Effects of Hyperbaric Oxygen Intervention on the Degenerated Intervertebral Disc: From Molecular Mechanisms to Animal Models.

MicroRNA (miRNA) 107 expression is downregulated but Wnt3a protein and ß-catenin are upregulated in degenerated intervertebral disc (IVD). We investigated mir-107/Wnt3a-ß-catenin signaling in vitro and in vivo following hyperbaric oxygen (HBO) intervention. Our results showed 96 miRNAs were upregulated and 66 downregulated in degenerated nucleus pulposus cells (NPCs) following HBO treatment. The 3' untranslated region (UTR) of the Wnt3a mRNA contained the "seed-matched-sequence" for miR-107. MiR-107 was upregulated and a marked suppression of Wnt3a was observed simultaneously in degenerated NPCs following HBO intervention. Knockdown of miR-107 upregulated Wnt3a expression in hyperoxic cells. HBO downregulated the protein expression of Wnt3a, phosphorylated LRP6, and cyclin D1. There was decreased TOP flash activity following HBO intervention, whereas the FOP flash activity was not affected. HBO decreased the nuclear translocation of ß-catenin and decreased the secretion of MMP-3 and -9 in degenerated NPCs. Moreover, rabbit serum KS levels and the stained area for Wnt3a and ß-catenin in repaired cartilage tended to be lower in the HBO group. We observed that HBO inhibits Wnt3a/ß-catenin signaling-related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment.

Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative disc cells following hyperbaric oxygen treatment.

BACKGROUND: MicroRNA (miRNA) plays a vital role in the intervertebral disc (IVD) degeneration. The expression level of miR-573 was downregulated whereas Bax was upregulated notably in human degenerative nucleus pulposus cells. In this study, we aimed to investigate the role of miR-573 in human degenerative nucleus pulposus (NP) cells following hyperbaric oxygen (HBO) treatment. METHODS: NP cells were separated from human degenerated IVD tissues. The control cells were maintained in 5% CO2/95% air and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The mRNA and protein levels of Bax were measured. The proliferation of NPCs was detected using MTT assay. The protein expression levels of Bax, cleaved caspase 9, cleaved caspase 3, pro-caspase 9, and pro-caspase 3 were examined. RESULTS: Bioinformatics analysis indicated that the 3' untranslated region (UTR) of the Bax mRNA contained the "seed-matched-sequence" for hsa-miR-573, which was validated via reporter assays. MiR-573 was induced by HBO and simultaneous suppression of Bax was observed in NP cells. Knockdown of miR-573 resulted in upregulation of Bax expression in HBO-treated cells. In addition, overexpression of miR-573 by HBO increased cell proliferation and coupled with inhibition of cell apoptosis. The cleavage of pro-caspase 9 and pro-caspase 3 was suppressed while the levels of cleaved caspase 9 and caspase 3 were decreased in HBO-treated cells. Transfection with anti-miR-573 partly suppressed the effects of HBO. CONCLUSION: Mir-573 regulates cell proliferation and apoptosis by targeting Bax in human degenerative NP cells following HBO treatment.

Upregulation of miR-107 expression following hyperbaric oxygen treatment suppresses HMGB1/RAGE signaling in degenerated human nucleus pulposus cells.

