Bilayer osteochondral graft in rabbit xenogeneic transplantation model comprising sintered 3D-printed bioceramic and human adipose-derived stem cells laden biohydrogel.

Reconstruction of severe osteochondral defects in articular cartilage and subchondral trabecular bone remains a challenging problem. The well-integrated bilayer osteochondral graft design expects to be guided the chondrogenic and osteogenic differentiation for stem cells and provides a promising solution for osteochondral tissue repair in this study. The subchondral bone scaffold approach is based on the developed finer and denser 3D ß-tricalcium phosphate (ß-TCP) bioceramic scaffold process, which is made using a digital light processing (DLP) technology and the novel photocurable negative thermo-responsive (NTR) bioceramic slurry. Then, the concave-top disc sintered 3D-printed bioceramic incorporates the human adipose-derived stem cells (hADSCs) laden photo-cured hybrid biohydrogel (HG + 0.5AFnSi) comprised of hyaluronic acid methacryloyl (HAMA), gelatin methacryloyl (GelMA), and 0.5% (w/v) acrylate-functionalized nano-silica (AFnSi) crosslinker. The 3D ß-TCP bioceramic compartment is used to provide essential mechanical support for cartilage regeneration in the long term and slow biodegradation. However, the apparent density and compressive strength of the 3D ß-TCP bioceramics can be obtained for ~ 94.8% theoretical density and 11.38 ± 1.72 MPa, respectively. In addition, the in vivo results demonstrated that the hADSC + HG + 0.5AFnSi/3D ß-TCP of the bilayer osteochondral graft showed a much better osteochondral defect repair outcome in a rabbit model. The other word, the subchondral bone scaffold of 3D ß-TCP bioceramic could accelerate the bone formation and integration with the adjacent host cancellous tissue at 12 weeks after surgery. And then, a thicker cartilage layer with a smooth surface and uniformly aligned chondrocytes were observed by providing enough steady mechanical support of the 3D ß-TCP bioceramic scaffold.

Detection of micro-plasma-induced exosomes secretion in a fibroblast-melanoma co-culture model.

BACKGROUND: Melanoma is a highly aggressive tumor and a significant cause of skin cancer-related death. Timely diagnosis and treatment require identification of specific biomarkers in exosomes secreted by melanoma cells. In this study, label-free surface-enhanced Raman spectroscopy (SERS) method with size-matched selectivity was used to detect membrane proteins in exosomes released from a stimulated environment of fibroblasts (L929) co-cultured with melanoma cells (B16-F10). To promote normal secretion of exosomes, micro-plasma treatment was used to gently induce the co-cultured cells and slightly increase the stress level around the cells for subsequent detection using the SERS method. RESULTS AND DISCUSSION: Firstly, changes in reactive oxygen species/reactive nitrogen species (ROS/RNS) concentrations in the cellular microenvironment and the viability and proliferation of healthy cells are assessed. Results showed that micro-plasma treatment increased extracellular ROS/RNS levels while modestly reducing cell proliferation without significantly affecting cell survival. Secondly, the particle size of secreted exosomes isolated from the culture medium of L929, B16-F10, and co-cultured cells with different micro-plasma treatment time did not increase significantly under single-cell conditions at short treatment time but might be changed under co-culture condition or longer treatment time. Third, for SERS signals related to membrane protein biomarkers, exosome markers CD9, CD63, and CD81 can be assigned to significant Raman shifts in the range of 943-1030 and 1304-1561 cm-1, while the characteristics SERS peaks of L929 and B16-F10 cells are most likely located at 1394/1404, 1271 and 1592 cm-1 respectively. SIGNIFICANCE AND NOVELTY: Therefore, this micro-plasma-induced co-culture model provides a promising preclinical approach to understand the diagnostic potential of exosomes secreted by cutaneous melanoma/fibroblasts. Furthermore, the label-free SERS method with size-matched selectivity provides a novel approach to screen biomarkers in exosomes secreted by melanoma cells, aiming to reduce the use of labeling reagents and the processing time traditionally required.

Synergic Effect of Combined Therapy of Hyperbaric Oxygen and Adipose-Derived Mesenchymal Stem Cells on Improving Locomotor Recovery After Acute Traumatic Spinal Cord Injury in Rat Mainly Through Downregulating Inflammatory and Cell-Stress Signalings.

