Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic. Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation

Bovine and human articular chondrocytes were seeded in 2% alginate constructs and cultured for up to 19 days in a rotating-wall-vessel (RWV) and under static conditions. Culture within the RWV enhanced DNA levels for bovine chondrocyte-seeded constructs when compared with static conditions but did not produce enhancement for human cells. There was a significant enhancement of glycosaminoglycans and hydroxyproline synthesis for both bovine and human chondrocytes. In all cases, histological analysis revealed enhanced Safranin-O staining in the peripheral regions of the constructs compared with the central region. There was an overall increase in staining intensity after culture within the RWV compared with static conditions. Type-II collagen was produced by both bovine and human chondrocytes in the peripheral and central regions of the constructs and the staining intensity was enhanced by culture within the RWV. A capsule of flattened cells containing type-I collagen developed around the constructs maintained under static conditions when seeded with either bovine or human chondrocytes, but not when cultured within the RWV bioreactor

We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups. These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic

Human bone-marrow mesenchymal stem cells have an important role in the repair of musculoskeletal tissues by migrating from the bone marrow into the injured site and undergoing differentiation. We investigated the use of autologous human serum as a substitute for fetal bovine serum in the ex vivo expansion medium to avoid the transmission of dangerous transfectants during clinical reconstruction procedures. Autologous human serum was as effective in stimulating growth of bone-marrow stem cells as fetal bovine serum. Furthermore, medium supplemented with autologous human serum was more effective in promoting motility than medium with fetal bovine serum in all cases. Addition of B-fibroblast growth factor to medium with human serum stimulated growth, but not motility. Our results suggest that autologous human serum may provide sufficient ex vivo expansion of human bone-marrow mesenchymal stem cells possessing multidifferentiation potential and may be better than fetal bovine serum in preserving high motility

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors. Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes. There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism

The gelatin-based haemostyptic compound Spongostan was tested as a three-dimensional (3D) chondrocyte matrix in an in vitro model for autologous chondrocyte transplantation using cells harvested from bovine knees. In a control experiment of monolayer cultures, the proliferation or de-differentiation of bovine chondrocytes was either not or only marginally influenced by the presence of Spongostan (0.3 mg/ml). In monolayers and 3-D Minusheet culture chambers, the cartilage-specific differentiation markers aggrecan and type-II collagen were ubiquitously present in a cell-associated fashion and in the pericellular matrix. The Minusheet cultures usually showed a markedly higher mRNA expression than monolayer cultures irrespective of whether Spongostan had been present or not during culture. Although the de-differentiation marker type-I collagen was also present, the ratio of type-I to type-II collagen or aggrecan to type-I collagen remained higher in Minusheet 3-D cultures than in monolayer cultures irrespective of whether Spongostan had been included in or excluded from the monolayer cultures. The concentration of GAG in Minusheet cultures reached its maximum after 14 days with a mean of 0.83 ± 0.8 μg/10. 6. cells; mean ±, . sem. , but remained considerably lower than in monolayer cultures with/without Spongostan. Our results suggest that Spongostan is in principle suitable as a 3-D chondrocyte matrix, as demonstrated in Minusheet chambers, in particular for a culture period of 14 days. Clinically, differentiating effects on chondrocytes, simple handling and optimal formability may render Spongostan an attractive 3-D scaffold for autologous chondrocyte transplantation

Surgical reconstruction of articular surfaces by transplantation of osteochondral autografts has shown considerable promise in the treatment of focal articular lesions. During mosaicplasty, each cylindrical osteochondral graft is centred over the recipient hole and delivered by impacting the articular surface. Impact loading of articular cartilage has been associated with structural damage, loss of the viability of chondrocytes and subsequent degeneration of the articular cartilage. We have examined the relationship between single-impact loading and chondrocyte death for the specific confined-compression boundary conditions of mosaicplasty and the effect of repetitive impact loading which occurs during implantation of the graft on the resulting viability of the chondrocytes. Fresh bovine and porcine femoral condyles were used in this experiment. The percentage of chondrocyte death was found to vary logarithmically with single-impact energy and was predicted more strongly by the mean force of the impact rather than by the number of impacts required during placement of the graft. The significance of these results in regard to the surgical technique and design features of instruments for osteochondral transplantation is discussed

