Effect of Hyperbaric Oxygen on Proliferation and Gene Expression of Human Chondrocytes: An In Vitro Study.

PURPOSE: The present study investigated the effects of hyperbaric oxygen (HBO) on human chondrocyte proliferation and gene expression patterns. METHODS: Chondrocyte cultures were transferred to a HBO chamber and exposed to 100% oxygen for 7 consecutive days. Within groups, pressure was varied between 1 and 2 atm and duration of HBO administration was varied among 60, 90, and 120 minutes. Cell counts were performed using the WST-1 assay at 1, 3, 5, and 7 days after initiation of HBO treatment to obtain data to plot a growth curve. Gene expression of apoptosis markers PARP and caspase 3, as well as cartilage specific proteins collagen II and COMP, were detected by reverse transcription polymerase chain reaction. RESULTS: The experiments showed that in vitro administration of HBO inhibit chondrocyte growth. When applied compression was increased up to 2 atm, chondrocyte cell count was reduced by half at days 3 and 7 in association with an upregulation of the apoptosis markers PARP and caspase 3 as well as the cartilage specific proteins collagen II and COMP. No significant differences were monitored from varied duration of daily treatment. CONCLUSION: Chondrocyte growth was inhibited in vitro by treatment of HBO. This inhibitory effect was even increased by elevating the applied pressure, while molecular testing showed reduced chondrocyte growth. Higher levels of HBO inhibited cell growth even more, but up-regulation of apoptosis specific markers and cartilage specific proteins were seen during administration of high oxygen levels. Thus, it has to be evaluated that there is a critical level of hypo-/hyperoxia required to stimulate or at least maintain chondrocyte cell proliferation.

Allogenic Myocytes and Mesenchymal Stem Cells Partially Improve Fatty Rotator Cuff Degeneration in a Rat Model.

PURPOSE: Rotator cuff (RC) tears result not only in functional impairment but also in RC muscle atrophy, muscle fattening and eventually to muscle fibrosis. We hypothesized that allogenic bone marrow derived mesenchymal stem cells (MSC) and myocytes can be utilized to improve the rotator cuff muscle fattening and increase the atrophied muscle mass in a rat model. METHODS: The right supraspinatus (SSP) tendons of 105 inbred rats were detached and muscle fattening was provoked over 4 weeks; the left side remained untouched (control group). The animals (n = 25) of the output group were euthanized after 4 weeks for reference purposes. The SSP-tendon of one group (n = 16) was left unoperated to heal spontaneously. The SSP-tendons of the remaining 64 rats (4 groups with n = 16) were repaired with transosseous sutures. One group received a saline solution injection in the SSP muscle belly, two other groups received 5 × 106 allogenic myocytes and 5 × 106 allogenic MSC injections from donor rats, respectively, and one group received no additional treatment. After 4 weeks of healing, the supraspinatus muscle mass was compared quantitatively and histologically to all the treated groups and to the untreated contralateral side. RESULTS: In the end of the experiments at week 8, the myocyte and MCS treated groups showed a significantly higher muscle mass with 0.2322 g and 0.2257 g, respectively, in comparison to the output group (0.1911 g) at week 4 with p < 0.05. There was no statistical difference between the repaired, treated, or spontaneous healing groups at week 8. Supraspinatus muscle mass of all experimental groups of the right side was significantly lower compared to the untreated contralateral muscle mass. CONCLUSION: This defect model shows that the injection of allogenic mycocytes and MSC in fatty infiltrated SSP muscles is better than no treatment and can partially improve the SSP muscle belly fattening. Nevertheless, a full restoration of the degenerated and fattened rotator cuff muscle to its original condition is not possible using myocytes and MSC in this model.

Fentanyl is less toxic on adult human mesenchymal stem cells compared to ropivacaine when used intraarticularly. A controlled in vitro study.