This study tested whether combined hyperbaric oxygen (HBO) and allogenic adipose-derived mesenchymal stem cells (ADMSCs) would be superior to either one for improving the locomotor recovery in rat after acute traumatic spinal cord injury (TSCI) in rat. Adult-male Sprague-Dawley rats were equally categorized into group 1 (sham-operated control), group 2 (TSCI), group 3 (TSCI + HBO for 1.5 h/day for 14 consecutive days after TSCI), group 4 (TSCI + ADMSCs/1.2 × 106 cells by intravenous injection at 3 h and days 1/2 after TSCI), and group 5 (TSCI + HBO + ADMSCs), euthanized, and spinal cord tissue was harvested by day 49 after TSCI. The protein expressions of oxidative-stress (NOX-1/NOX-2), inflammatory-signaling (TLR-4/MyD88/IL-1ß/TNF-α/substance-p), cell-stress signaling (PI3K/p-AKT/p-mTOR), and the voltage-gated sodium channel (Nav1.3/1.8/1.9) biomarkers were highest in group 2, lowest in group 1, and significantly lower in group 5 than in groups 3/4 (all P <0.0001), but they did not differ between groups 3 and 4. The spinal cord damaged area, the cellular levels of inflammatory/DNA-damaged biomarkers (CD68+/GFAP+/γ-H2AX+ cells), mitogen-activated protein kinase family biomarkers (p-P38/p-JNK/p-ERK1/2), and cellular expressions of voltage-gated sodium channel (Nav.1.3, Nav.1.8, and Nav.1.9 in NF200+ cells) as well as the pain-facilitated cellular expressions (p-P38+/peripherin+ cells, p-JNK+/peripherin+ cells, p-ERK/NF200+ cells) exhibited an identical pattern of inflammation, whereas the locomotor recovery displayed an opposite pattern of inflammation among the groups (all P < 0.0001). Combined HBO-ADMSCs therapy offered additional benefits for preserving the neurological architecture and facilitated the locomotor recovery against acute TSCI.

Additional benefit of induced pluripotent stem cell-derived mesenchymal stem cell therapy on sepsis syndrome-associated acute kidney injury in rat treated with antibiotic.

BACKGROUND: This study tested whether human induced-pluripotent stem-cell-derived mesenchymal-stem-cells (iPS-MSCs) would offer an additional benefit to the rodent with acute kidney injury (AKI) (ischemia for 1 h followed by reperfusion for 120 h) associated sepsis syndrome (SS) (by cecal-ligation-puncture immediately after AKI-induction) undergoing ciprofloxacin therapy. RESULTS: Male-adult SD rats (n = 80) were categorized into group 1 (sham-operated-control, n = 10), group 2 (AKI + SS, n = 24), group 3 (AKI + SS + ciprofloxacin/3 mg/kg, orally for 120 h, n = 12), group 4 (AKI + SS + iPS-MSCs/1.2 × 106/intravenously administered by 3 h after AKI, n = 12), group 5 (AKI + SS + iPS-MSCs/1.2 × 106/intravenously administered by 18 h after AKI, n = 12), group 6 (AKI + SS + iPS-MSCs/1.2 × 106/intravenously administered by 3 h after AKI induction + ciprofloxacin, n = 10] and euthanized by 120 h. The result showed that the mortality was significantly higher in group 2 than in other groups (all p < 0.01). The creatinine level was highest in group 2, lowest in group 1, significantly lower in group 6 than in groups 3, 4 and 5, (all p < 0.0001), but it showed no difference among the latter 3 groups. Flow cytometric analysis showed that the circulatory inflammatory cells (Ly6G/CD11b/c), early (AN-V+/PI-)/late (AN-V+/PI+) apoptosis, and circulatory/splenic immune cells (CD3+/CD4+, CD3+/CD8a+) were highest in group 2, lowest in group 1, significantly lower in group 6 than in groups 3/4/5 and significantly lower in group 4 than in groups 3/5 (all p < 0.0001), but they showed no difference between groups 3/5. Protein expressions of oxidative-stress (NOX-1/NOX2/oxidized protein), apoptotic (cleaved-caspase3/cleaved-PARP/mitochondrial-Bax), fibrotic (TGF-ß/Smad3), inflammatory (MMP-9/IL-6/TNF-α) and autophagic (Atg5/Beclin) biomarkers in kidney exhibited an identical pattern of circulatory inflammatory cells (all p < 0.0001). CONCLUSION: Combined iPS-MSCs-ciprofloxacin therapy was superior to either one alone for protecting AKI complicated by SS.

