Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

Recombinant human FGF18 preserves depth-dependent mechanical inhomogeneity in articular cartilage.

Articular cartilage is a specialised tissue that has a relatively homogenous endogenous cell population but a diverse extracellular matrix (ECM), with depth-dependent mechanical properties. Repair of this tissue remains an elusive clinical goal, with biological interventions preferred to arthroplasty in younger patients. Osteochondral transplantation (OCT) has emerged for the treatment of cartilage defects and osteoarthritis. Fresh allografts stored at 4 °C have been utilised, though matrix and cell viability loss remains an issue. To address this, several studies have developed media formulations to maintain cartilage explants in vitro. One promising factor for these applications is sprifermin, a human-recombinant fibroblast growth factor-18, which stimulates chondrocyte proliferation and matrix synthesis and is in clinical trials for the treatment of osteoarthritis. The study hypothesis was that addition of sprifermin during storage would maintain the unique depth-dependent mechanical profile of articular cartilage explants, a feature not often evaluated. Explants were maintained for up to 6 weeks with or without a weekly 24 h exposure to sprifermin (100 ng/mL) and the compressive modulus was assessed. Results showed that sprifermin-treated samples maintained their depth-dependent mechanical profile through 3 weeks, whereas untreated samples lost their mechanical integrity over 1 week of culture. Sprifermin also affected ECM balance by maintaining the levels of extracellular collagen and suppressing matrix metalloproteinase production. These findings support the use of sprifermin as a medium additive for OCT allografts during in vitro storage and present a potential mechanism where sprifermin may impact a functional characteristic of articular cartilage in repair strategies.

Measurement of the Q

We measured the g_{1} spin structure function of the deuteron at low Q^{2}, where QCD can be approximated with chiral perturbation theory (χPT). The data cover the resonance region, up to an invariant mass of W≈1.9 GeV. The generalized Gerasimov-Drell-Hearn sum, the moment Γ_{1}^{d} and the spin polarizability γ_{0}^{d} are precisely determined down to a minimum Q^{2} of 0.02 GeV^{2} for the first time, about 2.5 times lower than that of previous data. We compare them to several χPT calculations and models. These results are the first in a program of benchmark measurements of polarization observables in the χPT domain.

A large animal model that recapitulates the spectrum of human intervertebral disc degeneration.

OBJECTIVE: The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. DESIGN: Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (µCT), and measurement of disc biomechanical properties. RESULTS: Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. CONCLUSIONS: A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.

Towards a resolution of the proton form factor problem: new electron and positron scattering data.

There is a significant discrepancy between the values of the proton electric form factor, G(E)(p), extracted using unpolarized and polarized electron scattering. Calculations predict that small two-photon exchange (TPE) contributions can significantly affect the extraction of G(E)(p) from the unpolarized electron-proton cross sections. We determined the TPE contribution by measuring the ratio of positron-proton to electron-proton elastic scattering cross sections using a simultaneous, tertiary electron-positron beam incident on a liquid hydrogen target and detecting the scattered particles in the Jefferson Lab CLAS detector. This novel technique allowed us to cover a wide range in virtual photon polarization (ϵ) and momentum transfer (Q(2)) simultaneously, as well as to cancel luminosity-related systematic errors. The cross section ratio increases with decreasing ϵ at Q(2)=1.45 GeV(2). This measurement is consistent with the size of the form factor discrepancy at Q(2)≈1.75 GeV(2) and with hadronic calculations including nucleon and Δ intermediate states, which have been shown to resolve the discrepancy up to 2-3 GeV(2).

A high-throughput model of post-traumatic osteoarthritis using engineered cartilage tissue analogs.

OBJECTIVE: A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. METHOD: We utilized a high-throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. RESULTS: Engineered cartilage response to injury was strain dependent, with a 2-fold increase in glycosaminoglycan (GAG) loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in lactate dehydrogenase (LDH) release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor [Z-VAD-FMK (ZVF)] was effective at reducing cell death, while the amphiphilic polymer [Poloxamer 188 (P188)] and the free-radical scavenger [N-Acetyl-L-cysteine (NAC)] reduced GAG loss as compared to injury alone. CONCLUSIONS: The injury response in this engineered cartilage model replicated key features of the response of cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA.

Measurement of the neutron F2 structure function via spectator tagging with CLAS.