BACKGROUND: The expression of both high-mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) is upregulated in degenerated discs. HMGB1 is known to function as a coupling factor between hypoxia and inflammation in arthritis, and this inflammatory response is modulated by microRNAs (miRNAs), with miR-107 expression downregulated during hypoxia. In this study, we investigated the regulation of the miR-107/HMGB1/RAGE pathway in degenerated nucleus pulposus cells (NPCs) after hyperbaric oxygen (HBO) treatment. METHODS: NPCs were separated from human degenerated intervertebral disc tissues. The control cells were maintained in 5% CO2/95% air, and the hyperoxic cells were exposed to 100% O2 at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The cellular protein and mRNA levels of HMGB1, RAGE, and inducible nitric oxide synthase (iNOS) were assessed, and the phosphorylation of MAPK (p38MAPK, ERK, and JNK) was evaluated. Additionally, cytosolic and nuclear fractions of the IκBα and NF-κB p65 proteins were analyzed, and secreted HMGB1 and metalloprotease (MMP) levels in the conditioned media were quantified. RESULTS: Using microarray analyses, 96 miRNAs were identified as upregulated and 66 downregulated following HBO treatment. Based on these results, miR-107 was selected for further investigation. Bioinformatics analyses indicated that the 3' untranslated region of the HMGB1 mRNA contained the "seed-matched-sequence" for hsa-miR-107, which was validated via dual-luciferase reporter assays. MiR-107 was markedly induced by HBO, and simultaneous suppression of HMGB1 was observed in NPCs. Knockdown of miR-107 resulted in upregulation of HMGB1 expression in HBO-treated cells, and HBO treatment downregulated the mRNA and protein levels of HMGB1, RAGE, and iNOS and the secretion of HMGB1. In addition, HBO treatment upregulated the protein levels of cytosolic IκBα and decreased the nuclear translocation of NF-κB in NPCs. Moreover, HBO treatment downregulated the phosphorylation of p38MAPK, ERK, and JNK and significantly decreased the secretion of MMP-3, MMP-9, and MMP-13. CONCLUSIONS: HBO inhibits pathways related to HMGB1/RAGE signaling via upregulation of miR-107 expression in degenerated human NPCs.

Correlation of blood bone turnover biomarkers and Wnt signaling antagonists with AS, DISH, OPLL, and OYL.

BACKGROUND: Wnt signaling plays an important role in development and maintenance of many organs and tissues. The most-studied secreted Wnt inhibitors are sclerostin (SOST), Dickkopf-related protein 1 (DKK-1), and secreted frizzled related protein 1 (SFRP-1) which play important roles in bone turnover. The present study investigated the relationship between serum Wnt inhibitors and diseases with excessive ossification structures, such as ossification of posterior longitudinal ligament (OPLL), ankylosing spondylitis (AS), diffuse idiopathic skeletal hyperostosis (DISH), and ossification of yellow ligament (OYL). METHODS: Twenty-five patients with AS, DISH, OPLL, or OYL were recruited in this study. Fasting peripheral blood samples were collected from all patients and nine controls. Various biomarkers of bone turnover including osteocalcin (OSC), osteoprotegerin (OPG), SFRP-1, DKK-1, and SOST were investigated. RESULTS: Our data showed that serum levels of OSC were higher, but Dkk-1 levels were lower in AS, DISH, OPLL, and OYL patients than those in the controls. Serum levels of SFRP-1 were significantly higher in DISH patients than those in the controls. Serum levels of SOST were significantly higher in DISH and OPLL patients than both levels in the controls. Serum levels of OPG were lower in AS patients than those in the controls. Serum levels of OSC were higher in the OPLL patients than those in the AS patients. Serum levels of DKK-1, SFRP-1, SOST, and OPG were not significantly different between the different disease groups. CONCLUSIONS: In this exploratory study, both OSC and DKK-1 levels are correlated with the clinical conditions associated with excessive ossification, indicating that blood OSC and DKK-1 levels may serve as diagnostic biomarkers for AS, DISH, OPLL, and OYL. These findings may also help discover potential drug therapies for management of these diseases in the future.

Efficacy of vancomycin-releasing biodegradable poly(lactide-co-glycolide) antibiotics beads for treatment of experimental bone infection due to Staphylococcus aureus.