Double overexpression of miR-19a and miR-20a in induced pluripotent stem cell-derived mesenchymal stem cells effectively preserves the left ventricular function in dilated cardiomyopathic rat.

BACKGROUND: This study tested the hypothesis that double overexpression of miR-19a and miR-20a (dOex-mIRs) in human induced pluripotent stem cell (iPS)-derived mesenchymal stem cells (MSCs) effectively preserved left ventricular ejection fraction (LVEF) in dilated cardiomyopathy (DCM) (i.e., induced by doxorubicin) rat. METHODS AND RESULTS: In vitro study was categorized into groups G1 (iPS-MSC), G2 (iPS-MSCdOex-mIRs), G3 (iPS-MSC + H2O2/100uM), and G4 (iPS-MSCdOex-mIRs + H2O2/100uM). The in vitro results showed the cell viability was significantly lower in G3 than in G1 and G2, and that was reversed in G4 but it showed no difference between G1/G2 at time points of 6 h/24 h/48 h, whereas the flow cytometry of intra-cellular/mitochondrial oxidative stress (DCFA/mitoSOX) and protein expressions of mitochondrial-damaged (cytosolic-cytochrome-C/DRP1/Cyclophilin-D), oxidative-stress (NOX-1/NOX2), apoptotic (cleaved-caspase-3/PARP), fibrotic (p-Smad3/TGF-ß), and autophagic (ratio of LC3B-II/LC3BI) biomarkers exhibited an opposite pattern of cell-proliferation rate (all p< 0.001). Adult-male SD rats (n=32) were equally divided into groups 1 (sham-operated control), 2 (DCM), 3 (DCM + iPS-MSCs/1.2 × 106 cells/administered by post-28 day's DCM induction), and 4 (DCM + iPS-MSCdOex-mIRs/1.2 × 106 cells/administered by post-28 day's DCM induction) and euthanized by day 60 after DCM induction. LV myocardium protein expressions of oxidative-stress signaling (p22-phox/NOX-1/NOX-2/ASK1/p-MMK4,7/p-JNK1,2/p-cJUN), upstream (TLR-4/MAL/MyD88/TRIF/TRAM/ TFRA6/IKKα/ß/NF-κB) and downstream (TNF-α/IL-1ß/MMP-9) inflammatory signalings, apoptotic (cleaved-PARP/mitochondrial-Bax), fibrotic (Smad3/TGF-ß), mitochondrial-damaged (cytosolic-cytochrome-C/DRP1/cyclophilin-D), and autophagic (beclin1/Atg5) biomarkers were highest in group 2, lowest in group 1 and significantly lower in group 4 than in group 3, whereas the LVEF exhibited an opposite pattern of oxidative stress (all p< 0.0001). CONCLUSION: iPS-MSCdOex-mIRs therapy was superior to iPS-MSC therapy for preserving LV function in DCM rat.

Circulatory Rejuvenated EPCs Derived from PAOD Patients Treated by CD34+ Cells and Hyperbaric Oxygen Therapy Salvaged the Nude Mouse Limb against Critical Ischemia.

This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice (n = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood prior to CD34+ cell and HBO treatment (EPCPr-T) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 105) derived from PAOD patient's circulatory blood after CD34+ cell and HBO treatment (EPCAf-T) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 (p < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all p < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all p < 0.0010). The number of small vessels and endothelial cell surface markers (CD31+/vWF+) in the CLI area displayed an identical pattern of INBF (all p < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all p < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.

Alpha-5 Integrin Mediates Simvastatin-Induced Osteogenesis of Bone Marrow Mesenchymal Stem Cells.

