Effects of bioactive glass nanoparticles on the mechanical and biological behavior of composite coated scaffolds.

Biphasic calcium phosphates (BCP) scaffolds are widely used for bone tissue regeneration. However, brittleness, low mechanical properties and compromised bioactivities are, at present, their major disadvantages. In this study we coated the struts of a BCP scaffold with a nanocomposite layer consisting of bioactive glass nanoparticles (nBG) and polycaprolactone (PCL) (BCP/PCL-nBG) to enhance its mechanical and biological behavior. The effect of various nBG concentrations (1-90 wt.%) on the mechanical properties and in vitro behavior of the scaffolds was comprehensively examined and compared with that for a BCP scaffold coated with PCL and hydroxyapatite nanoparticles (nHA) (BCP/PCL-nHA) and a BCP scaffold coated with only a PCL layer (BCP/PCL). Introduction of 1-90 wt.% nBG resulted in scaffolds with compressive strengths in the range 0.2-1.45 MPa and moduli in the range 19.3-49.4 MPa. This trend was also observed for BCP/PCL-nHA scaffolds, however, nBG induced even better bioactivity and a faster degradation rate. The maximum compressive strength (increased ∼14 times) and modulus (increased ∼3 times) were achieved when 30 wt.% nBG was added, compared with BCP scaffolds. Moreover, BCP/PCL-nBG scaffolds induced the differentiation of primary human bone-derived cells (HOBs), with significant up-regulation of osteogenic gene expression for Runx2, osteopontin and bone sialoprotein, compared with the other groups.

Modulation of aggrecan and ADAMTS expression in ovine tendinopathy induced by altered strain.

OBJECTIVE: To evaluate histologic, immunohistochemical, and molecular changes in tendon induced by altered strain in a large-animal model. METHODS: A full-thickness partial-width laceration of the infraspinatus tendon was created in 5 sheep, while 5 sham-operated sheep were used as controls. Sheep were killed after 4 weeks, and 4 differentially stressed tendon regions (tensile or near bone attachment from overstressed or stress-deprived halves) were evaluated for histopathology, proteoglycan (PG) accumulation, and characterization of glycosaminoglycans and aggrecan catabolites. Gene expression of matrix components, enzymes, and inhibitors was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: Histopathologic changes were detected in both overstressed and stress-deprived tensile tendon, but only in stress-deprived tendon near bone. In overstressed and stress-deprived tensile tendon, levels of keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate were increased. In overstressed tensile tendon, levels of ADAMTS-generated aggrecan catabolites were increased. There was increased matrix metalloproteinase 13 (MMP-13) and decreased fibromodulin and decorin expression in all regions. Increased MMP-1, MMP-9, MMP-14, and ADAMTS-1 expression, and decreased type II collagen expression were restricted to stress-deprived tendon. In stress-deprived bone-attachment regions, messenger RNA (mRNA) for aggrecan was decreased, and ADAMTS was increased. In overstressed tensile tendon, aggrecan mRNA was increased, and ADAMTS was decreased. CONCLUSION: The distinct molecular changes in adjacent tissue implicate altered strain rather than humoral factors in controlling abnormal tenocyte metabolism, and highlight the importance of regional sampling. Tendon abnormalities induced by increased strain are accompanied by increased aggrecan, decreased ADAMTS, and low PG expression, which may negatively impact the structural integrity of the tissue and predispose to rupture.

Reliability of a new shoulder laxometer to assess inferior glenohumeral joint translation.