BACKGROUND: Clinical experience and animal studies have suggested that positron emission tomography (PET) using fluorine-18-labeled fluorodeoxyglucose ((18)F-FDG) may be promising for imaging of bone infections. In this study, we aimed to establish the accuracy of (18)F-FDG PET scanning for monitoring the response to poly(lactide-co-glycolide) (PLGA) vancomycin beads for treatment of bone infection. METHODS: PLGA was mixed with vancomycin and hot-compress molded to form antibiotic beads. In vitro, elution assays and bacterial inhibition tests were employed to characterize the released antibiotics. In vivo, cylindrical cavities were made in six adult male New Zealand white rabbits, and Staphylococcus aureus or saline was injected into the cavity to create a bone infection. After 2 weeks, the infection was confirmed by bacterial cultures, and the defect was filled with PLGA vancomycin beads. The treatment response was monitored by (18)F-FDG PET. RESULTS: The biodegradable beads released high concentrations of vancomycin (well above the breakpoint sensitivity concentration) for treatment of bone infection. In bacterial inhibition tests, the diameter of the sample inhibition zone ranged from 6.5 to 10 mm, which was equivalent to 12.5-100 % relative activity. (18)F-FDG PET results showed that uncomplicated bone healing was associated with a temporary increase in (18)F-FDG uptake at 2 weeks, with return to near baseline at 6 weeks. In the infected animals, localized infection resulted in intense continuous uptake of (18)F-FDG, which was higher than that in uncomplicated healing bones. Bone infection was confirmed with positive bacterial cultures. In vancomycin-treated animals, data showed rapidly decreasing amounts of (18)F-FDG uptake after treatment. CONCLUSIONS: In vitro and in vivo analyses showed that the use of biodegradable PLGA vancomycin beads successfully eradicated S. aureus infection in damaged bone.

Benefits of biphasic calcium phosphate hybrid scaffold-driven osteogenic differentiation of mesenchymal stem cells through upregulated leptin receptor expression.

BACKGROUND: The use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation. METHODS: MSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT). RESULTS: Tested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group. CONCLUSIONS: The BCP hybrid scaffold for osteogenesis of MSCs is better than the coralline HA hybrid scaffold by upregulating expression of leptin-R. This was consistent with in vivo data, which indicated that BCP hybrid scaffolds induced more bone formation in a spinal fusion model.

Identification of mesenchymal stem cells and osteogenic factors in bone marrow aspirate and peripheral blood for spinal fusion by flow cytometry and proteomic analysis.

BACKGROUND: An in vivo animal study and a prospective clinical study have indicated that bone marrow aspirate (BMA) augments spinal arthrodesis. However, there is no quantified data to explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion. METHODS: To analyze the proportion of mesenchymal stem cells (MSCs) and osteogenic factors in human BMA and peripheral blood (PB) of the same patient. Autologous BMA and PB from the patients were analyzed by flow cytometry (FACS) using cell markers for MSCs. The osteogenic potential of MSCs was determined by alkaline phosphatase (ALP) activity and calcium level quantification. Proteomics were used for the qualitative and quantitative mapping of the whole proteome from BMA and PB plasma. The mass-to-charge ratio was calculated by time-of-flight mass spectrometry (TOF-MS). The overexpression of protein was confirmed using Western blot analysis. RESULTS: The proportion of MSCs (CD34-/CD29+/CD105+) was higher in the BMA than that in the PB. Colony-forming cell (CFC) assays suggested that fewer colonies were formed in PB cultures than in BMA culture. There was no significant difference in the osteogenic potential of the MSCs between the PB and BMA. Proteomic mass spectrometry assays suggested that the levels of catalase (osteoclast inhibitor) and glutathione peroxidase 3 (osteogenic biomarker) were higher in the BMA than those in the PB, and this was confirmed by Western blot analysis. CONCLUSIONS: The proportions of MSCs and osteogenic factors were higher in the BMA than in the PB. This may explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion.

Effects of hyperbaric oxygen on the osteogenic differentiation of mesenchymal stem cells.

BACKGROUND: Hyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling. METHODS: The phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment. RESULTS: Cells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx 2 were up-regulated, while GSK-3ß was down-regulated after HBO treatment. Western blot analysis showed an increased ß-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment. CONCLUSIONS: HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes.

