Osteoarthritis early-, mid- and late-stage progression in the rat medial meniscus transection model.

Osteoarthritis is a degenerative disease of synovial joints affecting all tissues, including articular cartilage and subchondral bone. Osteoarthritis animal models can recapitulate aspects of human disease progression and are used to test efficacy of drugs, biomaterials, and cell therapies. The rat medial meniscus transection (MMT) model is a surgically induced posttraumatic osteoarthritis model commonly used for preclinical therapeutic screening. We describe herein, the qualitative and quantitative changes to articular cartilage, subchondral bone, and formation of osteophytes at early-, mid-, and late-stages of osteoarthritis progression. Tibia of MMT-operated animals showed proteoglycan loss and fibrillation along articular cartilage surfaces as early as 3-weeks post-surgery. With contrast-enhanced micro-CT technique, quantitative, 3-dimensional analysis of the tibia showed that the articular cartilage thickened at 3- and 6-weeks post-surgery and decreased at 12-weeks post-surgery. This decreased cartilage thickness corresponded with increased lesions in the articular cartilage that led to its full degradation and exposing the subchondral bone layer. Further, subchondral bone thickening was significant at 6-weeks post-surgery and followed cartilage damage. Osteophytes were found as early as 3-weeks post-surgery and coincided with articular cartilage degradation. Cartilaginous osteophytes preceded mineralization, suggesting endochondral ossification. The rat MMT model has predominantly been used out to 3-weeks, and most studies determined the effect of therapies to delay or prevent the onset of osteoarthritis. We provide evidence that an extension of the rat MMT model out to 6- and 12-weeks more resembled severe phenotypes of human osteoarthritis. Thus, evaluating novel therapeutics at late-stage will be important for eventual clinical translation.

Early signs of osteoarthritis in differing rat osteochondral defects.

Preclinical models of osteochondral defects (OCDs) are fundamental test beds to evaluate treatment modalities before clinical translation. To increase the rigor and reproducibility of translational science for a robust "go or no-go," we evaluated disease progression and pain phenotypes within the whole joint for two OCD rat models with same defect size (1.5 x 0.8 mm) placed either in the trochlea or medial condyle of femur. Remarkably, we only found subtle transitory changes to gaits of rats with trochlear defect without any discernible effect to allodynia. At 8-weeks post-surgery, anatomical evaluations of joint showed early signs of osteoarthritis with EPIC-microCT. For the trochlear defect, cartilage attenuation was increased in trochlear, medial, and lateral compartments of the femur. For condylar defect, increased cartilage attenuation was isolated to the medial condyle of the femur. Further, the medial ossicle showed signs of deterioration as indicated with decreased bone mineral density and increased bone surface area to volume ratio. Thus, OCD in a weight-bearing region of the femur gave rise to more advanced osteoarthritis phenotype within a unilateral joint compartment. Subchondral bone remodeling was evident in both models without any indication of closure of the articular cartilage surface. We conclude that rat OCD, placed in the trochlear or condylar region of the femur, leads to differing severity of osteoarthritis progression. As found herein, repair of the defect with fibrous tissue and subchondral bone is insufficient to alleviate onset of osteoarthritis. Future therapies using rat OCD model should address joint osteoarthritis in addition to repair itself.

Improved Cartilage Protection with Low Molecular Weight Hyaluronic Acid Hydrogel.

Traumatic joint injuries are common, leading to progressive tissue degeneration and the development of osteoarthritis. The post-traumatic joint experiences a pro-inflammatory milieu, initiating a subtle but deteriorative process in cartilage tissue. To prevent or even reverse this process, our group previously developed a tissue-penetrating methacrylated hyaluronic acid (MeHA) hydrogel system, crosslinked within cartilage to restore and/or protect the tissue. In the current study, we further optimized this approach by investigating the impact of biomaterial molecular weight (MW; 20, 75, 100 kDa) on its integration within and reinforcement of cartilage, as well as its ability to protect tissue degradation in a catabolic state. Indeed, the low MW MeHA integrated and reinforced cartilage tissue better than the high MW counterparts. Furthermore, in a 2 week IL-1ß explant culture model, the 20 kDa MeHA demonstrated the most protection from biphasic mechanical loss, best retention of proteoglycans (Safranin O staining), and least aggrecan breakdown (NITEGE). Thus, the lower MW MeHA gels integrated better into the tissue and provided the greatest protection of the cartilage matrix. Future work will test this formulation in a preclinical model, with the goal of translating this therapeutic approach for cartilage preservation.