The aim of this study was to determine whether subchondral bone influences in situ chondrocyte survival. Bovine explants were cultured in serum-free media over seven days with subchondral bone excised from articular cartilage (group A), subchondral bone left attached to articular cartilage (group B), and subchondral bone excised but co-cultured with articular cartilage (group C). Using confocal laser scanning microscopy, fluorescent probes and biochemical assays, in situ chondrocyte viability and relevant biophysical parameters (cartilage thickness, cell density, culture medium composition) were quantified over time (2.5 hours vs seven days). There was a significant increase in chondrocyte death over seven days, primarily within the superficial zone, for group A, but not for groups B or C (p < 0.05). There was no significant difference in cartilage thickness or cell density between groups A, B and C (p > 0.05). Increases in the protein content of the culture media for groups B and C, but not for group A, suggested that the release of soluble factors from subchondral bone may have influenced chondrocyte survival. In conclusion, subchondral bone significantly influenced chondrocyte survival in articular cartilage during explant culture. The extrapolation of bone-cartilage interactions in vitro to the clinical situation must be made with caution, but the findings from these experiments suggest that future investigation into in vivo mechanisms of articular cartilage survival and degradation must consider the interactions of cartilage with subchondral bone

External fixation of distal tibial fractures is often associated with delayed union. We have investigated whether union can be enhanced by using recombinant bone morphogenetic protein-7 (rhBMP-7). Osteoinduction with rhBMP-7 and bovine collagen was used in 20 patients with distal tibial fractures which had been treated by external fixation (BMP group). Healing of the fracture was compared with that of 20 matched patients in whom treatment was similar except that rhBMP-7 was not used. Significantly more fractures had healed by 16 (p = 0.039) and 20 weeks (p = 0.022) in the BMP group compared with the matched group. The mean time to union (p = 0.002), the duration of absence from work (p = 0.018) and the time for which external fixation was required (p = 0.037) were significantly shorter in the BMP group than in the matched group. Secondary intervention due to delayed healing was required in two patients in the BMP group and seven in the matched group. RhBMP-7 can enhance the union of distal tibial fractures treated by external fixation

Aims

The intra-articular administration of tranexamic acid (TXA) has been shown to be effective in reducing blood loss in unicompartmental knee arthroplasty and anterior cruciate reconstruction. The effects on human articular cartilage, however, remains unknown. Our aim, in this study, was to investigate any detrimental effect of TXA on chondrocytes, and to establish if there was a safe dose for its use in clinical practice. The hypothesis was that TXA would cause a dose-dependent damage to human articular cartilage.

Systemic factors are believed to be pivotal for the development of heterotopic ossification in severely-injured patients. In this study, cell cultures of putative target cells (human fibroblastic cells, osteoblastic cells (MG-63), and bone-marrow stromal cells (hBM)) were incubated with serum from ten consecutive polytraumatised patients taken from post-traumatic day 1 to day 21 and with serum from 12 healthy control subjects.

The serum from the polytraumatised patients significantly stimulated the proliferation of fibroblasts, MG-63 and of hBM cells. The activity of alkaline phosphatase in MG-63 and hBM cells was significantly decreased when exposed to the serum of the severely-injured patient. After three weeks in 3D cell cultures, matrix production and osteogenic gene expression of hBM cells were equal in the patient and control groups. However, the serum from the polytraumatised patients significantly decreased apoptosis of hBM cells compared with the control serum (4.3% vs 19.1%, p = 0.031).

Increased proliferation of osteoblastic cells and reduced apoptosis of osteoprogenitors may be responsible for increased osteogenesis in severely-injured patients.

The biological significance of cobalt-chromium wear particles from metal-on-metal hip replacements may be different to the effects of the constituent metal ions in solution. Bacteria may be able to discriminate between particulate and ionic forms of these metals because of a transmembrane nickel/cobalt-permease. It is not known whether wear particles are bacteriocidal.

