Chitosan in viscosupplementation: in vivo effect on rabbit subchondral bone.

BACKGROUND: To investigate the effect of intra-articular injection of Chitosan (Cs) added to hyaluronic acid (HA) on subchondral bone during osteoarthritis (OA), microarchitectural parameters and mineral density were measured in a rabbit model of early OA. A novel hybrid hydrogel adding reacetylated Cs of fungal origin to HA was compared to high molecular weight HA commercial formulation. METHOD: Eighteen rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into three groups (Saline-group, HA-group and Hybrid-group) depending on the intra-articular injection compound. Eight contralateral knees were used as non-operated controls (Contralateral-group). Micro-computed tomography was performed six weeks post-ACLT to study subchondral bone microarchitectural parameters and mineral density at an early stage of OA development. RESULTS: Cartilage thickness mean value was reduced only in Saline-group compared to Contralateral-group. When the Hybrid-group was compared to Saline-group, subchondral bone microarchitectural parameters (trabecular thickness and trabecular bone volume fraction) were significantly changed; subchondral bone plate and trabecular bone mineral densities (bone mineral density and tissue mineral density) were reduced. When the Hybrid-group was compared to HA-group, subchondral bone microarchitectural parameters (subchondral plate thickness and trabecular thickness) and trabecular bone mineral densities (bone mineral density and tissue mineral density) were significantly decreased. CONCLUSION: Conclusion: Compared to HA alone, the novel hybrid hydrogel, constituted of Cs added to HA, enhanced microarchitectural parameters and mineral density changes, leading to subchondral bone loss in a rabbit model of early experimental OA.

Effects of a viscosupplementation therapy on rabbit menisci in an anterior cruciate ligament transection model of osteoarthritis.

The aim of this study was to evaluate the morphological, microstructural, and mechanical effects of a viscosupplementation therapy on rabbit menisci at an early stage of osteoarthritis (OA). Anterior cruciate ligament transection (ACLT) was performed in twelve male New-Zealand White rabbits on the right knee joint. Six of these twelve rabbits received a mono intra-articular injection of high molecular weight hyaluronic acid (HA) two weeks after ACLT. Six additional healthy rabbits served as controls. Medial menisci were removed from all right knees (n=18) six weeks after ACLT and were graded macroscopically. Indentation-relaxation tests were performed in the anterior and posterior regions of the menisci. Collagen fiber organization and glycosaminoglycan (GAG) content were assessed by biphotonic confocal microscopy and histology, respectively. Viscosupplementation significantly (p=0.002) improved the surface integrity of the medial menisci compared to the operated non-treated group. Moreover, the injection seems to have an effect on the GAG distribution in the anterior region of the menisci. However, the viscoelastic properties of both operated groups were similar and significantly lower than those of the healthy group, which was explained by their modified collagen fiber organization. They displayed disruption of the tie fibers due to structural alterations of the superficial layers from which they emanate, leading to modifications in the deep zone. To conclude, the viscosupplementation therapy prevents macroscopic lesions of the menisci, but it fails to restore their collagen fiber organization and their viscoelastic properties. This finding supports the role of this treatment in improving the lubrication over the knee.

Viscoelastic properties of rabbit osteoarthritic menisci: A correlation with matrix alterations.

The aim of this study was to evaluate the effect of early osteoarthritis (OA) on the viscoelastic properties of rabbit menisci and to correlate the mechanical alterations with the microstructural changes. Anterior Cruciate Ligament Transection (ACLT) was performed in six male New-Zealand White rabbits on the right knee joint. Six healthy rabbits served as controls. Menisci were removed six weeks after ACLT and were graded macroscopically. Indentation-relaxation tests were performed in the anterior and posterior regions of the medial menisci. The collagen fibre organization and glycosaminoglycan (GAG) content were assessed by biphotonic confocal microscopy and histology, respectively. OA menisci displayed severe macroscopic lesions compared with healthy menisci (p=0.009). Moreover, the instantaneous and equilibrium moduli, which were 2.9±1.0MPa and 0.60±0.18MPa in the anterior region of healthy menisci, respectively, decreased significantly (p=0.03 and p=0.004, respectively) in OA menisci by 55% and 57%, respectively, indicating a global decrease in meniscal stiffness in this region. The equilibrium modulus alone decreased significantly (p=0.04) in the posterior region, going from 0.60±0.18MPa to 0.26±012MPa. This induced a loss of tissue elasticity. These mechanical changes were associated in the posterior region with a structural disruption of the superficial layers, from which the tie fibres emanate, and with a decrease in the GAG content in the anterior region. Consequently, the circumferential collagen fibres of the deep zone were dissociated and the collagen bundles were less compact. Our results demonstrate the strong meniscal modifications induced by ACLT at an early stage of OA and highlight the relationship between structural and chemical matrix alterations and mechanical properties.

Meniscal biomechanical alterations in an ACLT rabbit model of early osteoarthritis.