BACKGROUND: Although the individual effects of hyperbaric oxygen (HBO) and low-intensity pulsed ultrasound (LIPUS) on osteoarthritic (OA) chondrocytes have been reported, the effects of HBO combined with LIPUS treatment are unknown. METHODS: OA chondrocytes were obtained from patients undergoing knee replacement surgery. RNA was isolated for real-time polymerase chain reaction (PCR) analysis of inducible nitric oxide synthase (iNOS), type-II collagen, and aggrecan gene expression. The protein levels of MMP-3 and TIMP-1 were quantified by enzyme-linked immunosorbent assay (ELISA) after LIPUS or HBO treatment. The data are given as mean ± standard deviation (SD) of the results from three independent experiments. A p value less than 0.05 was defined as statistically significant. RESULTS: Our data suggested that ultrasound and HBO treatment increased cell bioactivity of OA chondrocytes. Real-time PCR analysis showed that HBO treatment increased the mRNA of type-II collagen, aggrecan, and TIMP-1 but suppressed the iNOS expression of OA chondrocytes. LIPUS treatment increased the type-II collagen and iNOS expression of OA chondrocytes. ELISA data showed that HBO or LIPUS treatment increased TIMP-1 production of OA chondrocyte. MMP-3 production was suppressed by HBO treatment. HBO combined with LIPUS treatments resulted in additive effect in TIMP-1 production and compensatory effect in iNOS expression. CONCLUSION: HBO combined with LIPUS treatment-induced increase of the anabolic factor (TIMP-1)/catabolic factor (MMP-3) ratio may provide an additive therapeutic approach to slow the course of OA degeneration.

Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/ß-catenin signaling--an in vitro and in vivo study.

We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is increased via osteogenic differentiation of bone marrow stromal cells (BMSCs), which is regulated by Wnt3a/ß-catenin signaling. Our in vitro data showed that HBO increased cell proliferation, Wnt3a production, LRP6 phosphorylation, and cyclin D1 expression in osteogenically differentiated BMSCs. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx2 were upregulated while those of GSK-3ß were downregulated after HBO treatment. The relative density ratio (phospho-protein/protein) of Akt and GSK-3ß was both up-regulated while that of ß-catenin was down-regulated after HBO treatment. We next investigated whether HBO affects the accumulation of ß-catenin. Our Western blot analysis showed increased levels of translocated ß-catenin that stimulated the expression of target genes after HBO treatment. HBO increased TCF-dependent transcription, Runx2 promoter/Luc gene activity, and the expression of osteogenic markers of BMSCs, such as alkaline phosphatase activity, type I collagen, osteocalcin, calcium, and the intensity of Alizarin Red staining. HBO dose dependently increased the bone morphogenetic protein (BMP2) and osterix production. We further demonstrated that HBO increased the expression of vacuolar-ATPases, which stimulated Wnt3a secretion from BMSCs. Finally, we showed that the beneficial effects of HBO on bone formation were related to Wnt3a/ß-catenin signaling in a rabbit model by histology, mechanical testing, and immunohistochemical assays. Accordingly, we concluded that HBO increased the osteogenic differentiation of BMSCs by regulating Wnt3a secretion and signaling.

Hyperbaric oxygen treatment prevents nitric oxide-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70.

Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia-induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on (1) interleukin-1ß (IL-1ß)-induced NO production and apoptosis of rabbit chondrocytes and (2) healing of articular cartilage defects. For the in vitro study, RT-PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL-1ß-treated chondrocytes. To clarify that the HSP70 was necessary for anti-iNOS and anti-apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO-induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70.

Hyperbaric oxygen treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells.

Nucleus pulposus cells (NPCs) from degenerating discs produce catabolic and inflammatory factors, including interleukin (IL)-1 and nitric oxide (NO). Enhanced production of NO has been implicated in the apoptosis of degenerating disc cells. This study evaluates the effects of hyperbaric oxygen (HBO) on degenerated human NPCs. All hyperoxic cells were exposed to 100% O(2) at 2.5 atmospheres absolute (ATA). Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) in NPCs was detected using the phosphor-kinase array kit. RNA was isolated for real-time polymerase chain reaction (PCR) analysis of aggrecan and type II collagen gene expression. The levels of IL- 1ß and NO were quantified by enzyme-linked immunosorbent assay (ELISA). To identify the HBO-induced anti-apoptotic pathways, expression of Bcl-2 and Bax proteins as well as activation of cysteine-containing aspartate-specific proteases (caspases) 3, 8, and 9 was evaluated using Western blotting after HBO treatment. Our data showed that HBO treatment decreased the expression of IL-1ß, suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, decreased synthesis of NO, and increased the gene expression of aggrecan and type II collagen in NPCs as compared with the atmospheric treatment. HBO up-regulated the ratio of Bcl-2 to Bax expression and reduced the activity of caspases 9 and 3 but not of caspase 8, indicating a selective effect over the mitochondrial apoptosis pathway in degenerated NPCs. These results support our hypothesis that HBO treatment suppresses MAPK signaling and mitochondrial apoptotic pathway in degenerated human intervertebral disc cells.