Effect of Human Synovial Fluid From Osteoarthritis Patients and Healthy Individuals on Lymphatic Contractile Activity.

The lymphatic system has been proposed to play a crucial role in preventing the development and progression of osteoarthritis (OA). As OA develops and progresses, inflammatory cytokines and degradation by-products of joint tissues build up in the synovial fluid (SF) providing a feedback system to exacerbate disease. The lymphatic system plays a critical role in resolving inflammation and maintaining overall joint homeostasis; however, there is some evidence that the lymphatics can become dysfunctional during OA. We hypothesized that the functional mechanics of lymphatic vessels (LVs) draining the joint could be directly compromised due to factors within SF derived from osteoarthritis patients (OASF). Here, we utilized OASF and SF derived from healthy (non-OA) individuals (healthy SF (HSF)) to investigate potential effects of SF entering the draining lymph on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractile activity of rat femoral LVs (RFLVs) ex vivo. Dilutions of both OASF and HSF containing serum resulted in a similar LEC migratory response to the physiologically endothelial basal medium-treated LECs (endothelial basal medium containing serum) in vitro. Ex vivo, OASF and HSF treatments were administered within the lumen of isolated LVs under controlled pressures. OASF treatment transiently enhanced the RFLVs tonic contractions while phasic contractions were significantly reduced after 1 h of treatment and complete ceased after overnight treatment. HSF treatment on the other hand displayed a gradual decrease in lymphatic contractile activity (both tonic and phasic contractions). The observed variations after SF treatments suggest that the pump function of lymphatic vessel draining the joint could be directly compromised in OA and thus might present a new therapeutic target.

Sodium alginate microencapsulation of human mesenchymal stromal cells modulates paracrine signaling response and enhances efficacy for treatment of established osteoarthritis.

Mesenchymal stromal cells (MSCs) have shown promise as osteoarthritis (OA) treatments; however, effective translation has been limited by high variability and heterogeneity of MSCs, suboptimal delivery strategies, and poor understanding of critical quality and potency attributes. Furthermore, most pre-clinical studies of MSC therapeutics for OA have focused on delaying OA development and not on treating established OA, which brings added clinical relevance. Thus, the objective of the current study was to assess the effects of sodium alginate microencapsulation on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. A Medial Meniscal Transection (MMT) pre-clinical model of OA was implemented. Three weeks post-surgery, after OA was established, intra-articular injections of encapsulated hMSCs or nonencapsulated hMSCs were administered. Six weeks post-surgery, microstructural changes in the knee joint were quantified using microCT. Encapsulated hMSCs reduced articular cartilage degeneration and subchondral bone remodeling. A multiplexed immunoassay panel was used to profile the in vitro secretome of hMSCs in response to IL-1ß. Nonencapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1ß while encapsulated hMSCs showed a targeted secretory response with increased expression of pro-inflammatory (IL-1ß, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that sodium alginate microencapsulation can modulate hMSC paracrine signaling and enhance the therapeutic efficacy of the hMSCs in treating established OA. This cytokine profile provides a foundation for the identification of key factors affecting the overall potency of hMSC therapeutics for OA. STATEMENT OF SIGNIFICANCE: While there has been considerable interest in material based MSC encapsulation for treatment of OA, there are critical gaps in our translational understanding of these biomaterial-based technologies for OA. More specifically, previous studies have several important limitations: (1) they have been largely focused on preventing OA development, which limits their translational utility and (2) little prior work has been done to delineate potential routes/mechanisms by which material encapsulation alters MSC therapeutic action. In our manuscript, we aimed to fill these gaps in knowledge by testing the hypotheses that: (1) hMSC encapsulation can attenuate established disease progression, which is a more clinically relevant scenario and (2) hMSC encapsulation significantly changes the secreted paracrine factors from hMSCs.

Multichromatic near-infrared imaging to assess interstitial lymphatic and venous uptake in vivo.

