Search

Meniscus-derived stem cells (MeSCs), a unique type of MSC, have outstanding advantages in meniscal cytotherapy and tissue engineering, but the effects and molecular mechanisms of PBM on MeSCs are still unclear. We used 660-nm LED light with different energy densities to irradiate six human MeSC samples and tested their proliferation rate via cell counting, chondrogenic differentiation capacity via the DMMB assay, mitochondrial activity via the MTT assay, and gene expression via qPCR. The proliferation ability, chondrogenic capacity and mitochondrial activity of the 18 J/cm 2 group were greater than those of the 4 J/cm 2 and control groups. The mRNA expression levels of Akt, PI3K, TGF-β3, Ki67 and Notch-1 in the 18 J/cm 2 group were greater than those in the other groups in most samples. After chondrogenic induction, the expression of Col2A1, Sox9 and Aggrecan in the 18 J/cm 2 group was significantly greater than that in the 4 J/cm 2 and control groups in most of the samples. The variation in the MTT values and Src, PI3K, Akt, mTOR and GSK3β levels decreased with time. The results showed that 660-nm LED red light promoted proliferation and chondrogenic differentiation and affected the gene expression of MeSCs, and the effects on gene expression and mitochondrial activity decreased with time., (© 2024. The Author(s).)

Due to their capacity to differentiate into the chondrogenic lineage, adipose-derived stromal/stem cells (ASC) are a promising source of therapeutically relevant cells for cartilage tissue regeneration. Their differentiation potential, however, varies between patients. In our study, we aim to stimulate ASC towards a more reliable chondrogenic phenotype using photobiomodulation (PBM). LED devices of either blue (475 nm), green (516 nm) or red (635 nm) light were used to treat human ASC from donors of varying chondrogenic potential. The treatment was applied either once during the 2D expansion phase or repeatedly during the 3D differentiation phase. Chondrogenic differentiation was assessed via pellet size, GAG/DNA content, histology and gene expression analysis. Reactions to PBM were found to be wavelength-dependent and more pronounced when the treatment was applied during expansion. Donors were assigned to responder categories according to their response to the treatment during expansion, whereby good responders were mainly donors with low intrinsic chondrogenic potential. Exposed to light, they revealed a particularly high relative increase in pellet size (more than twice the size of untreated controls after red light PBM), intense collagen type II immunostaining (low/absent in untreated controls) and activation of otherwise absent COL2A1 expression. Conversely, on a donor with high intrinsic chondrogenic potential, light had adverse effects. When applied with shorter wavelengths (blue, green), it led to reduced pellet size, GAG/DNA content and collagen type II immunostaining. However, when PBM was applied in 3D, the same donor was the only one to react with increased differentiation to all three wavelengths. We were able to demonstrate that PBM can be used to enhance or hamper chondrogenesis of ASC, and that success depends on treatment parameters and intrinsic cellular potential. The improvement of chondrogenesis in donors with low intrinsic potential highlights PBM as potent tool for cell-based cartilage regeneration. Its cost-effectiveness and ease of use make for an attractive treatment option to enhance the performance of ASC in cartilage tissue engineering., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background : There are different methods to develop in vitro neo-chondral tissues from adipose-derived stem cells (ADSCs). Application of electromagnetic field (EMF) on ADSCs is one of popular approaches, which results in chondrogenesis. If chondrogenic impact of EMF on ADSCs is supposed to be generalized as a protocol in translational medicine field, possible emergence of early or late hypertrophic maturation, mineralization and inflammatory side effects in chondrogenically differentiating ADSCs should be considered. Methods : The advent of chondrogenic and hypertrophic markers by differentiated cells under standard, platelet-rich plasma (PRP)-based or EMF treatments were monitored. Along with monitoring the expressions of chondrogenic markers, inflammatory and hypertrophic markers, VEGF/TNFα secretion, calcium deposition and ALP activity were evaluated. Results : Accordingly, treatment with %5 PRP results in higher GAG production, enhanced SOX9 transcription, lowered TNFα and VEGF secretions compared to other treatments. Although PRP up-regulates miR-146a and miR-199a in early and late stages of chondrogenesis, respectively, application of EMF + PRP down regulates miR-101 and -145 while up-regulates miR-140 and SOX9 expression. Conclusion : Comparing our results with previous reports suggests that presented EMF-ELF in this study with f = 50 Hz, EMF intensity of less than 30 mT, and 5% PRP (v/v), would facilitate chondrogenesis via mesenchymal stem cells with minor inflammation and hypertrophic maturation. Abbreviations: MSCs: mesenchymal stem cells; TGFβ: transforming growth factor-beta; PRP: platelet-rich plasma; ELF-EMF: extremely low-frequency electromagnetic fields; GAGs: glycosaminoglycans; ADSCs: adipose-derived stem cells; VEGF: vascular endothelial growth factor; TNFα: tumor necrosis factor alpha; ALP: alkaline phosphatase.