We compared the doubling time of coagulase negative staphylococcus, Staphylococcus aureus and methicillin resistant S. aureus when cultured in either wear particles from a metal-on-metal hip simulator, wear particles from a metal-on-polyethylene hip simulator, metal ions in solution or a control.

Doubling time halved in metal-on-metal (p = 0.003) and metal-on-polyethylene (p = 0.131) particulate debris compared with the control.

Bacterial nickel/cobalt-transporters allow metal ions but not wear particles to cross bacterial membranes. This may be useful for testing the biological characteristics of different wear debris. This experiment also shows that metal-on-metal hip wear debris is not bacteriocidal.

We analysed the effects of commonly used medications on human osteoblastic cell activity in vitro, specifically proliferation and tissue mineralisation. A list of medications was retrieved from the records of patients aged > 65 years filed in the database of the largest health maintenance organisation in our country (> two million members). Proliferation and mineralisation assays were performed on the following drugs: rosuvastatin (statin), metformin (antidiabetic), metoprolol (β-blocker), citalopram (selective serotonin reuptake inhibitor [SSRI]), and omeprazole (proton pump inhibitor (PPI)). All tested drugs significantly stimulated DNA synthesis to varying degrees, with rosuvastatin 5 µg/ml being the most effective among them (mean 225% (sd 20)), compared with metformin 10 µg/ml (185% (sd 10)), metoprolol 0.25 µg/ml (190% (sd 20)), citalopram 0.05 µg/ml (150% (sd 10)) and omeprazole 0.001 µg/ml (145% (sd 5)). Metformin and metoprolol (to a small extent) and rosuvastatin (to a much higher extent) inhibited cell mineralisation (85% (sd 5)). Our results indicate the need to evaluate the medications prescribed to patients in terms of their potential action on osteoblasts. Appropriate evaluation and prophylactic treatment (when necessary) might lower the incidence and costs associated with potential medication-induced osteoporosis.

Cite this article: Bone Joint J 2013;95-B:1575–80.

We have investigated in vitro the release kinetics and bioactivity of fibroblast growth factor-2 (FGF-2) released from a carrier of fibrin sealant. In order to evaluate the effects of the FGF-2 delivery mechanism on the repair of articular cartilage, full-thickness cylindrical defects, 5 mm in diameter and 4 mm in depth, which were too large to undergo spontaneous repair, were created in the femoral trochlea of rabbit knees. These defects were then filled with the sealant.

Approximately 50% of the FGF-2 was released from the sealant within 24 hours while its original bioactivity was maintained. The implantation of the fibrin sealant incorporating FGF-2 successfully induced healing of the surface with hyaline cartilage and concomitant repair of the subchondral bone at eight weeks after the creation of the defect.

Our findings suggest that this delivery method for FGF-2 may be useful for promoting regenerative repair of full-thickness defects of articular cartilage in humans.

Nanometre-sized particles of ultra-high molecular weight polyethylene have been identified in the lubricants retrieved from hip simulators. Tissue samples were taken from seven failed Charnley total hip replacements, digested using strong alkali and analysed using high-resolution field emission gun-scanning electron microscopy to determine whether nanometre-sized particles of polyethylene debris were generated in vivo. A randomised method of analysis was used to quantify and characterise all the polyethylene particles isolated.

We isolated nanometre-sized particles from the retrieved tissue samples. The smallest identified was 30 nm and the majority were in the 0.1 μm to 0.99 μm size range. Particles in the 1.0 μm to 9.99 μm size range represented the highest proportion of the wear volume of the tissue samples, with 35% to 98% of the total wear volume comprised of particles of this size. The number of nanometre-sized particles isolated from the tissues accounted for only a small proportion of the total wear volume. Further work is required to assess the biological response to nanometre-sized polyethylene particles.