Simvastatin (SVS) promotes the osteogenic differentiation of mesenchymal stem cells (MSCs) and has been studied for MSC-based bone regeneration. However, the mechanism underlying SVS-induced osteogenesis is not well understood. We hypothesize that α5 integrin mediates SVS-induced osteogenic differentiation. Bone marrow MSCs (BMSCs) derived from BALB/C mice, referred to as D1 cells, were used. Alizarin red S (calcium deposition) and alkaline phosphatase (ALP) staining were used to evaluate SVS-induced osteogenesis of D1 cells. The mRNA expression levels of α5 integrin and osteogenic marker genes (bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), collagen type I, ALP and osteocalcin (OC)) were detected using quantitative real-time PCR. Surface-expressed α5 integrin was detected using flow cytometry analysis. Protein expression levels of α5 integrin and phosphorylated focal adhesion kinase (p-FAK), which is downstream of α5 integrin, were detected using Western blotting. siRNA was used to deplete the expression of α5 integrin in D1 cells. The results showed that SVS dose-dependently enhanced the gene expression levels of osteogenic marker genes as well as subsequent ALP activity and calcium deposition in D1 cells. Upregulated p-FAK was accompanied by an increased protein expression level of α5 integrin after SVS treatment. Surface-expressed α5 integrin was also upregulated after SVS treatment. Depletion of α5 integrin expression significantly suppressed SVS-induced osteogenic gene expression levels, ALP activity, and calcium deposition in D1 cells. These results identify a critical role of α5 integrin in SVS-induced osteogenic differentiation of BMSCs, which may suggest a therapeutic strategy to modulate α5 integrin/FAK signaling to promote MSC-based bone regeneration.

Parathyroid hormone-(1-34) ameliorated knee osteoarthritis in rats via autophagy.

Anterior cruciate ligament (ACL) tear can lead to osteoarthritis (OA). However, parathyroid hormone (PTH)-(1-34) was found to alleviate OA progression in a papain-induced OA model. Autophagy is a protective mechanism in normal cartilage, and its aging-related loss is linked with chondrocyte death and OA. Thus we examined the roles of autophagy in PTH treatment in OA after ACL transection (ACLT). Thirty-six rats were randomized into three groups: control group, ACLT-induced OA (OA) group, and OA with intra-articular PTH-(1-34) treatment (OA+PTH) group. Weight-bearing and treadmill tests were evaluated. Cartilage matrix was determined by a histological evaluation of glycosaminoglycan (GAG), Osteoarthritis Research Society International (OARSI) score, chondrocyte apoptosis, and immunohistochemistry. Rats in the OA group had significantly decreased weight bearing and running endurance. The histological results indicated that GAG, collagen type II, and chondrocyte autophagy had decreased but that the OARSI score, terminal differentiation markers (collagen type X and Indian hedgehog), and chondrocyte apoptosis had increased in the OA group. Additionally, PTH-(1-34) treatment significantly improved weight bearing and treadmill endurance, preserved GAG and collagen type II, and reduced the OARSI score and terminal differentiation markers. Finally, PTH-(1-34) ameliorated chondrocyte apoptosis by regulating the expression of autophagy-related proteins, through reducing mechanistic target of rapamycin (mTOR) and p62 and enhancing microtubule-associated protein-1 light chain 3 (LC3) and beclin-1. Reconstructive surgery after ACL rupture cannot prevent OA occurrence. Intra-articular PTH-(1-34) treatment can alleviate OA progression after ACLT and histological molecular changes. Possible mechanisms are reducing chondrocyte terminal differentiation and apoptosis, with increasing autophagy. NEW & NOTEWORTHY Anterior cruciate ligament (ACL) tear can lead to osteoarthritis (OA). Intra-articular parathyroid hormone (PTH)-(1-34) significantly improved weight bearing and treadmill endurance, preserved glycosaminoglycan and collagen type II, and reduced Osteoarthritis Research Society International (OARSI) score and terminal differentiation. Finally, PTH-(1-34) ameliorated chondrocyte apoptosis by regulating the expression of autophagy-related proteins, through reducing mechanistic target of rapamycin (mTOR) and p62 and enhancing microtubule-associated protein-1 light chain 3 (LC3) and beclin-1. PTH-(1-34) can alleviate OA progression after ACL transection. Possible mechanisms are reducing chondrocyte terminal differentiation and apoptosis, with increasing autophagy.

Parathyroid hormone 1-34 reduces dexamethasone-induced terminal differentiation in human articular chondrocytes.