Background: Substantial evidence exists to show the positive effects of radialextracorporeal shock wave therapy (ESWT) on bone formation. However, it is unknown whether rESWT can act locally at the growth plate level to stimulate linear bone growth. One way to achieve this is to stimulate chondrogenesis in the growth plate without depending on circulating systemic growth factors. We wished to see whether rESWT would stimulate metatarsal rat growth plates in the absence of vascularity and associated systemic growth factors., Questions/purposes: To study the direct effects of rESWT on growth plate chondrogenesis, we asked: (1) Does rESWT stimulate longitudinal bone growth of ex vivo cultured bones? (2) Does rESWT cause any morphological changes in the growth plate? (3) Does rESWT locally activate proteins specific to growth plate chondrogenesis?, Methods: Metatarsal bones from rat fetuses were untreated (controls: n = 15) or exposed to a single application of rESWT at a low dose (500 impulses, 5 Hz, 90 mJ; n = 15), mid-dose (500 impulses, 5 Hz, 120 mJ; n = 14) or high dose (500 impulses, 10 Hz, 180 mJ; n = 34) and cultured for 14 days. Bone lengths were measured on Days 0, 4, 7, and 14. After 14 days of culturing, growth plate morphology was assessed with a histomorphometric analysis in which hypertrophic cell size (> 7 µm) and hypertrophic zone height were measured (n = 6 bones each). Immunostaining for specific regulatory proteins involved in chondrogenesis and corresponding staining were quantitated digitally by a single observer using the automated threshold method in ImageJ software (n = 6 bones per group). A p value < 0.05 indicated a significant difference., Results: The bone length in the high-dose rESWT group was increased compared with that in untreated controls (4.46 mm ± 0.75 mm; 95% confidence interval, 3.28-3.71 and control: 3.50 mm ± 0.38 mm; 95% CI, 4.19-4.72; p = 0.01). Mechanistic studies of the growth plate's cartilage revealed that high-dose rESWT increased the number of proliferative chondrocytes compared with untreated control bones (1363 ± 393 immunopositive cells per bone and 500 ± 413 immunopositive cells per bone, respectively; p = 0.04) and increased the diameter of hypertrophic chondrocytes (18 ± 3 µm and 13 ± 3 µm, respectively; p < 0.001). This was accompanied by activation of insulin-like growth factor-1 (1015 ± 322 immunopositive cells per bone and 270 ± 121 immunopositive cells per bone, respectively; p = 0.043) and nuclear factor-kappa beta signaling (1029 ± 262 immunopositive cells per bone and 350 ± 60 immunopositive cells per bone, respectively; p = 0.01) and increased levels of the anti-apoptotic proteins B-cell lymphoma 2 (718 ± 86 immunopositive cells per bone and 35 ± 11 immunopositive cells per bone, respectively; p < 0.001) and B-cell lymphoma-extra-large (107 ± 7 immunopositive cells per bone and 34 ± 6 immunopositive cells per bone, respectively; p < 0.001)., Conclusion: In a model of cultured fetal rat metatarsals, rESWT increased longitudinal bone growth by locally inducing chondrogenesis. To verify whether rESWT can also stimulate bone growth in the presence of systemic circulatory factors, further studies are needed., Clinical Relevance: This preclinical proof-of-concept study shows that high-dose rESWT can stimulate longitudinal bone growth and growth plate chondrogenesis in cultured fetal rat metatarsal bones. A confirmatory in vivo study in skeletally immature animals must be performed before any clinical studies.

Tissue engineering aims to take advantage of the ability of undifferentiated stem cells to differentiate into multiple cell types to repair damaged tissue. Photobiomodulation uses either lasers or light-emitting diodes to promote stem cell proliferation and differentiation. The present study aimed to investigate single and dual combinations of laser wavelengths on mesenchymal stem cells (MSCs). MSCs were derived from rabbit iliac bone marrow. One control and eight laser irradiated groups were designated as Infrared (IR, 810 nm), Red (R, 660 nm), Green (G, 532 nm), Blue (B, 485 nm), IR-R, IR-B, R-G, and B-G. Irradiation was repeated daily for 21 days and cell proliferation, osseous, or cartilaginous differentiation was then measured. RT-PCR biomarkers were SOX9, aggrecan, COL 2, and COL 10 expression for cartilage and ALP, COL 1, and osteocalcin expression for bone. Cellular proliferation was increased in all irradiated groups except G. All cartilage markers were significantly increased by IR and IR-B except COL 10 which was suppressed by IR-B combination. ALP expression was highest in R and IR groups during osseous differentiation. ALP was decreased by combinations of IR with B and with R, and also by G alone. R and B-G groups showed stimulated COL 1 expression; however, COL 1 was suppressed in IR-B, IR-R, and G groups. IR significantly increased osteocalcin expression, but in B, B-G, and G groups it was reduced. Cartilage differentiation was stimulated by IR and IR-B laser irradiation. The effects of single or combined laser irradiation were not clear-cut on osseous differentiation. Stimulatory effects on osteogenesis were seen for R and IR lasers, while G laser had inhibitory effects.