OBJECTIVE: The purpose of this study was to analyze the early biomechanical alterations of menisci during the early stage of osteoarthritis (OA) development and to correlate them with the chemical composition and matrix alteration. A particular focus was paid to pathological changes in glycosaminoglycan (GAG) content and collagen fiber architecture. DESIGN: Menisci (n = 24) were removed from rabbits' knee joints 6 weeks following surgical anterior cruciate ligament transection (ACLT). Both the anterior and posterior regions of medial and lateral menisci were characterized using indentation tests, Raman microspectroscopy (RM), biphotonic confocal microscopy (BCM) and histology. RESULTS: Mechanical and matrix alterations occurred in both regions of medial and lateral menisci. A significant decrease in the mechanical properties was observed in OA menisci, with a mean reduced modulus from 2.3 to 1.1 MPa. Microstructural observations revealed less organized and less compact collagen bundles in operated menisci than in contralateral menisci, as well as a loss of fiber tension. GAG content was increased in OA menisci, especially in the damaged areas. Neither changes in the secondary structure of collagen nor mineralization were detected through RM at this stage of OA. CONCLUSION: ACLT led to a disorganization of the collagen framework at the early stage of OA development, which decreases the mechanical resistance of the menisci. GAG content increases in response to this degradation. All of these results demonstrate the strong correlation between matrix and mechanical alterations.

A novel biocompatible hyaluronic acid-chitosan hybrid hydrogel for osteoarthrosis therapy.

A conventional therapy for the treatment of osteoarthrosis is intra-articular injection of hyaluronic acid, which requires repeated, frequent injections. To extend the viscosupplementation effect of hyaluronic acid, we propose to associate it with another biopolymer in the form of a hybrid hydrogel. Chitosan was chosen because of its structural similarity to synovial glycosaminoglycans, its anti-inflammatory effects and its ability to promote cartilage growth. To avoid polyelectrolyte aggregation and obtain transparent, homogeneous gels, chitosan was reacetylated to a 50% degree, and different salts and formulation buffers were investigated. The biocompatibility of the hybrid gels was tested in vitro on human arthrosic synoviocytes, and in vivo assessments were made 1 week after subcutaneous injection in rats and 1 month after intra-articular injection in rabbits. Hyaluronic acid-chitosan polyelectrolyte complexes were prevented by cationic complexation of the negative charges of hyaluronic acid. The different salts tested were found to alter the viscosity and thermal degradation of the gels. Good biocompatibility was observed in rats, although the calcium-containing formulation induced calcium deposits after 1 week. The sodium chloride formulation was further tested in rabbits and did not show acute clinical signs of pain or inflammation. Hybrid HA-Cs hydrogels may be a valuable alternative viscosupplementation agent.

A novel oxido-viscosifying Hyaluronic Acid-antioxidant conjugate for osteoarthritis therapy: biocompatibility assessments.

To overcome the problem of fast degradation of Hyaluronic Acid (HA) in the treatment of osteoarthritis (OA), HA was protected against the oxidative stress generated by the pathology. Antioxidant conjugated HAs were synthesized and tested in vitro for their resistance in an oxidative environment mimicking OA. HA-4-aminoresorcinol (HA-4AR) displayed the interesting property of increasing in viscosity under oxidative conditions because of crosslinking induced by electron transfer. The novel HA polymer conjugate was shown to be biocompatible in vitro on fibroblast-like synoviocytes extracted from an arthritic patient. This HA conjugate was also assessed in vivo by intra-articular injection in healthy rabbits and was found to be comparable to the native polymer in terms of biocompatibility. This study suggests that HA-4AR is a promising candidate for a next generation viscosupplementation formulation.

Intra-articular administration of xenogeneic neonatal Mesenchymal Stromal Cells early after meniscal injury down-regulates metalloproteinase gene expression in synovium and prevents cartilage degradation in a rabbit model of osteoarthritis.

OBJECTIVE: The anti-inflammatory and anti-catabolic effects of neonatal Mesenchymal Stromal Cell (MSC) were investigated in a xenogeneic model of mild osteoarthritis (OA). The paracrine properties of MSC on synoviocytes were further investigated in vitro. STUDY DESIGN: OA was induced by medial meniscal release (MMR) in 30 rabbit knees. A single early (day 3) or delayed (day 15) intra-articular (IA) injection of MSC isolated from equine Umbilical Cord Wharton's jelly (UC-MSC) was performed. Rabbits were euthanized on days 15 or 56. OA grading was performed and gene expression of inflammatory cytokines and metalloproteinases was measured in synovial tissue. Paracrine effects of UC-MSC were investigated using UC-conditioned vs control medium on rabbit primary synoviocytes stimulated with interleukin 1 beta in vitro. RESULTS: No adverse local or systemic responses were observed clinically after xenogeneic UC-MSC injection. At study end point, cartilage fibrillation was lower in early treatment than in delayed treatment group. Cellular infiltrate was observed in the synovium of both UC-MSC groups. OA synovium exhibited a reduced expression of metalloproteinases-1, -3, -13 in the early cell-treated group at d56. In vitro, UC-conditioned medium exerted anti-inflammatory and anti-catabolic effects on synoviocytes exposed to pro-inflammatory stimulus. CONCLUSIONS: Early IA injection of equine UC-MSC was effective in preventing OA signs in rabbit knees following MMR. UC-MSC target the synovium and modulate the gene expression pattern of synoviocytes to promote an anti-catabolic environment. This confirms the synovium is a major target and mediator of MSC therapy, modulating the expression of matrix-degrading enzymes.