OBJECTIVE: This study tested the reliability of the ORI laxometer, a newly developed non-invasive device for testing inferior translation of the humeral head in humans. DESIGN: The instrument was designed to measure displacement between the top of the acromion and the head of the humerus when loaded in a similar fashion to the sulcus sign. PARTICIPANTS AND INTERVENTIONS: Sixteen healthy subjects (32 shoulders) were measured for inferior glenohumeral joint laxity. One observer used the laxometer in 16 subjects on three separate occasions for the intra-observer trial. Three observers measured the inferior shoulder laxity of six subjects on one occasion in the inter-observer trial. Asymptomatic and unstable shoulders were also compared in 12 subjects with shoulder instability. MAIN OUTCOME MEASUREMENTS: Translation in the glenohumeral joint RESULTS: The range of inferior translation of glenohumeral joint in these subjects was between 0.01 mm and 6.5 mm with a mean of 1.5 mm. The intraclass correlation coefficient (ICC) for inter-observer reliability was 0.74. For intra-observer reliability the ICC was 0.76. These results are considered to be good to excellent. There was no advantage gained by using data from all five cycles of testing compared with three cycles. In patients with shoulder instability, laxometer measurements were significantly greater in their unstable shoulders than in their normal shoulders. CONCLUSIONS: The laxometer is easy to use, painless and gives objective measures for inferior glenohumeral laxity, with good intra- and inter-observer reliabilities. The ORI laxometer may be useful for assessing and monitoring global glenohumeral joint laxity which usually involves inferior laxity.

Arthroscopic estimation of the extent of chondropathy.

INTRODUCTION: Arthroscopy has been used to evaluate articular cartilage (AC) pathology in osteoarthritis (OA) for outcome measurement and validation of non-invasive imaging. However, many fundamental aspects of arthroscopic assessment remain un-validated. OBJECTIVES: This study evaluated arthroscopic estimates of extent of chondropathy. METHODS: Serial arthroscopic assessments were performed in a group of 15 sheep before and after bilateral stifle medial meniscectomy (MMx). Post-mortem assessments were performed in un-MMx sheep and 4 and 16 weeks post-MMx. Arthroscopic assessments of the extent of each grade of chondropathy were compared with a non-arthroscopic hybrid assessment that incorporated biomechanical, thickness and macroscopic assessments. RESULTS: Arthroscopy evaluated only 36% of AC and missed significant pathological changes, softening and chondro-osteophyte, occurring in peripheral regions. The patterns of change in arthroscopic assessments were similar to those of the non-arthroscopic assessment but there was a very strong tendency to over-estimate the extent of softened AC after MMx. In spite of these limitations arthroscopic assessments were responsive to change. Estimates of the extent of normal and softened AC were most responsive to change over time followed by estimates of superficial and deep fibrillation. Arthroscopy was as an excellent discriminator between normal and OA. Assessments of chondro-osteophyte and exposed bone were not responsive to change. CONCLUSIONS: Arthroscopic estimates of extent of chondropathy are prone to substantial error. While experience and training may reduce these errors other approaches may more effectively improve performance.

Overexpression of nitric oxide synthases in tendon overuse.

Tendon disorders with a chronic nature, including the rotator cuff, are extremely common, and represent a major clinical problem. Mechanical overload has been proposed as an important etiologic factor in tendinopathies. Nitric oxide (NO), a free radical produced by nitric oxide synthases (NOSs), is a potent regulator and stimulator of biological processes including tendon degeneration and healing. It is also involved in response to mechanical stimuli in different tissues. In an animal model of acutely injured tendon healing temporal and differential expression of NOS isoforms has been demonstrated, suggesting that different patterns of NOSs expression may have different biological functions. Therefore, we hypothesized that tendon overuse may result in a differential upregulation of NOSs, particularly iNOS. An animal model of supraspinatus tendon overuse was utilized, which consisted of treadmill running. A group of animals of the same strain and age subjected to normal cage activity were used as controls. Following a 4-week exercise protocol supraspinatus tendons were harvested, RNA was extracted, and subjected to competitive reverse transcription and polymerase chain reaction (RT-PCR) to determine the expression levels of inducible-, endothelial-, and neuronal-NOS isoforms (i-, e-, and nNOS). The mRNA expression of all three NOS isoforms increased in the supraspinatus tendons as a result of overuse exercise. iNOS and eNOS mRNA expression increased fourfold (p < 0.01), and there was an increase, but statistically not significant, in nNOS mRNA expression in the overused tendons when compared with the controls. This study is the first to show that NOS isoforms are upregulated in rotator cuff tendon as a result of chronic overuse, and suggests the involvement of nitric oxide in the response of tendon tissue to increased mechanical stress.

Spontaneous recovery of injured Achilles tendon in inducible nitric oxide synthase gene knockout mice.