Background: Low-intensity pulsed ultrasound (LIPUS) can induce mesenchymal stem cell (MSC) differentiation, although the mechanism of its potential effects on chondrogenic differentiation is unknown. Since autophagy is known to regulate the differentiation of MSCs, the aim of our study was to determine whether LIPUS induced chondrogenesis via autophagy regulation., Methods: MSCs were isolated from the rat bone marrow, cultured in either standard or chondrogenic medium, and stimulated with 3 MHz of LIPUS given in 20% on-off cycles, with or without prior addition of an autophagy inhibitor or agonist. Chondrogenesis was evaluated on the basis of aggrecan (AGG) organization and the amount of type II collagen (COL2) and the mRNA expression of AGG, COL2, and SRY-related high mobility group-box gene 9 (SOX9) genes., Results: LIPUS promoted the chondrogenic differentiation of MSCs, as shown by the changes in the extracellular matrix (ECM) proteins and upregulation of chondrogenic genes, and these effects were respectively augmented and inhibited by the autophagy inhibitor and agonist., Conclusions: Taken together, these results indicate that LIPUS promotes MSC chondrogenesis by inhibiting autophagy.

Purpose: Data on the response of chondrocytes differentiated from hiPSCs (hiPSC-DCHs) to ionizing radiation (IR) are lacking. The aim of present study was to assess DNA damage response (DDR) mechanisms of IR-treated hiPSC-DCHs., Methods and Materials: The following IR-response characteristics in irradiated hiPSC-DCHs were assessed: 1) the kinetics of DNA DSB formation; 2) activation of major DNA repair mechanisms; 3) cell cycle changes and 4) reactive oxygen species (ROS), level of key markers of apoptosis and senescence., Results: DNA DSBs were observed in 30% of the hiPSC-DCHs overall, and in 60% after high-dose (> 2 Gy) IR. Nevertheless, these cells displayed efficient DNA repair mechanisms, which reduced the DSBs over time until it reached 30% by activating key genes involved in homologous recombination and non-homologous end joining mechanisms. As similar to mature chondrocytes, irradiated hiPSC-DCH cells revealed accumulation of cells in G2 phase. Overall, the hiPSC-DCH cells were characterized by low levels of ROS, cPARP and high levels of senescence., Conclusions: The chondrocyte-like cells derived from hiPSC demonstrated features characteristic of both mature chondrocytes and "parental" hiPSCs. The main difference between hiPSC-derived chondrocytes and hiPSCs and mature chondrocytes appears to be the more efficient DDR mechanism of hiPSC-DCHs. The unique properties of these cells suggest that they could potentially be used safely in regenerative medicine if these preliminary findings are confirmed in future studies., Competing Interests: The authors have declared that no competing interests exist.

The effects of low intensity pulsed ultrasound (LIPUS) on proliferation and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) seeded on 3D printed poly-(ethylene glycol)-diacrylate (PEG-DA) scaffolds with varying pore geometries (square and hexagonal channels) were investigated. The scaffold with square pores resulted in higher hMSC growth and chondrogenic differentiation than a solid or a hexagonally porous scaffold. The optimal LIPUS parameters at 1.5 MHz were found to be 100 mW/cm 2 and 20% duty cycle. LIPUS stimulation increased proliferation by up to 60% after 24 hr. For chondrogenesis, we evaluated key cartilage biomarkers abundant in cartilage tissue; glycosaminoglycan (GAG), type II collagen and total collagen. LIPUS stimulation enhanced GAG synthesis up to 16% and 11% for scaffolds with square and hexagonal patterns, respectively, after 2 weeks. Additionally, type II collagen production increased by 60% and 40% for the same patterns, respectively under LIPUS stimulation after 3 weeks. These results suggest that LIPUS stimulation, which has already been approved by FDA for treatment of bone fracture, could be a highly efficient tool for tissue engineering in combination with 3D printing and hMSCs to regenerate damaged cartilage tissues., (© 2017 Wiley Periodicals, Inc.)