Ultrasonograph and clinical quantitative characterization of tendinopathy by modified splitting in a goat model.

A tendinopathy is a clinical condition characterized by activity-related pain, focal tendons tenderness, and intratendinous imaging changes. This study characterizes a surgically induced tendinopathy in a goat model with a noninvasive in vivo longitudinal followup based on physical examination and US. Cross-sectional area (CSA) is the most objective feature for the evaluation of tendinopathy in correlation with clinical findings. The deep digital flexor tendon (DDFT) of the left hind limb of six goats was isolated and scarified by a modified splitting. Pain and lameness at walk and trot were evaluated. External width and thickness of tendon region were measured by calipers. CSA and the ratio lesion/tendon CSA were obtained at days 0, 7, 21, 42, and 84 by US. The highest value of global functional score was obtained at day 7, then decreased until day 40 and was not significantly different from day 0 at the end of the study. The external width recovered a normal value at the end of the study, but the external thickness was still significantly increased (P < 0.05). Peritendinous oedema was observed at day 7, but intratendinous lesions were visible only at day 21 as a focal hypo to anechoic area. At day 84, two tendons still presented visible lesions. US examination was reproducible, specific, and provided complementary information to the global functional score. A standardized focal tendinopathy was induced in goats. This experimental model of focal tendinopathy could be used to study the effect of different treatments.

Knee joint ultrasonography of the ACLT rabbit experimental model of osteoarthritis: relevance and effectiveness in detecting meniscal lesions.

OBJECTIVES: To develop a protocol for rabbit knee joint ultrasonography (US); to grade ultrasonographically the meniscal injuries of the anterior cruciate ligament transection (ACLT) rabbit model of osteoarthritis (OA); to assess with US the effectiveness of the ACLT; to compare final US with macroscopy for the evaluation of medial and lateral meniscal injuries depending on the age and weight when ACLT is performed. METHODS: Twenty-two skeletally mature and adolescent New Zealand white rabbits were housed during the same period at the Institut Claude-Bourgelat, Lyon, France. Surgical ACLT was performed in the left knee of nine adolescent and five adult rabbits. Final US and macroscopic semi-quantitative grading of the meniscal injuries were compared 5 months after ACLT. RESULTS: A standardised protocol was developed to evaluate the rabbit knee joint. US was performed in both control and ACLT knees. Normal and abnormal meniscal US appearances were described. A semi-quantitative scale to grade US meniscal injuries was created. Macroscopic and US total meniscal scores were significantly positively correlated (P<0.001, r=0.70). US detection of meniscal injuries was 92% sensitive and 87.5% specific compared to macroscopy. Positive and negative predictive values of US were, respectively, 92% and 87.5%. US detection of the ACLT effectiveness was 100% specific and 78.5% sensitive. CONCLUSION: A significant relationship was found between ultrasonographic and macroscopic grading of meniscal injuries. US was both specific and sensitive in detecting meniscal lesions. We propose US as a non-invasive, non-expensive, in vivo imaging technique for preclinical studies in the ACLT rabbit OA model.

Non-invasive in vivo quantification of the medial tibial cartilage thickness progression in an osteoarthritis rabbit model with quantitative 3D high resolution micro-MRI.

OBJECTIVE: To develop a quantitative non-invasive in vivo three-dimensional (3D) high resolution (HR) micro-magnetic resonance imaging (microMRI) protocol to measure the medial tibial cartilage thickness (MT.ThC) in the normal rabbit and in the anterior cruciate ligament transection (ACLT) rabbit model of osteoarthritis and quantify the progression of MT.ThC. METHODS: The left knee of 10 control and 40 operated rabbits was imaged in vivo with a 7T microMRI system at 3 and 5 months after ACLT. A 3D fast low angle short (FLASH) fat-suppressed MRI protocol was implemented leading to 44x176 microm(3) spatial resolution and to 44 microm(3) isotropic voxel after cubic interpolation. Semi-automatic MT.ThC measurements were made in 3D, in four different locations, in vivo and longitudinally in both groups. At 5 months, gross macroscopy, visual analogical evaluation of the cartilage and histology were compared to the MR-based MT.ThC. RESULTS: At 3 and 5 months, the MT.ThC measured in the minimum interbone distance area was the thinnest MR-based MT.ThC. It was significantly lower in the operated group and among the four evaluated MT.ThC, it was the most discriminative between the normal and the operated groups (P<0.05). The MT.ThC measured in the minimum interbone distance area was also the most sensitive to change in the operated group (66.4% MT.ThC loss, P=0.003) while no significant changes were observed in the control group. CONCLUSION: Quantitative 3D HR microMRI allowed for non-invasive longitudinal MT.ThC measurements in four different locations in both the normal and the operated rabbits. We concluded the MT.ThC measured in the minimum interbone distance area reflected the severity of the disease and was the most effective to measure the progression of the medial tibial cartilage destruction.