SIGNIFICANCE: Changes in interstitial fluid clearance are implicated in many diseases. Using near-infrared (NIR) imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies. AIM: We investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces. APPROACH: We used a chemically inert IR Dye 800CW (D800) to target venous capillaries and a purified conjugate of IR dye 680RD with 40 kDa PEG (P40D680) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging's utility in two in vivo tissue beds: the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise. RESULTS: In an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. In the mouse tail, co-injected NIR tracers cleared from the interstitial space via distinct routes, suggestive of lymphatic and venous uptake mechanisms. In the rat knee, we determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, whereas exercise pre-injection exhibited a transient delay in clearance from the joint. CONCLUSIONS: NIR imaging enables simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure temporal changes in clearance rates and pathways.

Endothelin-1 inhibits size dependent lymphatic clearance of PEG-based conjugates after intra-articular injection into the rat knee.

Clearance of particles from the knee is an essential mechanism to maintain healthy joint homeostasis and critical to the delivery of drugs and therapeutics. One of the limitations in developing disease modifying drugs for joint diseases, such as osteoarthritis (OA), has been poor local retention of the drugs. Enhancing drug retention within the joint has been a target of biomaterial development, however, a fundamental understanding of joint clearance pathways has not been characterized. We applied near-infrared (NIR) imaging techniques to assess size-dependent in vivo clearance mechanisms of intra-articular injected, fluorescently-labelled polyethylene glycol (PEG-NIR) conjugates. The clearance of 2 kDa PEG-NIR (τ = 171 ±â€¯11 min) was faster than 40 kDa PEG-NIR (τ = 243 ±â€¯16 min). 40 kDa PEG-NIR signal was found in lumbar lymph node while 2 kDa PEG-NIR signal was not. Thus, these two conjugates may be cleared through different pathways, i.e. lymphatics for 40 kDa PEG-NIR and venous for 2 kDa PEG-NIR. Endothelin-1 (ET-1), a potent vasoconstrictor of vessels, is elevated in synovial fluid of OA patients but, its effects on joint clearance are unknown. Intra-articular injection of ET-1 dose-dependently inhibited the clearance of both 2 kDa and 40 kDa PEG-NIR. ET-1 caused a 1.63 ±â€¯0.17-fold increase in peak fluorescence for 2 kDa PEG-NIR and a 1.85 ±â€¯0.15-fold increase for 40 kDa PEG-NIR; and ET-1 doubled their clearance time constants. The effects of ET-1 were blocked by co-injection of ET receptor antagonists, bosentan or BQ-123. These findings provide fundamental insight into retention and clearance mechanisms that should be considered in the development and delivery of drugs and biomaterial carriers for joint diseases. STATEMENT OF SIGNIFICANCE: This study demonstrates that in vivo knee clearance can be measured using NIR technology and that key factors, such as size of materials and biologics, can be investigated to define joint clearance mechanisms. Therapies targeting regulation of joint clearance may be an approach to treat joint diseases like osteoarthritis. Additionally, in vivo functional assessment of clearance may be used as diagnostics to monitor progression of joint diseases.

Automated, quantitative screening assay for antiangiogenic compounds using transgenic zebrafish.

Pathologic angiogenesis has emerged as an important therapeutic target in several major diseases. Zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. We developed the first quantitative, automated assay for antiangiogenic compound identification using zebrafish embryos. This assay uses transgenic zebrafish with fluorescent blood vessels to facilitate image analysis. We developed methods for automated drugging and imaging of zebrafish in 384-well plates and developed a custom algorithm to quantify the number of angiogenic blood vessels in zebrafish. The assay was used to screen the LOPAC1280 compound library for antiangiogenic compounds. Two known antiangiogenic compounds, SU4312 and AG1478, were identified as hits. Additionally, one compound with no previously known antiangiogenic activity, indirubin-3'-monoxime (IRO), was identified. We showed that each of the hit compounds had dose-dependent antiangiogenic activity in zebrafish. The IC(50) of SU4312, AG1478, and IRO in the zebrafish angiogenesis assay was 1.8, 8.5, and 0.31 micromol/L, respectively. IRO had the highest potency of the hit compounds. Moreover, IRO inhibited human umbilical vein endothelial cell tube formation and proliferation (IC(50) of 6.5 and 0.36 micromol/L, respectively). It is therefore the first antiangiogenic compound discovered initially in a zebrafish assay that also has demonstrable activity in human endothelial cell-based angiogenesis assays.

Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons.