The purpose of this study was to evaluate the toxicity of ropivacaine and fentanyl on adult human mesenchymal stem cells (hMSC). hMSC's were seeded in monolayer triple-flasks and then plated into 96-well plates at a density of 5000 cells per well. After fully aspirating the culture medium, ropivacaine or fentanyl in its corresponding concentration (0.5%, 0.25%, 0.125% for ropivacaine and 0.05%, 0.025%, 0.0125% for fentanyl) or culture medium only was added to each well. After 30 min, the anaesthetic was removed and fresh culture medium was added. hMSCs mitochondrial activity as a marker of cell proliferation and apoptosis marker was evaluated after 1, 24 h and 7 days. Proliferation was significantly decreased after a 30 min exposure to 0.5% and 0.125% ropivacaine, respectively compared to the control group after 24 h (p < 0.001). Simultaneously, apoptosis was significantly induced. Proliferation of hMSC's was decreased after 24 h when exposed to 0.05%, 0.025% and 0.0125% fentanyl (p < 0.001). Apoptosis was only induced 24 h after an exposure to 0.05% fentanyl. Our data suggest that both drugs have a concentration-dependent effect on proliferation in adult hMSC's in vitro. This effect was more distinct with ropivacaine compared to fentanyl. Translating these results into clinical practice, this in vitro study suggests fentanyl as a potentially less toxic analgetic drug for intraarticular application after arthroscopic bone marrow stimulation or rotator cuff repair with comparable to prolonged pain reduction.

Comparison of ropivacaine and fentanyl toxicity in human fibroblasts.

INTRODUCTION: Although ropivacaine and fentanyl are commonly administered intra-articularly after knee or shoulder arthroscopy for postoperative pain control, there are no studies investigating the toxicity of ropivacaine and fentanyl on human fibroblasts (hF). MATERIAL AND METHODS: Human fibroblasts were seeded in monolayer triple flasks at a density of 10(4) cells/cm(2) and plated into 96 plates at a density of 5000 cells per well. After fully aspirating the culture medium 200 µl of ropivacaine or fentanyl in its corresponding concentration or culture medium only was added to each well. After 30 min ropivacaine or fentanyl was removed and fresh culture medium was added. Fibroblast mitochondrial activity and apoptosis marker level were evaluated after 1 h, 24 h and 7 days. RESULTS: We found a significant decrease in mitochondrial activity after 7 days when exposed to 0.5% ropivacaine. Mitochondrial activity after 1 h, 24 h and 7 days was significantly decreased when fibroblasts were exposed to 0.05% fentanyl. Also, a significant decrease in mitochondrial activity was observed 1 h after exposure to 0.025% fentanyl. Cell viability remained unchanged at any other point in time. A significant increase of caspase-3, as a marker of apoptosis, was only present after exposure to 0.5% ropivacaine after 7 days. CONCLUSIONS: These data suggest that both drugs have a concentration-dependent effect on mitochondrial activity in hF in vitro. This effect is more pronounced with fentanyl. Because the cytotoxicity of fentanyl, without the anticipated increase of caspase-3 as an apoptosis marker, remains unclear, we cannot support fentanyl as an alternative to ropivacaine.

Effects of selective paralysis of the supraspinatus muscle using botulinum neurotoxin a in rotator cuff healing in rats.

We hypothesized that a temporary rotator cuff paralysis using botulinum-neurotoxin A (BoNtA) would lead to an improved tendon-to-bone healing after repair of supraspinatus lesions. One hundred sixty Sprague-Dawley rats were randomly assigned to either the BoNtA or the control (saline) group. BoNtA/saline-solution was injected into the supraspinatus muscle 1 week prior to surgery. A supraspinatus defect was made; we distinguished between a lesion with normal and increased repair load. Furthermore, one subgroup had the operated shoulder immobilized in a cast. Histologic analysis and biomechanical testing followed. Specimens from the BoNtA-group, which were treated with an increased repair load, showed less cellularity and more organization in the interface tissue compared to the saline control group. In addition, we found that the collagen 1-3 quotient in the BoNtA specimen was significantly (p = 0.0051) higher than in the control group. Ultimate load at failure between the groups was not significantly different (p > 0.05). We did not observe any significant differences between the mobilized and immobilized specimen (p = 0.2079). The study shows that tendon-to-bone healing after rotator cuff repair can be altered positively using BoNtA pre-operatively. Tears with increased repair load seem to benefit the most-at least histologically.

Collagen type I and decorin expression in tenocytes depend on the cell isolation method.

