Modulation by 17,20S(OH)2pD of Fibrosis-Related Mediators in Dermal Fibroblast Lines from Healthy Donors and from Patients with Systemic Sclerosis.

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-ß1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)2pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)2pD or 1,25(OH)2D3 (positive control) with/without TGF-ß1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-ß1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-ß1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

17,20S(OH)2pD Can Prevent the Development of Skin Fibrosis in the Bleomycin-Induced Scleroderma Mouse Model.

Systemic sclerosis (SSc; scleroderma) is a chronic fibrotic disease involving TGF-ß1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-ß1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-ß1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 µg/100 µL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 µg/kg for 21 days. The injected skin was biopsied; analyzed histologically; examined for total collagen by Sircol; and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

Anti-inflammatory role of TPCA-1 encapsulated nanosomes in porcine chondrocytes against TNF-α stimulation.

In this study, we evaluated the hypothesis that immunonanosomes carrying the drug [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) will help in reducing nuclear factor-kappaB (NF-κB)-associated inflammation in porcine chondrocytes against tumor necrosis factor-alpha (TNF-α)-induced stress. The nanosomes were tagged with monoclonal anti-type II collagen (MabCII) antibody to specifically target the exposed type II collagen in cartilage matrix. TPCA-1 at a concentration of 10 µM significantly reduced expression of the matrix-degrading enzyme, Matrix metalloproteinase-13 (MMP-13) and blocked the p65 nuclear translocation. In comparison to the TPCA-1 solution alone, the TPCA-1 nanosomes were found to be more effective in reducing the cellular toxicity, oxidative stress and inflammation in chondrocytes treated with TNF-α. In addition, TPCA-1 nanosomes were more effective in reducing the gene expression of hypoxia-inducible factor-2alpha (HIF-2α) that in turn is associated with the regulation of MMP-13 gene. TPCA-1 nanosomes significantly reduced expression of both these genes. The data also showed that TPCA-1 did not attenuate the down-regulated gene expression levels of anabolic genes aggrecan (ACAN) and collagen type II alpha (COL2A1). In conclusion, this study showed that TPCA-1 nanosomes carrying a dose of 10 µM TPCA-1 can effectively increase the survival of cultured porcine chondrocytes against TNF-α-induced stress. The findings of this study could be used to develop nanosome-based drug delivery systems (DDSs) for animal model of OA. Moreover, the approach presented here can be further utilized in other studies for targeted delivery of the drug of interest at a cellular level.

Cytoprotective role of vitamin E in porcine adipose-tissue-derived mesenchymal stem cells against hydrogen-peroxide-induced oxidative stress.

Survival of mesenchymal stem cells (MSCs) against oxidative stress and inflammation is vital for effective stem cell therapy. The reactive oxygen species (ROS) result in apoptosis and release of inflammatory mediators. Adipose-derived stem cells (ASCs) have shown promise for stem cell therapy owing to their anti-inflammatory and anti-oxidant activity. Previously, we showed the benefits of vitamin E against hydrogen peroxide (H2O2)-induced oxidative stress in rat bone marrow-derived MSCs. In this study, we aim to evaluate the effect of vitamin E treatment on porcine adipose-derived mesenchymal stem cells (pASCs) against H2O2-induced oxidative stress. The oxidative stress was induced by treating pASCs with 500 µM H2O2 with or without vitamin E. Viability of pASCs is enhanced after vitamin E treatment. In addition, reduced cellular toxicity, total NO level, PGE2 production and caspase-3 activity were observed after vitamin E treatment. Gene expression analysis of vitamin E-treated pASCs showed down-regulated expression for the genes associated with oxidative stress and apoptosis, viz., NOS2, Casp3, p53, BAX, MDM2, NFκB, HIF1α and VEGF-A genes. On the other hand, expression of anti-apoptotic and survival genes was up-regulated, viz., BCL2, BCL2L1 and MCL1. Furthermore, phosphorylation of Akt was attenuated following vitamin E treatment. The findings of this study may help in developing effective stem cell therapy for the diseases characterized by the oxidative stress and inflammation.