Background: Low-intensity pulsed ultrasound (LIPUS) is a mechanical stimulus that plays a key role in regulating the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the way in which it affects the chondrogenic differentiation of BMSCs remains unknown. In this study, we aimed to investigate whether LIPUS is able to influence TGF-β1-induced chondrogenesis of BMSCs through the integrin-mechanistic target of the Rapamycin (mTOR) signaling pathway., Methods: BMSCs were isolated from rat bone marrow and cultured in either standard or TGF-β1-treated culture medium. BMSCs were then subjected to LIPUS at a frequency of 3 MHz and a duty cycle of 20%, and integrin and mTOR inhibitors added in order to analyze their influence on cell differentiation. BMSCs were phenotypically analyzed by flow cytometry and the degree of chondrogenesis evaluated through toluidine blue staining, immunofluorescence, and immunocytochemistry. Furthermore, expression of COL2, aggrecan, SOX9, and COL1 was assessed by qRT-PCR and western blot analysis., Results: We found that LIPUS promoted TGF-β1-induced chondrogenesis of BMSCs, represented by increased expression of COL2, aggrecan and SOX9 genes, and decreased expression of COL1. Notably, these effects were prevented following addition of integrin and mTOR inhibitors., Conclusions: Taken together, these results indicate that mechanical stimulation combined with LIPUS promotes TGF-β1-induced chondrogenesis of BMSCs through the integrin-mTOR signaling pathway.

Pulse electromagnetic fields (PEMFs) have been shown to recruit calcium-signaling cascades common to chondrogenesis. Here we document the effects of specified PEMF parameters over mesenchymal stem cells (MSC) chondrogenic differentiation. MSCs undergoing chondrogenesis are preferentially responsive to an electromagnetic efficacy window defined by field amplitude, duration and frequency of exposure. Contrary to conventional practice of administering prolonged and repetitive exposures to PEMFs, optimal chondrogenic outcome is achieved in response to brief (10 minutes), low intensity (2 mT) exposure to 6 ms bursts of magnetic pulses, at 15 Hz, administered only once at the onset of chondrogenic induction. By contrast, repeated exposures diminished chondrogenic outcome and could be attributed to calcium entry after the initial induction. Transient receptor potential (TRP) channels appear to mediate these aspects of PEMF stimulation, serving as a conduit for extracellular calcium. Preventing calcium entry during the repeated PEMF exposure with the co-administration of EGTA or TRP channel antagonists precluded the inhibition of differentiation. This study highlights the intricacies of calcium homeostasis during early chondrogenesis and the constraints that are placed on PEMF-based therapeutic strategies aimed at promoting MSC chondrogenesis. The demonstrated efficacy of our optimized PEMF regimens has clear clinical implications for future regenerative strategies for cartilage.

Background: Adipose-derived stem cells (ASCs) are important to homeostasis and the regeneration of subcutaneous fat. Hence, we examined the proliferation and differentiation capacity of irradiated ASCs over time., Methods: Two female pigs received a single 18 Gy dose of ionizing radiation to an 18 × 8 cm area on the dorsal body skin via a 6 MeV electron beam. After irradiation, the ASCs were cultured from adipose tissue harvested from a non-irradiated area and an irradiated area at 2, 4, and 6 weeks. The proliferation capacity of ASCs was evaluated by a colony-forming units-fibroblasts (CFUs-Fs) assay, a cholecystokinin (CCK) test with 10 % fetal bovine serum (FBS), and a 1 % FBS culture test. The senescence of ASCs was evaluated through morphological examination, immunophenotyping, and β-galactosidase activity, and the multipotent differentiation potential of ASCs was evaluated in adipogenic, osteogenic, and chondrogenic differentiation media., Results: Irradiated ASCs demonstrated significantly decreased proliferative capacity 6 weeks after irradiation. As well, the cells underwent senescence, which was confirmed by blunted morphology, weak mesenchymal cell surface marker expression, and elevated β-galactosidase activity. Irradiated ASCs also exhibited significant losses in the capacity for adipocyte and chondrocyte differentiation. In contrast, osteogenic differentiation was preserved in irradiated ASCs., Conclusions: We observed decreased proliferation and senescence of irradiated ASCs compared to non-irradiated ASCs 6 weeks after irradiation. Furthermore, irradiated ASCs demonstrated impaired adipocyte and chondrocyte differentiation but retained their osteogenic differentiation capacity. Our results could shed light on additional pathogenic effects of late irradiation, including subcutaneous fibrosis and calcinosis.