BACKGROUND: The treatment of rotator cuff tears is still challenging. Tendon tissue engineering (TTE) might be an alternative in future. Tenocytes seem to be the most suitable cell type as they are easy to obtain and no differentiation in vitro is necessary. The aim of this study was to examine, if the long head of the biceps tendon (LHB) can deliver viable tenocytes for TTE. In this context, different isolation methods, such as enzymatic digestion (ED) and cell migration (CM), are investigated on differences in gene expression and cell morphology. METHODS: Samples of the LHB were obtained from patients, who underwent surgery for primary shoulder arthroplasty. Using ED as isolation method, 0.2% collagenase I solution was used. Using CM as isolation method, small pieces of minced tendon were put into petri-dishes. After cell cultivation, RT-PCR was performed for collagen type I, collagen type III, decorin, tenascin-C, fibronectin, Scleraxis, tenomodulin, osteopontin and agreccan. RESULTS: The total number of isolated cells, in relation to 1 g of native tissue, was 14 times higher using ED. The time interval for cell isolation was about 17 hours using ED and approximately 50 days using CM. Cell morphology in vitro was similar for both isolation techniques. Higher expression of collagen type I could be observed in tenocyte-like cell cultures (TLCC) using ED as isolation method (p < 0.05), however decorin expression was higher in TLCC using CM as isolation method (p < 0.05). Dedifferentiation potential seemed to be similar for both isolation techniques. CONCLUSION: In summary tenocyte-like cells can be obtained with both isolation methods (ED and CM) from the LHB. As no obvious disadvantage could be seen using ED, this method is more suitable for clinical use, as time for cell isolation is shorter and a remarkably higher number of cells can be obtained. However, both isolation methods can further be improved.

Carbamazepine affects water and electrolyte homoeostasis in rat--similarities and differences to vasopressin antagonism.

BACKGROUND: Carbamazepine (CBZ) is a drug widely used in the therapy of epilepsy and mood disorders. One frequently observed side effect is hyponatraemia. The role of vasopressin in hyponatraemic action of CBZ is discussed controversially. In this study, we tested the influence of CBZ on water and salt homoeostasis in rat under different hydration states and under vasopressin 2 receptor (V2R) antagonism by satavaptan to elucidate the renal and vasopressin independent action of CBZ. METHODS: CBZ-treated rats were investigated on metabolic cages after (i) 6 day with ad libitum fluid intake, (ii) moderate water load and (iii) water restriction. The effect of satavaptan was tested in clearance experiments under continuous saline infusion in anaesthetized rats after CBZ pretreatment. RESULTS: Compared to controls, CBZ induced a higher urinary flow rate which was most pronounced (20-fold) after water load and significantly elevated (2-fold) after 10-h water restriction. In addition, CBZ consistently increased renal sodium loss but failed to decrease plasma sodium concentration. In the presence of satavaptan, urinary flow and natriuresis were further increased by CBZ, while there was no differential effect on urea excretion and anion gap. CONCLUSIONS: At the investigated dose (50 mg/kg body weight), CBZ did not induce hyponatraemia or antidiuresis in the rat. However, depending on the hydration state, it induced an increased water and electrolyte loss. Its enhanced influence on urinary flow and natriuresis in the presence of satavaptan suggests additional renal targets for CBZ, independent of vasopressin signalling.

Effects of low frequency electromagnetic fields on the chondrogenic differentiation of human mesenchymal stem cells.

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-ß(3) (TGF-ß(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.

An evaluation of medications commonly used for epidural neurolysis procedures in a human fibroblast cell culture model.

BACKGROUND AND OBJECTIVES: Epidural injections are popular therapies for sciatica and low back pain. Local anesthetics and corticosteroids are commonly used for most injections techniques, but some treatments use a specific combination of several agents. The epidural lysis of adhesions procedure (Racz) uses a combination of bupivacaine, hyaluronidase, a corticosteroid, and hypertonic saline. Because severe complications, some with permanent neurologic deficits, have been observed, we considered the possibility that individual agents or a combination thereof might be capable of damaging or destroying cells in primarily the epidural tissues. METHODS: We used monolayer cell cultures of human fibroblasts in Dulbecco modified Eagle medium to study these pharmacological agents alone or in combination. Cell viability and proliferation were assessed by Trypan blue staining, cell counts, and the WST-1 assay. Time and concentration series were performed. RESULTS: With the corticosteroid, we observed the previously described proliferation-retarding effects. Hyaluronidase was not found to have a relevant effect on fibroblast proliferation. Bupivacaine and hypertonic saline were found to have a time- and concentration-dependent effect on cell viability and proliferation. Both were found to be toxic at concentrations well below the ones used clinically. CONCLUSIONS: We identified a potential for harm caused by commonly used pharmacological agents when applied epidurally. Animal studies will have to show whether the same can be observed in living tissues.

Stimulation of bone growth factor synthesis in human osteoblasts and fibroblasts after extracorporeal shock wave application.