OBJECTIVE AND DESIGN: To determine if inducible nitric oxide synthase (iNOS) gene could affect Achilles tendon healing using iNOS gene knockout mice. METHODS: 21 iNOS knockout (iNOS(-/-)) mice and 8 of the wild type (iNOS(+/+)) mice were utilized in this study. Group 1: iNOS(+/+) mice (n = 8), group 2: iNOS(-/-) mice (n = 11) and group 3: iNOS(-/-) with a NOS inhibitor, (aminoguanidine, 500 mg/kg/day, via an intraperitoneal mini-osmotic pump for 7 days, n = 10). The right Achilles tendon was transected in all mice and harvested on day 7 for cross-sectional area and biomechanical properties. Serum nitrate concentration of the mice was measured by gas chromatography mass spectrometry (GC/MS). RESULTS: A significant reduction in cross-sectional area of the healing Achilles tendon was observed in group 3 mice compared to group 2 mice (p < 0.01). The serum nitrate concentration in both group 2 and group 3 mice was lower than that in group 1 mice (p < 0.01) iNOS gene deletion and inhibition of NOS did not affect the biomechanical properties of the healing tendons. CONCLUSIONS: iNOS gene is not solely responsible for the beneficial effects of nitric oxide (NO) on tendon healing.

Ovariectomy alters the structural and biomechanical properties of ovine femoro-tibial articular cartilage and increases cartilage iNOS.

OBJECTIVE: To examine the effect of oestrogen depletion produced by surgical ovariectomy on the structural and biomechanical properties of ovine femoro-tibial articular cartilage (AC), and the production of inducible nitric oxide synthase (iNOS) and nitrotyrosine by these tissues. METHODS: Six aged ewes were surgically ovariectomised (OVX), while six were used as unoperated controls. Dynamic biomechanical indentation testing of tibial plateau AC was performed at 26 weeks post-op. Histological sections of medial tibial plateau and lateral tibial plateau (LTP), medial and lateral femoral condyles (MFC, LFC) and patellar AC were examined for histopathology, toluidine blue staining intensity, and patterns of collagen birefringence intensity. Immunoreactivity for iNOS and nitrotyrosine was assessed in full-thickness biopsy plugs of LFC and patellar AC, and patellar AC explants were cultured to determine in vitro NO release. RESULTS: Phase lag was reduced overall in LTP-AC of OVX sheep (10.9+/-2.2 degrees vs 12.1+/-2.3 degrees ; P<0.0001). Cartilage thickness was reduced in the LTP of OVX sheep (P=0.0002), in association with localised changes in dynamic shear modulus. Toluidine blue staining intensity was reduced in the patella, LFC, and MFC. Histological examination revealed greater histopathology scores in the MFC of OVX animals, and altered collagen birefringence intensity plots in the LTP. Immunostaining for iNOS was increased in patella AC (P=0.008), whilst nitrotyrosine immunoreactivity was increased in patella (P=0.03) and LFC (P<0.0001) AC. NO release by patellar AC explants was also elevated. CONCLUSIONS: Oestrogen depletion induced by OVX caused regional thinning of femoro-tibial cartilage, with biomechanical and histological changes suggestive of a disturbance in the content and/or structural organisation of the proteoglycan and collagen macromolecular assembly. The observed up-regulation of cartilage iNOS suggests a possible mechanism for these matrix changes.

Arthroscopy -- a potential "gold standard" for the diagnosis of the chondropathy of early osteoarthritis.