We report on the first measurement of the F(2) structure function of the neutron from the semi-inclusive scattering of electrons from deuterium, with low-momentum protons detected in the backward hemisphere. Restricting the momentum of the spectator protons to ≲100 MeV/c and their angles to ≳100° relative to the momentum transfer allows an interpretation of the process in terms of scattering from nearly on-shell neutrons. The F(2)(n) data collected cover the nucleon-resonance and deep-inelastic regions over a wide range of Bjorken x for 0.65

Tensor correlations measured in 3He(e,e' pp)n.

We have measured the 3He(e,e' pp)n reaction at an incident energy of 4.7 GeV over a wide kinematic range. We identified spectator correlated pp and pn nucleon pairs by using kinematic cuts and measured their relative and total momentum distributions. This is the first measurement of the ratio of pp to pn pairs as a function of pair total momentum p(tot). For pair relative momenta between 0.3 and 0.5 GeV/c, the ratio is very small at low p(tot) and rises to approximately 0.5 at large p(tot). This shows the dominance of tensor over central correlations at this relative momentum.

Measurement of single- and double-spin asymmetries in deep inelastic pion electroproduction with a longitudinally polarized target.

We report the first measurement of the transverse momentum dependence of double-spin asymmetries in semi-inclusive production of pions in deep-inelastic scattering off the longitudinally polarized proton. Data have been obtained using a polarized electron beam of 5.7 GeV with the CLAS detector at the Jefferson Lab (JLab). Modulations of single spin asymmetries over the azimuthal angle between lepton scattering and hadron production planes ϕ have been measured over a wide kinematic range in Bjorken x and virtual photon squared four-momentum Q2. A significant nonzero sin2ϕ single spin asymmetry was observed for the first time indicating strong spin-orbit correlations for transversely polarized quarks in the longitudinally polarized proton.

Measurement of the differential cross section for the reaction gamman-->pi- p from deuterium.

We report a measurement of the differential cross section for the gamman-->pi- p process from the CLAS detector at Jefferson Laboratory in Hall B for photon energies between 1.0 and 3.5 GeV and pion center-of-mass (c.m.) angles (thetac.m.) between 50 degrees and 115 degrees. We confirm a previous indication of a broad enhancement around a c.m. energy ([sqrt]s) of 2.1 GeV at thetac.m.=90 degrees in the scaled differential cross section s7dsigma/dt and a rapid falloff in a center-of-mass energy region of about 400 MeV following the enhancement. Our data show an angular dependence of this enhancement as the suggested scaling region is approached for thetac.m. from 70 degrees to 105 degrees.

Bayesian analysis of pentaquark signals from CLAS data.

We examine the results of two measurements by the CLAS collaboration, one of which claimed evidence for a Theta(+) pentaquark, while the other found no such evidence. The unique feature of these two experiments was that they were performed with the same experimental setup. Using a Bayesian analysis, we find that the results of the two experiments are in fact compatible with each other, but that the first measurement did not contain sufficient information to determine unambiguously the existence of a Theta(+). Further, we suggest a means by which the existence of a new candidate particle can be tested in a rigorous manner.

Search for the Theta+ pentaquark in the reaction gammad --> pK-K+n.

A search for the Theta+ in the reaction gammad --> pK-K+n was completed using the CLAS detector at Jefferson Lab. A study of the same reaction, published earlier, reported the observation of a narrow Theta+ resonance. The present experiment, with more than 30 times the integrated luminosity of our earlier measurement, does not show any evidence for a narrow pentaquark resonance. The angle-integrated upper limit on Theta+ production in the mass range of 1.52-1.56 GeV/c2 for the gammad --> pK-Theta+ reaction is 0.3 nb (95% C.L.). This upper limit depends on assumptions made for the mass and angular distribution of Theta+ production. Using Lambda(1520) production as an empirical measure of rescattering in the deuteron, the cross section upper limit for the elementary gamman --> K-Theta+ reaction is estimated to be a factor of 10 higher, i.e., approximately 3 nb (95% C.L.).

Measurement of two- and three-nucleon short-range correlation probabilities in nuclei.

The ratios of inclusive electron scattering cross sections of 4He, 12C, and 56Fe to 3He have been measured at 1 < xB <. At Q2 > 1.4 GeV2, the ratios exhibit two separate plateaus, at 1.5 < xB < 2 and at xB > 2.25. This pattern is predicted by models that include 2- and 3-nucleon short-range correlations (SRC). Relative to A = 3, the per-nucleon probabilities of 3-nucleon SRC are 2.3, 3.1, and 4.4 times larger for A = 4, 12, and 56. This is the first measurement of 3-nucleon SRC probabilities in nuclei.