The use of fluorescence-labeled mesenchymal stem cells in poly(lactide-co-glycolide)/hydroxyapatite/collagen hybrid graft as a bone substitute for posterolateral spinal fusion.

BACKGROUND: Posterolateral spinal fusion is used to treat patients with degenerative spinal disorders. In this study, we investigated the effectiveness of a mesenchymal stem cell (MSC)/hydroxyapatite/type I collagen hybrid graft for posterolateral spinal fusion in a rabbit model. METHODS: In vitro study, the hybrid graft was cultured in complete or osteogenic medium for 7 days and 14 days and examined by scanning electron microscopy. The alkaline phosphatase activity of the MSCs was assessed and the expression of osteogenic gene was determined by reverse transcription polymerase chain reaction. In vivo investigation, spinal fusion was examined using radiography, manual palpation, computed tomography, torsional loading tests, and histologic analysis. Furthermore, using a PKH fluorescence labeling system, we examined whether the newly formed bone was derived from the transplanted MSCs. RESULTS: Our data suggested that the MSCs differentiated into osteoblasts and produced extracellular matrix in the hybrid graft. Increased alkaline phosphatase activity was noted and mRNA of Cbfa-1 and osteopontin were detected. Radiographs and computed tomography images showed a continuous bone bridge and a satisfactory fusion mass incorporated into the transverse processes. The results of manual palpation and biomechanical data did not significantly differ between the two groups. Histologic examination of both groups revealed the presence of cartilage and endochondral ossification in the gaps between the grafted fragments. In situ tracing of the PKH 67-labeled MSCs indicated that the transplanted MSCs were partly responsible for the new bone formation. CONCLUSION: The hybrid graft could be effectively used to achieve posterolateral spinal fusion.

In vitro and in vivo analysis of a biodegradable poly(lactide-co-glycolide) copolymer capsule and collagen composite system for antibiotics and bone cells delivery.

The authors investigated poly(lactide-co-glycolide) (PLGA) capsule and collagen composite system for antibiotics and bone cells delivery to treat infected bone defects. Poly(lactide-co-glycolide) was mixed with vancomycin and hot compressing molded to form an antibiotic capsule. Rabbit mesenchymal stem cells (MSCs) were entrapped in collagen gel phase and dispersed throughout the void volume of capsule. In vitro study, the composite systems were cultured in complete or osteogenic medium for 21 days. The profiles of vancomycin released from the systems were evaluated using the high-performance liquid chromatography method. Relative activity of vancomycin against Staphylococcus aureus was determined by an antibiotic disk diffusion method. The expression of osteogenic gene was determined by reverse-transcription polymerase chain reaction. The alkaline phosphatase activity and calcium level of the MSCs were assessed. Analytical results demonstrated that the concentrations of vancomycin eluted from the composite system were above the minimal inhibitory concentration for 21 days. Sample inhibition zone was 10 to 24 mm, and the relative activity was 17.6% to 100%. mRNA of Cbfa1 and osteocalcin were detected, and increased alkaline phosphatase activity and calcium levels were noted. In in vivo investigation, the PKH 26-labeled MSCs and composite systems were implanted in the distal femoral cavities of four rabbits. The local concentration of vancomycin was above the minimal inhibitory concentration for 56 days. Sample inhibition zone was 9 mm to 24 mm, and the relative activity was 11.8% to 100%. Implanted PKH 26-labeled MSCs were identified in the newly formed bony trabeculae in specimens at 2 and 4 months after implantation. The results offer a potential approach to meet clinical requirements in the treatment of infected bone defects.

Effect of hyperbaric oxygenation on intervertebral disc degeneration: an in vitro study with human lumbar nucleus pulposus.