OBJECTIVES: The aims of this study were to: 1. Evaluate the performance of arthroscopy for the diagnosis of chondropathy and to compare it to that of direct non-arthroscopic assessments; 2. Determine intra-observer reliability of arthroscopic assessments; 3. Evaluate the effects of the arthroscopic video quality and probing upon diagnostic performance. DESIGN: The ovine medial meniscectomy (MMx) model of early osteoarthritis (OA) was used assuming that pre-MMx articular cartilage (AC) was "normal" and post-MMx AC "chondropathic". Video recordings of arthroscopic assessments of each stifle compartment were evaluated. Scores were given for the quality of the video and the amount of probing. The diagnostic performances of dynamic shear modulus (G), light microscopic assessment and superficial zone collagen birefringence assessments were evaluated and compared to that of arthroscopy. Intra-observer reliability of arthroscopic assessments was also evaluated. RESULTS: Arthroscopic assessments had high sensitivity (91-100%), specificity (62-88%) and accuracy (75-93%) for the diagnosis of chondropathy 16 weeks after MMx. Arthroscopy compared favourably with the direct non-arthroscopic assessments in the lateral compartment and was found to have extremely high intra-observer reliability (kappa 0.78-1.00). The quality of arthroscopic video recordings and the amount of probing did not significantly influence accuracy or reliability. CONCLUSIONS: Arthroscopy performs as well as direct non-arthroscopic assessments of AC for diagnosis of early OA. These results suggest that arthroscopy can be used as a "gold standard" for the validation of non-invasive assessments like magnetic resonance imaging and that arthroscopic diagnosis can be based on small amounts of video footage without AC probing.

Biomechanical, histologic and macroscopic assessment of articular cartilage in a sheep model of osteoarthritis.

OBJECTIVES: Our primary objective was to explore the full potential of the ovine medial meniscectomy (MMx) model of early osteoarthritis (OA) for studies to validate non-destructive articular cartilage (AC) assessments and therapeutic interventions. Our secondary objective was to re-evaluate the relationships between the different types of AC assessment after MMx in sheep. METHODS: Macroscopic assessments, dynamic shear modulus (G*), phase lag and AC thickness measurements were performed at a total of 5437 reference points on all six articular surfaces in four normal joints and 16 MMx ovine stifle (knee) joints. Comparisons with histologic assessments of gross structural damage, collagen organisation (birefringence) and proteoglycan content were possible at 702 of these points. RESULTS: Histologic gross structural damage and proteoglycan loss were seen throughout the joint with greatest severity (fibrillation) in closest proximity to the MMx site. Increases in AC (30-50%) thickness, reductions in G* (30-40%) and collagen birefringence intensity (15-30%) occurred more evenly throughout the joint. Macroscopic softening was evident only when G* declined by 80%. G* correlated with AC thickness (rho=-0.47), collagen organisation rho=0.44), gross structural damage (rho=-0.44) and proteoglycan content (rho=0.42). Multivariate analysis showed that collagen organisation contributed twice as much to dynamic shear modulus (t=6.66 as proteoglycan content (t=3.21). Collagen organisation (rho=0.11) and proteoglycan content (rho=0.09) correlated only weakly to phase lag. CONCLUSIONS: Macroscopic assessments were insensitive to AC softening suggesting that arthroscopic assessments of AC status might also perform poorly. Collagen integrity was more important for the maintenance of AC stiffness (G*) than proteoglycan content. The development of major AC softening and thickening throughout the joint following MMx suggested involvement of non-mechanical (e.g., protein and biochemical) chemical and cytokine mediated processes in addition to the disturbance in biomechanical loading. The ovine MMx model provides a setting in which the spectrum of AC changes associated with the initiation and progression of OA may be evaluated.

Topical administration of the nitric oxide donor glyceryl trinitrate modifies the structural and biomechanical properties of ovine articular cartilage.

OBJECTIVE: To examine the effect of topical administration of glyceryl trinitrate (GTN), an exogenous nitric oxide (NO) donor, on the structural and biomechanical properties of uncalcified articular cartilage (UCC) in aged ewes. DESIGN: Twelve ewes were used for this study. Six of these were treated with 2% GTN ointment (0.7 mg/kg) twice per week (GTN), and the remaining six were used as normal controls (NOC). After sacrifice at 26 weeks, dynamic biomechanical indentation testing and thickness determination (by needle penetration) were performed on tibial plateau articular cartilage at 18 locations. Using histological sections prepared from the lateral and medial femoral condyles (LFC, MFC) and tibial plateau (LTP, MTP), the thickness of UCC, cartilage proteoglycan content (intensity of toluidine blue staining; LFC, MFC only), and collagen birefringence (LTP, MTP, LFC only) were quantified by computer-assisted image analysis. RESULTS: Phase lag of tibial plateau cartilage was reduced in GTN sheep relative to NOC (mean of all testing locations 11.0+/-1.9 degrees vs 12.1+/-2.3 degrees; P=0.0001). GTN treatment also globally reduced UCC thickness across the joint (ANOVA for all measured zones, P<0.0001). UCC thinning was most pronounced in the MFC (P=0.025) and LTP (P=0.0002). Proteoglycan content was reduced in the MFC(P=0.019), while collagen birefringence was increased in superficial cartilage zones of the LTP. CONCLUSIONS: NO donation via topical administration of GTN to normal ewes reduced the thickness and phase lag of femoro-tibial articular cartilage, suggesting a disturbance in chondrocyte metabolism. Regional alterations of collagen organisation and proteoglycan content were consistent with this interpretation.