Objective: To study the effects of pulsed ultrasound (PUS) and pulsed electromagnetic fields (PEMF) on the secretion of extracellular matrix from a culture complex during in vitro chondrogenesis., Methods: All the rat bone marrow mesen- chymal stem cell pellets were cultured in achondrogenic medium. Different intensities of PUS (100, 150, and 200 mW · cm⁻²) and PEMF (1, 2, and 5 mT) were applied to the cell pellets for 2 weeks. Group N was cultured without PUS and PEMF stimu- lation as control. The culture medium was collected after 2 weeks of culture. Enzyme-linked immunosorbent assay (ELISA) was used to detect the type of collagen and glycosaminoglycan (GAG) in the culture medium., Results: PUS increased the secreting-type collagen and GAG from cell pellets compared with group N (P < 0.05), whereas there was no difference in different intensities (P > 0.05). PEMF had no significant effect on the secretion of the type of collagen (P > 0.05). A PEMF of 1 mT had no significant effect on the secretion of GAG (P > 0.05). A PEMF 2 and 5 mT decreased the secretion of GAG (P < 0.05)., Conclusion: To prevent the secretary of extracellular matrix may play a role in chondrogenic effect of PEMF.

Repairing bone defect by recapitulation of endochondral bone formation, known as developmental engineering, has been a promising approach in bone tissue engineering. The critical issue in this area is how to effectively construct the hypertrophic cartilaginous template in vitro and enhance in vivo endochondral ossification process after implantation. Pulsed ultrasound stimulation has been widely used in the clinic for accelerating bone healing in fractures and nonunions. The aim of this study was to investigate whether ultrasound (US) could accelerate in vitro chondrogenesis and the hypertrophic process in certain microenvironments. Rat bone marrow mesenchymal stem cells were chondrogenic or hypertrophic differentiated in a three-dimensional pellet culture system with different media, and treated with different intensities of US. US exposure promoted chondrogenic differentiation of stem cells and inhibited their transition into the hypertrophic stage in a chondrogenic-friendly microenvironment. US significantly advanced hypertrophic differentiation of bone marrow stem cell pellets in hypertrophic medium after chondrogenesis. Our data indicated that pulsed US promoted in vitro chondrogenic and hypertrophic differentiation of stem cell pellets in specific culture conditions. The present study proves the potential application of US in the in vitro stage of "developmental engineering" for bone development and repair., (Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts., (© 2015 Wiley Periodicals, Inc.)

Electromagnetic fields (EMF) have been shown to exert beneficial effects on cartilage tissue. Nowadays, differentiated human mesenchymal stem cells (hMSCs) are discussed as an alternative approach for cartilage repair. Therefore, the aim of this study was to examine the impact of EMF on hMSCs during chondrogenic differentiation. HMSCs at cell passages five and six were differentiated in pellet cultures in vitro under the addition of human fibroblast growth factor 2 (FGF-2) and human transforming growth factor-β(3) (TGF-β(3) ). Cultures were exposed to homogeneous sinusoidal extremely low-frequency magnetic fields (5 mT) produced by a solenoid or were kept in a control system. After 3 weeks of culture, chondrogenesis was assessed by toluidine blue and safranin-O staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR) for cartilage-specific proteins, and a DMMB dye-binding assay for glycosaminoglycans. Under EMF, hMSCs showed a significant increase in collagen type II expression at passage 6. Aggrecan and SOX9 expression did not change significantly after EMF exposure. Collagen type X expression decreased under electromagnetic stimulation. Pellet cultures at passage 5 that had been treated with EMF provided a higher glycosaminoglycan (GAG)/DNA content than cultures that had not been exposed to EMF. Chondrogenic differentiation of hMSCs may be improved by EMF regarding collagen type II expression and GAG content of cultures. EMF might be a way to stimulate and maintain chondrogenesis of hMSCs and, therefore, provide a new step in regenerative medicine regarding tissue engineering of cartilage., (Copyright © 2010 Wiley-Liss, Inc.)

Noninvasive monitoring of matrix development in tissue-engineered cartilage constructs would permit ongoing assessment with the ability to modify culture conditions during development to optimize tissue characteristics. In this study, chondrocytes seeded in a collagen hydrogel were exposed for 20 min/day to pulsed low-intensity ultrasound (PLIUS) at 30 mWcm(-2) and cultured for up to 5 weeks. Biochemical assays, histology, immunohistochemistry, Fourier transform infrared spectroscopy, and magnetic resonance imaging (MRI) were performed at weeks 3 and 5 after initiation of growth. The noninvasive MRI measurements were correlated with those from the invasive studies. In particular, MRI transverse relaxation time (T2) and magnetization transfer rate (k(m)) correlated with macromolecular content, which was increased by application of PLIUS. This indicates the sensitivity of MR techniques to PLIUS-induced changes in matrix development, and highlights the potential for noninvasive assessment of the efficacy of anabolic interventions for engineered tissue.