Intra-articular injection of dexamethasone (Dex) is occasionally used to relieve pain and inflammation in osteoarthritis (OA) patients. Dex induces terminal differentiation of chondrogenic mesenchymal stem cells in vitro and causes impaired longitudinal skeletal growth in vivo. Parathyroid hormone 1-34 (PTH 1-34) has been shown to reverse terminal differentiation of osteoarthritic articular chondrocytes. We hypothesized that Dex induces terminal differentiation of articular chondrocytes and that this effect can be mitigated by PTH 1-34 treatment. We tested the effect of Dex on terminal differentiation in human articular chondrocytes and further tested if PTH 1-34 reverses the effects. We found that Dex treatment downregulated chondrogenic-induced expressions of SOX-9, collagen type IIa1 (Col2a1), and aggrecan and reduced synthesis of cartilaginous matrix (Col2a1 and sulfated glycosaminoglycan) synthesis. Dex treatment upregulated chondrocyte hypertrophic markers of collagen type X and alkaline phosphatase at mRNA and protein levels, and it increased the cell size of articular chondrocytes and induced cell death. These results indicated that Dex induces terminal differentiation of articular chondrocytes. To test whether PTH 1-34 treatment reverses Dex-induced terminal differentiation of articular chondrocytes, PTH 1-34 was co-administered with Dex. Results showed that PTH 1-34 treatment reversed both changes of chondrogenic and hypertrophic markers in chondrocytes induced by Dex. PTH 1-34 also decreased Dex-induced cell death. PTH 1-34 treatment reduces Dex-induced terminal differentiation and apoptosis of articular chondrocytes, and PTH 1-34 treatment may protect articular cartilage from further damage when received Dex administration.

Combination of adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes for protecting kidney from acute ischemia-reperfusion injury.

BACKGROUND: In this study, we tested the hypothesis that a combined adipose-derived mesenchymal stem cell (ADMSC) and ADMSC-derived exosome therapy protected rat kidney from acute ischemia-reperfusion (IR) injury (i.e., ligation of both renal arteries for 1h and reperfusion for 72h prior to euthanization). METHODS AND RESULTS: Adult-male SD rats (n=40) were equally categorized into group 1 (sham control), group 2 (IR), group 3 [IR+exosome (100µg)], group 4 [IR+ADMSC (1.2×10(6) cells)], and group 5 (IR-exosome-ADMSC). All therapies were performed at 3h after IR procedure from venous administration. By 72h, the creatinine level and kidney injury score were the lowest in group 1 and the highest in group 2, significantly higher in group 3 than in groups 4 and 5, and significantly higher in group 4 than in group 5 (all P<0.0001). The protein expression of inflammatory (TNF-α/NF-κB/IL-1ß/MIF/PAI-1/Cox-2), oxidative-stress (NOX-1/NOX-2/oxidized protein), apoptotic (Bax/caspase-3/PARP), and fibrotic (Smad3/TGF-ß) biomarkers showed an identical pattern, whereas the anti-apoptotic (Smad1/5, BMP-2) and angiogenesis (CD31/vWF/angiopoietin) biomarkers and mitochondrial cytochrome-C showed an opposite pattern of creatinine level among the five groups (all P<0.001). The microscopic findings of glomerular-damage (WT-1), renal tubular-damage (KIM-1), DNA-damage (γ-H2AX), inflammation (MPO/MIF/CD68) exhibited an identical pattern, whereas the podocyte components (podocin/p-cadherin/synaptopodin) displayed a reversed pattern of creatinine level (all P<0.0001). CONCLUSION: Combined exosome-ADMSC therapy was superior to either one for protecting kidney from acute IR injury.

Suppression of discoidin domain receptor 1 expression enhances the chondrogenesis of adipose-derived stem cells.

Effectively directing the chondrogenesis of adipose-derived stem cells (ADSCs) to engineer articular cartilage represents an important challenge in ADSC-based articular cartilage tissue engineering. The discoidin domain receptor 1 (DDR1) has been shown to affect cartilage homeostasis; however, little is known about the roles of DDR1 in ADSC chondrogenesis. In this study, we used the three-dimensional culture pellet culture model system with chondrogenic induction to investigate the roles of DDR1 in the chondrogenic differentiation of human ADSCs (hADSCs). Real-time polymerase chain reaction and Western blot were used to detect the expression of DDRs and chondrogenic genes. Sulfated glycosaminoglycan (sGAG) was detected by Alcian blue and dimethylmethylene blue (DMMB) assays. Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was used to assess cell death. During the chondrogenesis of hADSCs, the expression of DDR1 but not DDR2 was significantly elevated. The depletion of DDR1 expression in hADSCs using short hairpin RNA increased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and cartilaginous matrix deposition (collagen type II and sGAG) and only slightly increased cell death (2-8%). DDR1 overexpression in hADSCs decreased the expression of chondrogenic genes (SOX-9, collagen type II, and aggrecan) and sGAG and enhanced hADSC survival. Moreover, DDR1-depleted hADSCs showed decreased expression of the terminal differentiation genes runt-related transcription factor 2 (Runx2) and matrix metalloproteinase 13 (MMP-13). These results suggest that DDR1 suppression may enhance ADSC chondrogenesis by enhancing the expression of chondrogenic genes and cartilaginous matrix deposition. We proposed that the suppression of DDR1 in ADSCs may be a candidate strategy of genetic modification to optimize ADSC-based articular cartilage tissue engineering.