STUDY DESIGN: An in vitro study with degenerated human lumbar intervertebral disc specimens cultured under hyperbaric oxygenation (HBO). OBJECTIVE: To observe the changes in interleukin (IL)-1ß, prostaglandin (PG)-E2, nitric oxide (NO), cell growth, and apoptosis of the human nucleus pulposus cell (NPC) after HBO. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration has been demonstrated as related to IL-1ß, PG-E2, NO, and O2 concentration but the actual mechanism is not clear. HBO also has also been reported in the literature to influence changes in IL-1ß, prostaglandin E2, NO, and O2 concentration. However, the direct effect of HBO on the disc cells has not been previously reported. METHODS: We collected 12 human lumbar degenerated disc specimens and evaluated the effects of HBO on the cultured NPCs. The amounts of IL-1ß, PG-E2, and NO in the conditioned medium were quantified by enzyme-linked immunosorbent assay and high performance liquid chromatography. Cell growth was measured by increase in cell number. Cell viability and proteoglycan content were evaluated by histologic study using safranin O staining. In situ analysis of apoptosis was performed using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. RESULTS: Our data indicated that HBO treatment inhibited IL-1ß, PG-E2, and NO production but increased cell number and matrix synthesis of cultured NPCs. TUNEL staining showed that HBO treatment suppressed the apoptosis of cultured NPCs. CONCLUSION: HBO provides a potential treatment modality for disc degeneration.

Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disk cells via suppression of IL-1ß and p38 MAPK signal.

Nucleus pulposus cells (NPCs) from degenerating disks produce catabolic and inflammatory factors, including interleukin (IL)-1, nitric oxide (NO), prostaglandin E2 (PGE-2), and matrix metalloproteinaes (MMPs). An imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) has been proposed to exist in the degenerating disk. This study evaluates the effects of hyperbaric oxygen (HBO) on the human degenerated NPCs. NPCs were maintained in alginate bead culture. All hyperoxic cells were exposed to 100% O(2) at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber. p38 MAPK phosphorylation of the NPCs was detected using the phosphor-kinase array kit. RNA was isolated for real-time quantitative polymerase chain reaction (Q-PCR) analysis of aggrecan and type II collagen gene expression. The amounts of IL-1ß, NO, PGE-2, MMP-3, and TIMP-1 in the conditioned media were quantified by enzyme-linked immunosorbent assay (ELISA). Our data showed that HBO treatment decreased expression of IL-1ß, increased the gene expression of aggrecan and type II collagen, suppressed the phosphorylation of p38 MAPK, decreased NO, PGE-2, and MMP-3, and increased TIMP-1 expression in NPCs as compared with the atmospheric treatment. These results support the hypothesis that IL-1ß and the p38 MAPK signal may be responsible for many of the inflammatory and catabolic changes seen in the human disk degeneration, and support our proposal that HBO treatment-induced increase of the anabolic factor (TIMP-1)/catabolic factor (MMP-3) ratio may provide a therapeutic approach to slow the course of intervertebral disk degeneration.

Arthroscopic ganglionectomy through an intrafocal cystic portal for wrist ganglia.

PURPOSE: A retrospective study was conducted on arthroscopic ganglionectomy in wrists using a novel intrafocal cystic portal. The safety and efficacy of this technique were assessed by treatment of 15 wrists in 15 patients. METHODS: Arthroscopic ganglionectomy was performed by the same surgeon with the patient under general anesthesia or regional block. Preoperative complaints, intraoperative findings, and postoperative results of all the patients were reported. The mean follow-up was 15.3 months. Functional assessment by use of modified Mayo wrist scores, patient satisfaction, and recurrence were included in the follow-up evaluation. RESULTS: Two thirds of the patients acquired good to excellent results, whereas the results for the remaining third were fair. Complications included 1 recurrence and 1 case of transient paresthesia sensation. The most common arthroscopic findings were capsular and ligament lesions, rather than ganglionic stalks. CONCLUSIONS: Arthroscopic ganglionectomy through an intrafocal cystic portal is a safe and efficacious option for the treatment of painful wrist ganglia. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Use of fluorescence labeled mesenchymal stem cells in pluronic F127 and porous hydroxyapatite as a bone substitute for posterolateral spinal fusion.