Low-level laser therapy (LLLT) has been used as a method for biostimulation. Cartilage develops through the differentiation of mesenchymal cells into chondrocytes, and differentiated chondrocytes in articular cartilage maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix. The aim of this study is to evaluate the enhancement of chondrocyte differentiation and the expression levels of chondrogenic mRNA in prechondrogenic ATDC5 cells after laser irradiation. For chondrogenic induction, ATDC5 cells were irradiated with a blue laser (405 nm, continuous wave) at 100 mW/cm(2) for 180 s following incubation in chondrogenic differentiation medium. Differentiation after laser irradiation was quantitatively evaluated by the measurement of total collagen contents and chondrogenesis-related mRNAs. The total amount of collagen and mRNA levels of aggrecan, collagen type II, SOX-9, and DEC-1 were increased relative to those of a non-laser irradiated group after 14 days of laser irradiation. On the other hand, Ap-2alpha mRNA, a negative transcription factor of chondrogenesis, was dramatically decreased after laser irradiation. In addition, intracellular reactive oxygen species (ROS) were generated after laser irradiation. These results, for the first time, provide functional evidence that mRNA expression relating to chondrogenesis is increased, and Ap-2alpha is decreased immediately after laser irradiation. As this technique could readily be applied in situ to control the differentiation of cells at an implanted site within the body, this approach may have therapeutic potential for the restoration of damaged or diseased tissue., (Copyright 2010 Elsevier B.V. All rights reserved.)

Purpose: Potential targets for selective radiorecovery modulation were investigated via the identification of late upregulated genes and pathways during growth plate chondrocyte recovery., Methods and Materials: Three groups of six 5-week-old male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days totaling 17.5 Gy and were then killed at 7, 11, and 16 days following the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones. Differential gene expression was analyzed between irradiated right and nonirradiated left tibiae using RAE230 2.0 GeneChip microarray, compared between zones and time points, and subjected to functional pathway cluster analysis with real-time polymerase chain reaction (PCR) to confirm selected results., Results: The reserve zone showed the greatest number of differentially expressed genes and enriched pathways: 259 and 134, respectively. Differentially expressed genes included: Timp3, Gpx1, Gas6, Notch2, VEGF, and HIF-1. Enriched pathways included the developmental processes of regeneration, antiapoptosis, developmental growth, tissue regeneration, mesenchymal cell proliferation, negative regulation of immune response, and determination of symmetry. The reserve zone late upregulation of genes was validated using real-time PCR for Mgp, Gas6, and Eef1a1., Conclusions: A significant difference in late upregulated genes between growth plate zones exists. The reserve zone shows the greatest change, containing a 10-fold increase in the total number of genes differentially expressed between days 7 and 16. These findings suggest that reserve zone chondrocytes may play a later role in growth plate recovery response following irradiation., (Copyright © 2010 S. Karger AG, Basel.)

Phocomelia is a devastating, rare congenital limb malformation in which the long bones are shorter than normal, with the upper portion of the limb being most severely affected. In extreme cases, the hands or fingers are attached directly to the shoulder and the most proximal elements (those closest to the shoulder) are entirely missing. This disorder, previously known in both autosomal recessive and sporadic forms, showed a marked increase in incidence in the early 1960s due to the tragic toxicological effects of the drug thalidomide, which had been prescribed as a mild sedative. This human birth defect is mimicked in developing chick limb buds exposed to X-irradiation. Both X-irradiation and thalidomide-induced phocomelia have been interpreted as patterning defects in the context of the progress zone model, which states that a cell's proximodistal identity is determined by the length of time spent in a distal limb region termed the 'progress zone'. Indeed, studies of X-irradiation-induced phocomelia have served as one of the two major experimental lines of evidence supporting the validity of the progress zone model. Here, using a combination of molecular analysis and lineage tracing in chick, we show that X-irradiation-induced phocomelia is fundamentally not a patterning defect, but rather results from a time-dependent loss of skeletal progenitors. Because skeletal condensation proceeds from the shoulder to fingers (in a proximal to distal direction), the proximal elements are differentially affected in limb buds exposed to radiation at early stages. This conclusion changes the framework for considering the effect of thalidomide and other forms of phocomelia, suggesting the possibility that the aetiology lies not in a defect in the patterning process, but rather in progenitor cell survival and differentiation. Moreover, molecular evidence that proximodistal patterning is unaffected after X-irradiation does not support the predictions of the progress zone model.

Ovine bone marrow mesenchymal cells (BMMCs) were seeded on to non-woven filamentous plasma-treated polyester scaffolds and cultured in a chondrogenic medium for 4 weeks. Thereafter a pulsatile hydrostatic pressure (PHP) was applied to these cell-scaffolds constructs at an amplitude of 0.1 MPa and frequency of 0.25 Hz, for 30 min a day, over a period of 10 days. Samples (n = 6) were removed 24 h after PHP stimulation at days 1, 4, 7, and 10 for biochemical analysis. Similar analyses were conducted, at the same time points, on control samples that were not subjected to a PHP. The results showed that the glycosaminoglycan (GAG) content did not significantly increase until after the application of a PHP for 7 days. The GAG content was 1.5 and 2.7 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The deoxyribonucleic acid (DNA) content was 1.5 times greater in the PHP group than in the control group at day 10 (p<0.01). GAG synthesis amounts, expressed as the total GAG contents per microgram of DNA, were 1.6 and 1.8 times higher in the PHP group than in the control group at days 7 and 10 respectively (p<0.01). The total collagen content in the medium did not change until after PHP application for 10 days, when it was 1.9 times higher than the control (p < 0.05). The results suggest that a light PHP applied at a low frequency has a cumulative stimulatory effect on the BMMCs' metabolic activities including cell proliferation and synthesis of the extracellular matrix.