BACKGROUND: Nonunion is a common problem in Orthopedic Surgery. In the recent years alternatives to the standard surgical procedures were tested clinically and in vitro. Extracorporeal shock wave therapy (ESWT) showed promising results in both settings. We hypothesized that in target tissue cells from nonunions like fibroblasts and osteoblasts ESWT increases the release of bone growth factors. METHODS: Fibroblasts and osteoblasts were suspended in 3 ml cryotubes and subjected to 250/500 shock waves at 25 kV using an experimental electrohydraulic lithotripter. After ESWT, cell viability was determined and cells were seeded at 1 × 10(5) cells in 12 well plates. After 24, 48, and 72 h cell number was determined and supernatant was frozen. The levels of growth factors FGF-2 and TGF-ß(1) were examined using ELISA. A control group was treated equally without receiving ESWT. RESULTS: After 24 h there was a significant increase in FGF-2 levels (p < 0.05) with significant correlation to the number of impulses (p < 0.05) observed. TGF-ß(1) showed a time-dependent increase with a peak at 48 h which was not significantly different from the control group. CONCLUSIONS: FGF-2, an important growth factor in new bone formation, was shown to be produced by human fibroblasts and osteoblasts after treatment with ESWT. These findings demonstrate that ESWT is able to cause bone healing through a molecular way by inducing growth factor synthesis.

Inflammatory response against different carbon fiber-reinforced PEEK wear particles compared with UHMWPE in vivo.

Poly(ether ether ketone) (PEEK) and its composites are recognized as alternative bearing materials for use in arthroplasty because of their mechanical properties. The objective of this project was to evaluate the biological response of two different kinds of carbon fiber-reinforced (CFR) PEEK compared with ultra-high molecular weight polyethylene (UHMWPE) in vivo as a standard bearing material. Wear particles of the particulate biomaterials were injected into the left knee joint of female BALB/c mice. Assessment of the synovial microcirculation using intravital fluorescence microscopy as well as histological evaluation of the synovial layer were performed 7 days after particle injection. Enhanced leukocyte-endothelial cell interactions and an increase in functional capillary density as well as histological investigations revealed that all tested biomaterials caused significantly (P < 0.05) increased inflammatory reactions compared with control animals (injected with sterile phosphate-buffered saline), without any difference between the tested biomaterials (P > 0.05). These data suggest that wear debris of CFR-PEEK is comparable with UHMWPE in its biological activity. Therefore, CFR-PEEK represents an alternative bearing material because of its superior mechanical and chemical behavior without any increased biological activity of the wear particles, compared with a standard bearing material.

Human beta-defensin-2 increases cholinergic response in colon epithelium.

The human beta-defensin-2 (hBD-2) is expressed in epithelial cells of skin and respiratory and gastrointestinal tracts. Defensins are arginine-rich small cationic peptides with six intramolecular disulfide bonds and are antimicrobially active against a broad spectrum of pathogens. In addition, they have cytokine-like immunomodulatory properties. We hypothesized that hBD-2 also might influence epithelial cells themselves, thereby altering fluid composition in the gastrointestinal tract. We therefore tested its impact on electrogenic ion transport properties of distal colon in Ussing chamber experiments. Application of hBD-2 did not affect transepithelial voltage or resistance in cAMP-stimulated distal colon. However, it increased cholinergic Ca(2+)-dependent Cl(-) secretion. After 20 min of incubation with hBD-2, the effect of carbachol (CCh) on the equivalent short circuit current (I'(sc)) was enhanced twofold compared to vehicle-treated colon. Modulation of Ca(2+) signaling by hBD-2 was validated by Fura-2 measurements in human colon carcinoma HT29 cells. Twenty-minute incubation with hBD-2 increased the CCh-induced Ca(2+) transient by 20-30% compared to either vehicle-treated cells or cells treated with the defensins hBD-1, hBD-3, or HD-5. This effect was concentration-dependent, with an EC(50) of 0.043 microg/ml, and still present in the absence of extracellular Ca(2+). Also, the ionomycin-induced Ca(2+) transient was increased by hBD-2 treatment. We conclude that hBD-2 facilitates cholinergic Ca(2+)-regulated epithelial Cl(-) secretion. These findings contribute to the concept of a specific interaction of antimicrobial peptides with epithelial function.

The effect of high-energy extracorporeal shock waves on hyaline cartilage of adult rats in vivo.