Parkinson's disease is characterized by a severe loss of dopaminergic neurons resulting in a range of motor deficits. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to cause a similar loss of dopaminergic neurons in the human midbrain with corresponding Parkinsonian symptoms. Several animal species have also shown sensitivity to MPTP, including primates, mice, goldfish, and, most recently, zebrafish. This study demonstrates that the effect of MPTP on dopaminergic neurons in zebrafish larvae is mediated by the same pathways that have been demonstrated in mammalian species. MPTP-induced neurodegeneration was prevented by co-incubation with either the monoamine oxidase-B (MAO-B) inhibitor l-deprenyl or the dopamine transporter (DAT) inhibitor nomifensine. Furthermore, targeted inactivation of the DAT gene by antisense morpholinos also protected neurons from MPTP damage. Thus, the mechanism for MPTP-induced dopaminergic neuron toxicity in mammals is conserved in zebrafish larvae. Effects on swimming behavior and touch response that result from MPTP damage are partially ameliorated by both l-deprenyl and DAT knockdown.

Angiotensin II-induced hypertension in mice caused an increase in insulin secretion.

OBJECTIVE AND DESIGN: Insulin action was determined in a mouse model of human hypertension via chronic angiotensin II administration followed by a glucose tolerance test. METHODS: Angiotensin II or saline was infused systemically into mice via osmotic pump for 2 or 4 weeks. In angiotensin II-treated mice versus saline controls we compared blood pressure, blood glucose, and serum insulin concentrations during an intravenous glucose tolerance test and assessed glucose transport and insulin signaling in muscle. RESULTS: Blood pressure increased at 2 and 4 weeks following angiotensin II treatment. Mice treated with angiotensin II for 4 weeks cleared a glucose bolus faster than mice treated with saline despite similar basal serum insulin concentrations. Upon glucose administration, the increase in serum insulin was greater in angiotensin II-treated mice, 38.8+/-6.5 pmol/l, compared to saline-treated mice, 21.8+/-2.9 pmol/l, but only at 4 weeks of angiotensin II treatment while no difference was observed at 2 weeks of angiotensin II administration. At 4 weeks of angiotensin II treatment, insulin signaling in the liver and in the skeletal muscle was not affected, since both the number of insulin receptors and phosphorylation of Akt were unchanged. Also at 4 weeks of angiotensin II treatment, ex vivo soleus muscle did not exhibit any change in basal and insulin-stimulated glucose uptake. CONCLUSIONS: This study suggests that long-term angiotensin II treatment for 4 weeks enhances glucose-stimulated insulin secretion in mice. Angiotensin II-induced hyperinsulinemia may play a role in the development of insulin resistance in patients with hypertension.

Differential distribution and function of hyperpolarization-activated channels in sensory neurons and mechanosensitive fibers.

Sensory neurons express hyperpolarization-activated currents (I(H)) that differ in magnitude and kinetics within the populations. We investigated the structural basis for these differences and explored the functional role of the I(H) channels in sensory neurons isolated from rat nodose ganglia. Immunohistochemical studies demonstrated a differential distribution of hyperpolarization-activated cyclic nucleotide-gated (HCN) protein (HCN1, HCN2, HCN4) in sensory neurons and peripheral terminals. HCN2 and HCN4 immunoreactivity was present in all nodose neurons. In contrast, only 20% of the total population expressed HCN1 immunoreactivity. HCN1 did not colocalize with IB4 (a marker for C-type neurons), and only 15% of HCN1-positive neurons colocalized with immunoreactivity for the vanilloid receptor VR1, another protein associated primarily with C-type neurons. Therefore, most HCN1-containing neurons were A-type neurons. In further support, HCN1 was present in the mechanosensitive terminals of myelinated but not unmyelinated sensory fibers, whereas HCN2 and HCN4 were present in receptor terminals of both myelinated and unmyelinated fibers. In voltage-clamp studies, cell permeant cAMP analogs shifted the activation curve for I(H) to depolarized potentials in C-type neurons but not A-type neurons. In current-clamp recording, CsCl, which inhibits only I(H) in nodose neurons, hyperpolarized the resting membrane potential from -63 +/- 1 to -73 +/- 2 mV and nearly doubled the input resistance from 1.3 to 2.2 GOmega. In addition, action potentials were initiated at lower depolarizing current injections in the presence of CsCl. At the sensory receptor terminal, CsCl decreased the threshold pressure for initiation of mechanoreceptor discharge. Therefore, elimination of the I(H) increases excitability of both the soma and the peripheral sensory terminals.