The purpose of this study was to evaluate the benefits of in vitro preconditioning of mesenchymal stem cells (MSCs) using low-intensity ultrasound (US) in the induction of chondrogenic differentiation of MSCs in vivo. After rabbit bone marrow-derived MSCs were seeded onto a polyglycolic acid (PGA) scaffold, the PGA-MSCs constructs were divided into 4 subgroups: untreated control, low-intensity US group, transforming growth factor-beta [TGF]-treated group and low-intensity US/TGF group. The chondrocyte-seeded PGA construct served as a positive control. For 1 week before implantation, the low-intensity US groups were subjected to ultrasound treatment for 20 min daily at an intensity of 200 mW/cm(2). The TGF groups were treated with 10 ng/mL TGF-beta1. The cells were then implanted into the nude mouse subcutaneously. Retrieved 1, 2, 4, and 6 weeks after implantation, each construct underwent gross examination, histology, biochemical assays, mechanical testing, and reverse transcriptase polymerase chain reaction (RT-PCR). Substantial size reduction and blood invasion were found much earlier in the groups that did not undergo low-intensity US than in those that did. Safranin O/Fast green staining revealed that the chondrogenic differentiation of MSCs was more widespread throughout the constructs in the low-intensity US groups. In the biochemical and mechanical analyses, the low-intensity US and low-intensity US/TGF groups were significantly better in forming hyaline cartilage-like tissue by 4 weeks than the non-low-intensity US groups. Presented by von Kossa staining, the development of osteogenic phenotypes was highly suppressed until 4 weeks in the low-intensity US groups, along with compressive strength comparable to the positive control. In the RT-PCR analysis before implantation, the messenger RNA levels of Sox-9, aggrecan, and tissue inhibitors of metalloproteinase-2 were higher in the low-intensity US groups, while those of type I and type X collagens and matrix metalloproteinase-13 were higher in the non-low-intensity US groups. Blood invasion into the constructs was also considerably hindered in the low-intensity US groups. These results strongly indicate that low-intensity US preconditioning in vitro could be an effective cue to upregulate chondrogenic differentiation of MSCs in vivo.

Introduction: The growth plate response following radiotherapy is poorly understood. In particular, little is known about the changes in growth plate growth factors and cytokines following irradiation. The hypothesis was that a limited number of growth factors and cytokines play a role in growth plate proliferative and hypertrophic chondrocyte radio-recovery., Methods: The right limbs of 6 rats were irradiated (17.5 Gy), leaving the left limbs as controls. Limbs were harvested 1 (n = 3) and 2 (n = 3) weeks later. Microarrays were constructed from chondrocytes obtained by laser microdissection from the proliferative zone (PZ) and the hypertrophic zone (HZ) of normal and irradiated tibia growth plates. Real-time PCR was used to confirm the expression of parathyroid hormone receptor 1 (Pthr1), connective tissue growth factor (CTGF), insulin-like growth factor I receptor (IGF1R), insulin-like growth factor II (IGF2), interleukin 17beta (IL17b) and chemokine ligand 12 (CXCL12)., Results and Conclusions: IGF2 is upregulated in the PZ and CTGF is upregulated in both the PZ and HZ 1 week after irradiation, prior to the histomorphometric appearance of growth plate recovery in this immature animal radiation model, supporting their role in stimulating early return of the growth plate. By 2 weeks after irradiation, a number of growth factors and cytokines, including CTGF and Pthr1 in both zones, CXCL12 and its receptor in the PZ, and IL17b and bone morphogenetic protein 2 in the HZ, show upregulation, suggesting a possible later role in radiorecovery. The effects of irradiation on Pthr1, CTGF, IGF2 and CXCL12 in PZ and Pthr1, CTGF, IL17b and IGF1R in the HZ determined by microarray and real-time RT-PCR was highly correlated (r = 0.797, p < 0.05 in the PZ and r = 0.875, p < 0.01 in the HZ, respectively)., (Copyright 2007 S. Karger AG, Basel.)

A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplete.

