electromagnetic field stimulation for treating delayed union or non-union of long bone fractures in adults. Cochrane Database Syst Rev 4:CD008471. Hattori T, Müller C, Gebhard S, Bauer E, Pausch F, Schlund B, Bösl MR, Hess A, Surmann-Schmitt C, von der Mark H, de Crombrugghe B, von der Mark K. 2010. SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development 137:901–911. Komori T. 2010. Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell Tissue Res 339:189– 195. Mackie EJ, Ahmed YA, Tatarczuch L, Chen KS, Mirams M. 2008. Endochondral ossification: How cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol 40:46–62.
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