Posterolateral spinal fusion is used to treat patients with degenerative spinal disorders. We investigated the effectiveness of a mesenchymal stem cell (MSC)/Pluronic F127/Interpore hybrid graft for spinal fusion in rabbits. Spinal fusion was examined using radiography, manual palpation, computed tomography (CT), torsional loading tests, and histological analysis. Using a PKH fluorescence labeling system, we also examined whether the newly formed bone was derived from the transplanted MSCs. We found that the MSCs adhered to the Interpore surface and within its pores, and differentiated into osteoblasts. Radiographs and CT images showed a continuous bone bridge and a satisfactory fusion mass incorporated into the transverse processes. The results of manual palpation and biomechanical data did not differ significantly from an autograft group. Histology from both groups revealed the presence of fibrous tissue, cartilage, and endochondral ossification in the gaps between the grafted fragments. In both groups, the degree of mature bone formation was greater at 12 weeks than at 6 weeks after grafting. Quantitative histomorphometry revealed no significant differences between the two groups at either time point. In situ tracing of the PKH 67-labeled MSCs indicated that the transplanted MSCs were partly responsible for the new bone formation in both the repaired transverse processes and the grafted fragments. Thus, the MSC/Pluronic F127/Interpore hybrid graft could be used effectively to achieve posterolateral spinal fusion.

Additive effects of hyperbaric oxygen and platelet-derived growth factor-BB in chondrocyte transplantation via up-regulation expression of platelet-derived growth factor-beta receptor.

The present study investigated the effects of hyperbaric oxygen (HBO) and platelet-derived growth factor-BB (PDGF-BB) in chondrocyte transplantation. In vitro, chondrocytes were treated with HBO, PDGF-BB, and HBO combined with PDGF-BB (H+P). Cell growth was analyzed using cell counting, MTT assay, and FACS analysis. mRNA expression of the PDGF-alpha receptor (PDGFR-alpha) and beta receptor (PDGFR-beta) was detected by RT-PCR. Protein expression of PDGFR-beta was detected by Western blotting. In vivo, chondrocytes and PDGF-BB were suspended in alginate as a transplantation system. Cartilage defects were grafted with this system and with or without HBO treatment. Released PDGF-BB concentration was quantified by ELISA. After 8 weeks, animals were sacrificed and the repaired tissues were examined. In vitro data suggested that each treatment increased cell growth via the up-regulated mRNA expression of PDGFR-alpha and increased cell accumulation in the S-phase. The H+P treatment was more additive in cell growth and in mRNA and protein expression of PDGFR-beta than HBO or PDGF-BB. In vivo results suggested that PDGF-BB delivery lasted for more than 5 weeks. Scoring results showed that each treatment significantly increased the cartilage repair. Safranin-O and type II collagen staining confirmed the hyaline-like cartilage regeneration in the repaired tissues. In situ up-regulation of PDGFR-beta expression partially explains the additive effect of H+P treatment in cartilage repair. Accordingly, H+P offers a potential treatment method for cartilage repair.

Mesenchymal stem cell and nucleus pulposus cell coculture modulates cell profile.

Spontaneous cell fusion can occur in cocultured stem cells. We examined whether telomerase activity change and cell fusion occurred in mesenchymal stem cell (MSC) and nucleus pulposus cell (NPC) coculture. MSCs and NPCs were labeled with PKH26 and PKH67 dyes and cocultured at a 50:50 ratio. An equal number of MSCs or NPCs were used as the control. After 14 days, cells were evaluated by cell growth, telomerase activity, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and histologic observation. Cell fusion was confirmed by microscopic observation and fluorescence-activated cell sorter (FACS) analysis. The results suggested cell growth rate and telomerase activity were higher in cocultured cells than in NPCs cultured alone. The mRNA expression levels of the Type II collagen and aggrecan were elevated in cocultured cells. Immunohistochemical analysis revealed positive staining for Type II collagen and keratan sulfate in NPCs cultured alone and in a proportion of cocultured cells. Histologic observation revealed binucleated cocultured cells expressed both PKH dyes in the same location and slide focus. The FACS analysis revealed 42% of cocultured cells were double-stained. Cocultured cells partially maintained the NPC phenotype. The partially maintained phenotype of the NPCs may be attributable to spontaneous cell fusion in association with increased telomerase activity.