Addition of nitric oxide via nitroflurbiprofen enhances the material properties of early healing of young rat Achilles tendons.

OBJECTIVE AND DESIGN: To determine if the addition of nitric oxide (NO) via nitroflurbiprofen (NO-flurbiprofen) would enhance rat Achilles tendon healing. MATERIALS AND METHODS: Sixty-five male Sprague-Dawley rats were randomly divided into NO-flurbiprofen, flurbiprofen and vehicle groups, given drugs or vehicle subcutaneously, and their right Achilles tendon divided. Histological assessment was carried out at day 5, 10, and 15 post-operation. Healing tendon biomechanical properties and hydroxyproline content were measured at day 10. RESULTS: The healing Achilles tendon from the NO-flurbiprofen and flurbiprofen groups showed a better organization of extracellular collagenous matrix than that from the vehicle group. Flurbiprofen and NO-flurbiprofen decreased healing tendon cross-sectional area by 30% and 20%. This reduction was accompanied by a decreased failure load in the flurbiprofen group, but not the NO-flurbiprofen group. NO-flubiprofen prevented the reduction of body weight gain observed in the flubiprofen group. CONCLUSION: Both flurbiprofen and NO-flurbiprofen promoted better collagen reorganization during tendon healing. NO-flurbiprofen further improved tendon healing by increasing tendon stress and reducing the side effects (body weight loss) of flurbiprofen. The enhanced tendon healing by NO-flurbiprofen is likely due to the release of NO from the compound.

Topographical analysis of the structural, biochemical and dynamic biomechanical properties of cartilage in an ovine model of osteoarthritis.

OBJECTIVE: The relationship between the topographical variations in the structural, biochemical and dynamic biomechanical properties of articular cartilage (AC) before and 6 months after meniscectomy has not been previously reported but is clearly relevant to our understanding of the role of mechanical factors on the pathogenesis of osteoarthritis (OA). The objective of this study was to address this deficiency using an ovine model of OA induced by bilateral lateral meniscectomy. DESIGN: The dynamic effective shear modulus (G*) and phase lag were determined ex vivo at 26 individual locations over the medial and lateral tibial plateaux of non-operated and meniscectomized ovine joints 6 months after surgery using a novel hand-held dynamic indentation probe. AC thickness was measured with a needle penetration probe. The AC from the same topographical locations as indented was then analysed for sulfated glycosaminoglycans (S-GAG) as a measure of proteoglycan (PG) levels, collagen and water content. Histological evaluation of the collagen organization using quantitative analysis of birefringence intensity was performed on stained tissue sections from the same topographical locations of each animal. RESULTS: It was demonstrated that the AC of the entire lateral tibial compartment of the meniscectomized joints underwent significant local degenerative and compensatory changes as indicated by a decreased G* and an increase in phase lag and water content. This was accompanied by a decrease in PG content of the AC of the middle and inner regions. While the AC of the outer region of the lateral meniscectomized compartment showed a marked increase in PG content and a more than two-fold increase in thickness, these tissues were also found to be structurally inferior, as indicated by a decreased G* and abnormal collagen birefringence intensity. The AC thickness was elevated at all locations of the lateral and medial tibial plateau of the meniscectomized joints. Strong and significant correlations between the biomechanical and biochemical data were established for a number of the parameters examined, especially between collagen content and G*, collagen content and AC thickness, and G* and AC thickness. An inverse correlation between S-GAG content and G* was only apparent in non-operated control tissues, whereas correlations between collagen and water content, water content and G*, and water content and thickness were evident for AC of the meniscectomized tibial plateaux. Less striking changes were noted in the medial compartment where the intact meniscus remained in place. However, elevated PG content, thicker AC together with slight changes in G* suggested an early hypertrophic response in these tissues. CONCLUSION: This study has highlighted the variable response of AC in different topographical regions of meniscectomized joints to the altered mechanical stresses introduced by this surgical procedure. The AC at the joint margins, while thicker and richer in PG, was found to be biomechanically softer (lower shear modulus) than normal AC, and because of this, would be expected to undergo degenerative changes with time leading to the onset of OA.