The aim of this study was to determine if extracorporeal shock wave therapy (ESWT) in vivo affects the structural integrity of articular cartilage. A single bout of ESWT (1500 shock waves of 0.5 mJ/mm(2)) was applied to femoral heads of 18 adult Sprague-Dawley rats. Two sham-treated animals served as controls. Cartilage of each femoral head was harvested at 1, 4, or 10 weeks after ESWT (n = 6 per treatment group) and scored on safranin-O-stained sections. Expression of tenascin-C and chitinase 3-like protein 1 (Chi3L1) was analyzed by immunohistochemistry. Quantitative real-time polymerase chain reaction (PCR) was used to examine collagen (II)alpha(1) (COL2A1) expression and chondrocyte morphology was investigated by transmission electron microscopy no changes in Mankin scores were observed after ESWT. Positive immunostaining for tenascin-C and Chi3L1 was found up to 10 weeks after ESWT in experimental but not in control cartilage. COL2A1 mRNA was increased in samples 1 and 4 weeks after ESWT. Alterations found on the ultrastructural level showed expansion of the rough-surfaced endoplasmatic reticulum, detachment of the cell membrane and necrotic chondrocytes. Extracorporeal shock waves caused alterations of hyaline cartilage on a molecular and ultrastructural level that were distinctly different from control. Similar changes were described before in the very early phase of osteoarthritis (OA). High-energy ESWT might therefore cause degenerative changes in hyaline cartilage as they are found in initial OA.

Membrane-based cultures generate scaffold-free neocartilage in vitro: influence of growth factors.

Scaffold-free cultures provide promising potential in chondrogenic differentiation of human mesenchymal stem cells (hMSCs). In this study, a novel scaffold-free membrane-based culture system, in which hMSCs were cultivated on a cellulose acetate membrane filter at medium-gas interface, was evaluated for chondrogenesis under the addition of growth factors. Chondrogenic differentiation of hMSCs has been described in scaffold-free pellet cultures with good results. In our study membrane-based cultures (1 x 10(6) hMSCs) were produced, maintained at the medium-gas interface and cultured for 21 days. Results were compared with findings from standard pellet cultures (2.5 x 10(5) hMSCs). The effects of the following growth factors were examined: human transforming growth factor-beta(3) (TGF-beta(3)) +/- insulin-like growth factor-1 or +/- human fibroblast growth factor 2. After 3 weeks of culture, chondrogenesis was assessed by Safranin-O staining, immunohistochemistry, a dimethylmethylene blue dye binding assay for glycosaminoglycans, and quantitative real-time polymerase chain reaction for cartilage-specific proteins. Membrane-based cultures containing growth factors formed hemispherical structures with a large surface area (65 mm(2)). When removed from the membrane they showed a histologically smooth cartilage-like surface. Membrane-based cultures stained positive for Safranin-O and collagen type II and contained a high content of glycosaminoglycans. Expression of cartilage-specific markers like collagen type II, aggrecan, and SOX9 was observed under the addition of TGF-beta(3), whereas combinations of growth factors let to a significant increase of collagen type II expression. A markedly reduced expression of collagen type X was found in membrane-based cultures when only TGF-beta(3) was added. Pellet cultures showed similar results besides an increased expression of collagen type X and type II that were observed. Membrane-based cultures provide a differentiation system, comparable in chondrogenesis to pellet cultures, which is able to generate scaffold-free neocartilage. The key benefit factors of membrane-based cultures are a histologically smooth cartilage-like surface and reduced expression of collagen type X, both of which are suitable features for its future application in cartilage regeneration.

Cell quality affects clinical outcome after MACI procedure for cartilage injury of the knee.

The aim of our study was to test the hypothesis that in early follow up after matrix guided autologous chondrocyte implantation (MACI), clinical results do not correlate with radiological and histological results, and that MACI as first line procedure and treatment of traumatic cartilage defects leads to better results compared to second line treatment and treatment of degenerative defects. Six and twelve months after MACI, patients IKDC-score was analysed, as well as the results of MRI-examinations. Specimens of the scaffold were histologically assessed at the time of implantation. The IKDC-score as well as the MRI-score improved significantly during follow up. The number of morphological abnormal cells was correlated with a poor clinical outcome. Defect aetiology proved to be a decisive factor for good clinical outcome. Patients with a short history of trauma (<1 year) and an osteochondritis dissecans were found to have better scores 1 year after MACI than patients with a trauma more than 1 year ago. Defect-size, patients age and -gender did not significantly influence the clinical outcome. No differences were seen when MACI was used as first- or second-line procedure. Defect aetiology and quality of the cells are decisive for the clinical outcome. MACI can produce good and very good clinical results even when used as second-line procedure.