The negative irradiation complications of growth loss leading to limb length asymmetry and pathological fracture incurred following radiation therapy in pediatric patients has led to a renewed interest in understanding the specific effects of irradiation on the growth plate and the surrounding bone. In the present report, we examined the radiation therapy effects on primary rat growth cartilage chondrocytes in order to determine the chondrocyte radiosensitivity relative to other bone cell constituents and tumor cells, the postirradiation temporal progression of radiation-induced alterations in chondrocyte function, and the time course for the functional restoration of chondrocyte pathways that drive the eventual recovery in growth function. We employed an in vitro primary rat costochondral growth cartilage cell culture model system to evaluate the radiation therapy effects on proliferative chondrocytes using serial radiation doses (0-20 Gy) that are well within the clinically relevant range. Following irradiation, all of the following occurred in a dose-dependent manner: proliferation decreased, cytotoxicity increased, several markers of apoptosis increased, markers of radiation-induced cellular differentiation increased, and cell synthetic activity was disturbed. Alterations in proliferation, cell death, and induction of apoptosis are likely due to a transient radiation-induced derangement of the parathyroid hormone-related protein-Indian hedgehog proliferation-maturation pathway. Alterations in cellular differentiation and cell synthetic activity are novel observations for chondrocytes. Further, these results correspond very well to our previous work in an in vivo Sprague-Dawley rat model, making this model particularly relevant to researching the radiation therapy effects on longitudinal growth.

The purpose was to evaluate the effect of mandibular distraction osteogenesis (DO) on condylar cartilage after radiotherapy and hyperbaric oxygenation (HBO). Unilateral DO was performed on low- and high-dose irradiated rabbits with or without accompanying HBO, and non-irradiated animals. High-dose irradiated animals were given irradiation in the temporomandibular joint (TMJ) equivalent to 50 Gy in 25 fractions. Low-dose irradiated rabbits received scattered irradiation of 10% of that of high-dose irradiated animals. After radiotherapy, some of the animals were given HBO 18 times at 2.5 ATA for 90 min/day. One month after completion of radiotherapy, distraction osteotomy with distractor placement was performed. After a latency period, distraction was started at the rate of 1 mm/day, continued for 2 weeks, and the regenerate was allowed to consolidate for 1 month. Condyles of non-operated rabbits served as controls. Histological changes were more evident on the distracted than on the non-distracted side. In distracted, non-irradiated animals, condylar cartilage changes were minor and probably clinically insignificant. In irradiated rabbits, condylar cartilage changes on the lengthened side were severe, and often cartilage was either totally or partially sealed off by bone. Condylar heads were morphologically deformed. Even low doses of irradiation resulted in notable changes on the operated side, and HBO did not prevent disadvantageous effects.

Members of the TGFbeta/BMP gene family regulate cartilage and bone development. These genes are re-expressed in bone repair and are thought to mediate chondro- and osteoprogenitor cell differentiation. These observations have led to a therapeutic strategy of introducing these growth factors into experimental cartilage and bone defects. Therapeutic efficacy, however, has been limited by diffusion or inactivation of these growth factors from the desired site and by the inability to deliver sustained concentrations of growth factors. This study demonstrates an increase in basal TGFbeta mRNA and protein levels in association with chondrogenic differentiation in endochondral ossification. mRNA is increased by 158%; protein by 23%, and cells immunopositive for TGFbeta by 343% at maximal TGFbeta expression. Importantly, the pattern of TGFbeta expression is preserved throughout the developmental sequence. Our data suggest that the exposure to a specific electromagnetic field (EMF) enhances, but does not disorganize, chondrogenesis and endochondral calcification as well as the normal physiologic expression of TGFbeta. The ability to increase TGFbeta at a moderately low dose for sustained periods of time without disorganizing its physiology suggests the ability to establish temporal concentration gradients of growth factors for the purpose of stimulating skeletal repair.

Recent information from several laboratories suggest that power frequency fields may stimulate cell differentiation in a number of model systems. In this way, they may be similar to pulsed electromagnetic fields, which have been used therapeutically. However, the effects of power frequency fields on phenotypic or genotypic expression have not been explained. This study describes the ability of power frequency fields to accelerate cell differentiation in vivo and describes dose relationships in terms of both amplitude and exposure duration. No change in proliferation or cell content were observed. A clear dose relationship, in terms of both amplitude and duration of exposure, was determined with the maximal biological response occurring at 0.1 mT and 7-9 h/day. Because this study was designed to explore biological activity at environmental exposure levels, this exposure range does not necessarily define optimal dosing conditions from the therapeutic point of view. This study reports the stimulation by power frequency fields of transforming growth factor-beta, an important signalling cytokine known to regulate cell differentiation. The hypothesis is raised that the stimulation of regulatory cytokines by electromagnetic fields may be an intermediary mechanism by which these fields have their biological activity.