Search for Theta+ (1540) Pentaquark in High-Statistics Measurement of gammap-->K0K+n at CLAS.

The exclusive reaction gammap-->K0K+n was studied in the photon energy range between 1.6 and 3.8 GeV searching for evidence of the exotic baryon Theta+ (1540)-->nK+. The decay to nK+requires the assignment of strangeness S=+1 to any observed resonance. Data were collected with the CLAS detector at the Thomas Jefferson National Accelerator Facility corresponding to an integrated luminosity of 70 pb-1. No evidence for the Theta+ pentaquark was found. Upper limits were set on the production cross section as function of center-of-mass angle and nK+ mass. The 95% C.L. upper limit on the total cross section for a narrow resonance at 1540 MeV was found to be 0.8 nb.

Quasifree pi0 and pi- electroproduction on 4He in the Delta-resonance region.

The reactions 4He(e, e' p3He)pi- and 4He(e, e' p3He)pi0 were studied simultaneously, and for the first time, in a large kinematical domain including the Delta-resonance region. This was achieved by detecting the recoiling 3He and 3H nuclei instead of the emitted pions. The dependences of the cross section on the recoil momentum p(rec), the invariant mass WpiN, and the direction thetapi,q' and phipi,q' of the produced pion, are globally well described by the results of (quasifree) distorted-wave impulse approximation calculations. However, in the Delta-resonance region there are clear discrepancies, which point to medium modifications of the Delta in 4He.

Experimental determination of the evolution of the Bjorken integral at low Q2.

We extract the Bjorken integral Gamma1(p-n) in the range 0.17 < Q2 < 1.10 GeV2 from inclusive scattering of polarized electrons by polarized protons, deuterons, and 3He, for the region in which the integral is dominated by nucleon resonances. These data bridge the domains of the hadronic and partonic descriptions of the nucleon. In combination with earlier measurements at higher Q2, we extract the nonsinglet twist-4 matrix element f2.

Measurement of the generalized polarizabilities of the proton in virtual Compton scattering at Q2=0.92 and 1.76 GeV2.

We report a virtual Compton scattering study of the proton at low c.m. energies. We have determined the structure functions P(LL)-P(TT)/epsilon and P(LT), and the electric and magnetic generalized polarizabilities (GPs) alpha(E)(Q2) and beta(M)(Q2) at momentum transfer Q(2)=0.92 and 1.76 GeV2. The electric GP shows a strong falloff with Q2, and its global behavior does not follow a simple dipole form. The magnetic GP shows a rise and then a falloff; this can be interpreted as the dominance of a long-distance diamagnetic pion cloud at low Q2, compensated at higher Q2 by a paramagnetic contribution from piN intermediate states.

Measurement of ep-->e' ppi+ pi- and baryon resonance analysis.

The cross section for the reaction ep-->e(')ppi(+)pi(-) was measured in the resonance region for 1.4

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes.

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.

Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes.

OBJECTIVE AND DESIGN: To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture. MATERIAL OR SUBJECTS: Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses. TREATMENT: Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10(-10) to 10(-4) M. METHODS: Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Northern blots of total RNA from the treated cells were probed with equine specific cDNA probes for a number of cartilage matrix-related genes. Zymography, Western blotting, and fluorography were also performed to study the effects on protein synthesis. RESULTS: The glucocorticoids, dexamethasone, triamcinolone, and prednisolone, markedly decreased MMP1, MMP3, MMP13, TIMPI, and ferritin steady-state mRNA levels. There were no qualitative differences seen among the tested corticosteroids although dexamethasone and triamcinolone appeared to be slightly more potent than prednisolone. The effects of the glucocorticoids on MMP transcription occurred consistently at lower doses than those required to similarly downregulate type II collagen and aggrecan. Link protein and fibronectin mRNA were increased by the glucocorticoids, and biglycan and decorin were minimally affected. Fluorography of [14-C] proline-labeled media demonstrated that the decrease in type II collagen transcription (mRNA levels) was paralleled at the protein level. Zymography and Western blotting confirmed the decrease in functional metalloproteinases found in chondrocyte cultures following glucocorticoid treatment. CONCLUSIONS: The effects of glucocorticoids are complex inasmuch as they differentially affect numerous genes involved in the composition of cartilage matrix and the degradation of that matrix. This study provides new insight into the effects of glucocorticoids on the regulation of extra-cellular matrix and matrix-related genes by demonstrating that low doses of glucocorticoids can inhibit the degradative metalloproteinases with minimal negative effects on the transcription of extracellular matrix genes.