Critical size defects in ovine tibiae, stabilised with intramedullary interlocking nails, were used to assess whether the addition of carboxymethylcellulose to the standard osteogenic protein-1 (OP-1/BMP-7) implant would affect the implant’s efficacy for bone regeneration. The biomaterial carriers were a ‘putty’ carrier of carboxymethylcellulose and bovine-derived type-I collagen (OPP) or the standard with collagen alone (OPC). These two treatments were also compared to “ungrafted” negative controls. Efficacy of regeneration was determined using radiological, biomechanical and histological evaluations after four months of healing. The defects, filled with OPP and OPC, demonstrated radiodense material spanning the defect after one month of healing, with radiographic evidence of recorticalisation and remodelling by two months. The OPP and OPC treatment groups had equivalent structural and material properties that were significantly greater than those in the ungrafted controls. The structural properties of the OPP- and OPC-treated limbs were equivalent to those of the contralateral untreated limb (p > 0.05), yet material properties were inferior (p < 0.05). Histopathology revealed no residual inflammatory response to the biomaterial carriers or OP-1. The OPP- and OPC-treated animals had 60% to 85% lamellar bone within the defect, and less than 25% of the regenerate was composed of fibrous tissue. The defects in the untreated control animals contained less than 40% lamellar bone and more than 60% was fibrous tissue, creating full cortical thickness defects. In our studies carboxymethylcellulose did not adversely affect the capacity of the standard OP-1 implant for regenerating bone.

In an attempt to increase the life of cementless prostheses, an hydroxyapatite-coated implant which releases a bisphosphonate has been suggested as a drug-delivery system. Our in vitro study was designed to determine the maximum dose to which osteoblasts could be safely exposed.

Our findings demonstrated that zoledronate did not impair the proliferation of human osteoblasts when used at concentrations below 1 μm. Murine cells can be exposed to concentrations as high as 10 μm.

A concentration of 0.01% of titanium particles did not impair the proliferation of either cell line. Zoledronate affected the alkaline phosphatase activity of murine osteoblasts through a chelation phenomenon. The presence of titanium particles strongly decreased the alkaline phosphatase activity of murine osteoblasts. We did not detect any synergic effect of zoledronate and titanium particles on the behaviour of both human and murine osteoblasts.

The aim of our study was to investigate the effect of platelet-rich plasma on the proliferation and differentiation of rat bone-marrow cells and to determine an optimal platelet concentration in plasma for osseous tissue engineering. Rat bone-marrow cells embedded in different concentrations of platelet-rich plasma gel were cultured for six days. Their potential for proliferation and osteogenic differentiation was analysed. Using a rat limb-lengthening model, the cultured rat bone-marrow cells with platelet-rich plasma of variable concentrations were transplanted into the distraction gap and the quality of the regenerate bone was evaluated radiologically.

Cellular proliferation was enhanced in all the platelet-rich plasma groups in a dose-dependent manner. Although no significant differences in the production and mRNA expression of alkaline phosphatase were detected among these groups, mature bone regenerates were more prevalent in the group with the highest concentration of platelets.

Our results indicate that a high platelet concentration in the platelet-rich plasma in combination with osteoblastic cells could accelerate the formation of new bone during limb-lengthening procedures.

Although much has been published on the causes of slipped upper femoral epiphysis and the results of treatment, little attention has been given to the mechanism of the slip. This study presents the results of the analysis of 13 adolescent femora, and the attempts to reproduce the radiological appearances of a typical slip. The mean age of the skeletons was 13 years (11 to 15). It was found that the internal bony architecture in the zone of the growth plate was such that a slip of the epiphysis on the metaphysis (in the normal meaning of the word slip) could not take place, largely relating to the presence of a tubercle of bone projecting down from the epiphysis. The only way that the appearance of a typical slipped upper femoral epiphysis could be reproduced was by rotating the epiphysis posteromedially on the metaphysis. The presence and size of this peg-like tubercle was shown radiologically by CT scanning in one pair of intact adolescent femurs.

Implantation of autologous chondrocytes and matrix autologous chondrocytes are techniques of cartilage repair used in the young adult knee which require harvesting of healthy cartilage and which may cause iatrogenic damage to the joint. This study explores alternative sources of autologous cells.

Chondrocytes obtained from autologous bone-marrow-derived cells and those from the damaged cartilage within the lesion itself are shown to be viable alternatives to harvest-derived cells. A sufficient number and quality of cells were obtained by the new techniques and may be suitable for autologous chondrocyte and matrix autologous chondrocyte implantation.