Electromagnetic fields enhance chondrogenesis of human adipose-derived stem cells in a chondrogenic microenvironment in vitro.

We tested the hypothesis that electromagnetic field (EMF) stimulation enhances chondrogenesis in human adipose-derived stem cells (ADSCs) in a chondrogenic microenvironment. A two-dimensional hyaluronan (HA)-coated well (2D-HA) and a three-dimensional pellet culture system (3D-pellet) were used as chondrogenic microenvironments. The ADSCs were cultured in 2D-HA or 3D-pellet, and then treated with clinical-use pulse electromagnetic field (PEMF) or the innovative single-pulse electromagnetic field (SPEMF) stimulation. The cytotoxicity, cell viability, and chondrogenic and osteogenic differentiations were analyzed after PEMF or SPEMF treatment. The modules of PEMF and SPEMF stimulations used in this study did not cause cytotoxicity or alter cell viability in ADSCs. Both PEMF and SPEMF enhanced the chondrogenic gene expression (SOX-9, collagen type II, and aggrecan) of ADSCs cultured in 2D-HA and 3D-pellet. The expressions of bone matrix genes (osteocalcin and collagen type I) of ADSCs were not changed after SPEMF treatment in 2D-HA and 3D-pellet; however, they were enhanced by PEMF treatment. Both PEMF and SPEMF increased the cartilaginous matrix (sulfated glycosaminoglycan) deposition of ADSCs. However, PEMF treatment also increased mineralization of ADSCs, but SPEMF treatment did not. Both PEMF and SPEMF enhanced chondrogenic differentiation of ADSCs cultured in a chondrogenic microenvironment. SPEMF treatment enhanced ADSC chondrogenesis, but not osteogenesis, when the cells were cultured in a chondrogenic microenvironment. However, PEMF enhanced both osteogenesis and chondrogenesis under the same conditions. Thus the combination of a chondrogenic microenvironment with SPEMF stimulation can promote chondrogenic differentiation of ADSCs and may be applicable to articular cartilage tissue engineering.

Effects of anti-inflammatory drugs on proliferation, cytotoxicity and osteogenesis in bone marrow mesenchymal stem cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) were found to suppress proliferation and induce cell death in cultured osteoblasts, and steroids were found to decrease the osteogenesis potential of mesenchymal stem cells. In this study, we further tested the effects of anti-inflammatory drugs (AIDs) on the functions of bone marrow mesenchymal stem cells (BMSCs). The BMSCs from mice (D1-cells) and humans (hBMSCs) were treated with dexamethasone (10(-7) to 10(-6) M), cyclooxygenase-2 (COX-2) selective NSAIDs (10(-6) to 10(-5) M) and non-selective NSAIDs (10(-5) to 10(-4) M). Drug effects on proliferation, cell cycle kinetics, cytotoxicity and mRNA and protein expressions of cell cycle regulators were tested. The osteogenesis potential of D1-cells were evaluated by testing mRNA expressions of type Ialpha collagen and osteocalcin 2-8 days after treatments, and testing mineralization 1-3 weeks after treatments. The results showed that all the tested drugs suppressed proliferation and arrested cell cycle of D1-cells, but no significant cytotoxic effects was found. Prostaglandin E1, E2 and F2alpha couldn't rescue the effects of AIDs on proliferation. The p27kip1 expression was up-regulated by indomethacin, celecoxib and dexamethasone in both D1-cells and hBMSCs. Higher concentrations of indomethacin and dexamethasone also up-regulated p21Cip1/Waf1 expression in hBMSCs, and so did celecoxib on D1-cells. Expressions of cyclin E1 and E2 were down-regulated by these AIDs in D-cells, while only cyclin E2 was down-regulated by dexamethasone in hBMSCs. All the tested NSAIDs revealed no obvious detrimental effects on osteogenic differentiation of D1-cells. These results suggest that the proliferation suppression of AIDs on BMSCs may act via affecting expressions of cell cycle regulators, but not prostaglandin-related mechanisms.