The accuracy and reliability of a novel handheld dynamic indentation probe for analysing articular cartilage.

This study investigates the accuracy and reliability of a novel handheld indentation system designed to ascertain the dynamic biomechanical properties of articular cartilage. A series of standard elastomers were assessed with both the handheld indentation system and a bench-top dynamic indentation system to assess the accuracy of the instrument. Interoperator and intraoperator experiments were undertaken to investigate the reliability of the system when used by an individual operator and by five different operators. Intraclass coefficients (Rho) were derived using a random effects model. The system was then used to ascertain the topographical variation in the shear moduli and phase lag of articular cartilage across normal ovine tibial plateaux. The system was shown to be highly accurate (R2 = 0.97), and had excellent reliability when measuring the dynamic shear modulus of articular cartilage (interoperator Rho = 0.75, intraoperator Rho = 0.79). Measurement of static shear modulus was less reliable (interoperator Rho = 0.15, intraoperator Rho = 0.52), but may be improved by monitoring the load applied to the instrument by the operator. The instrument was used to differentiate between different regions of cartilage and generated a topographical map of an ovine tibial plateau. The cartilage located beneath the menisci was 200-500% stiffer than the cartilage that was not covered by the menisci, while the phase lag was almost constant (10+/-2 SD) over the entire tibial plateau. The system was shown to be an accurate and reliable tool for rapidly assessing the dynamic biomechanical properties of articular cartilage, while being small enough to be used arthroscopically.

Biomechanical, histological and immunohistological studies of patellar cartilage in an ovine model of osteoarthritis induced by lateral meniscectomy.

OBJECTIVE: To evaluate the biomechanical, histological and immunohistochemical changes induced in patellar articular cartilage (AC) in ovine stifle joints 3 months after bilateral lateral meniscectomy, a procedure known to induce experimental osteoarthritis (OA) in the femoro-tibial joint (FTJ). METHODOLOGY: Fifteen mature adult Merino female sheep were used in this study. Ten were subjected to bilateral-lateral meniscectomy, while the remaining five were used as 'non-operated controls' (NOC). All animals were killed 3 months post-surgery. Topographical biomechanical indentation tests were performed on each patellae using a UMIS-2000 micro-indentation system. Initial load, relaxed and unload shear moduli were determined using an elastic analytical model, while the permeability was assessed by comparing the indentation response to a simulated indentation test conducted using a poroelastic finite element model. Immunohistochemical, normal and polarized histological studies were performed on each specimen after biomechanical testing. RESULTS: Patellar AC from meniscectomized joints exhibited an overall decrease in initial (-34%), relaxed (-32%) and unload shear modulus (-22%), and an increase in the permeability (+72%) relative to NOC cartilage (P< 0.01). The most significant differences in mechanical properties occurred on the lateral and central aspects of the patellae. There were no significant histological difference in staining between sections from NOC and meniscectomized joint AC using Toluidine Blue, a dye which binds to proteoglycans. However immunohistochemical staining with monoclonal antibody MAb 3B3(-), a putative marker of early OA change in PGs, demonstrated increased binding in the lateral and central regions of patellar sections from meniscectomized joints relative to the same regions of NOC AC. Moreover polarized light microscopy of Picro Sirius red stained sections revealed a significant decrease in birefringence intensity in the superficial-middle zones of the lateral and central regions of the patellar cartilage derived from the meniscectomized joints. CONCLUSION: This study has demonstrated that lateral meniscectomy is a procedure which was known to induce classical OA like changes in AC and subchondral bone of the FTJ also produced an early pathological response in the patellar AC.