Decreased expression levels of Ifi genes is associated to the increased resistance to spontaneous arthritis disease in mice deficiency of IL-1RA.

BACKGROUND: The mouse strain BALB/c deficient in IL-1 receptor antagonist protein (Il-1ra) develops spontaneous arthritis disease (SAD) while the strain DBA/1 IL1rn (-/-) with the same deficiency does not. Previously, we mapped a QTL on chromosome 1 for SAD and then developed a congenic mouse strain BALB.D1-1(-/-) that contains the QTL genomic fragment associated with resistance from DBA/1(-/-) on a BALB/c(-/-) background. The congenic strain was relatively resistant to spontaneous arthritis and had delayed onset and reduced severity of disease. We obtained whole genome expression profiles from the spleen of the congenic strain BALB.D1-1(-/-) and four other strains, the wild type BALB/c, DBA/1 and the deficient DBA/1 IL1rn (-/-) and the BALB/c IL1rn (-/-). We then compared the similarities and differences between the congenic strain and the four parental strains. Here we report the selected potential causal genes based on differential expression levels as well as function of genes. RESULTS: There is a considerable number of genes that are differentially expressed between the congenic strain and the three parental strains, BALB/c, DBA/1, and DBA/1(-/-). However there only a few differentially expressed genes were identified by comparing the congenic strain and the BALB/c(-/-)strain. These differentially expressed genes are mainly from T-cell receptor beta chain (Tcrb) and interferon-activatable protein (Ifi) genes. These genes are also differentially expressed between congenic strain and BALB/c strains. However, their expression levels in the congenic strain are similar to that in DBA/1 and DBA/1(-/-). The expression level of Tcrb-j gene is positively associated with two genes of Ifi gene 200 cluster. CONCLUSIONS: Decreased expression levels of Ifi genes is associated to the increased resistance to spontaneous arthritis disease and with down regulation of expressions of Tcrb genes in the mouse congenic strain. Ifi genes may play an important role in the susceptibility to SAD in mice.

In Vivo Dual Fluorescence Imaging to Detect Joint Destruction.

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near-infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP-1 and MMP-13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro-orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune-reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis.

The Effects of Platelet-Rich Plasma on Halting the Progression in Porcine Intervertebral Disc Degeneration.

Disc degeneration and the subsequent herniation and/or rupture of the intervertebral disc (IVD) are due to a failure of the extracellular matrix of the annulus to contain the contents of the nucleus. This results from inadequate maintenance of the matrix components as well as the proteolytic activity of matrix metalloproteinases (MMPs) that degrade matrix molecules. Arresting progression of disc degeneration in the annulus holds greater clinical potential at this point than prevention of its onset in the nucleus. Therefore, in this study, we have therapeutic aims that would decrease levels of the cytokines and growth factors that indirectly lead to disc degeneration via stimulating MMP and increase levels of several beneficial growth factors, such as transforming growth factor-ß, with the addition of platelet-rich plasma (PRP) that would stimulate cell growth and matrix synthesis. For this study, we attempted to address these imbalances of metabolism by using tumor necrosis factor-α treated annulus fibrosus cells isolated from porcine IVD tissue and incubating the cells in a growth factor rich environment with PRP. These results indicate that the PRP in vitro increased the production of the major matrix components (type II collagen and aggrecan) and decreased the inhibitory collagenase MMP-1. This application will address a therapeutic approach for intervening early in the degenerative process.

Detection of early cartilage damage using targeted nanosomes in a post-traumatic osteoarthritis mouse model.

Osteoarthritis (OA) is a major cause of pain and disability in the US. A problem with early intervention is that it is very difficult to detect OA before irreversible damage has already occurred. This study characterizes a novel method of early OA detection in a mouse model of post-traumatic osteoarthritis (PTOA) using fluorescent nanosomes. In this investigation, knee injury was induced in mice by compressive loading. Nanosomes encapsulating fluorescent dye and conjugated to collagen type II antibody were utilized to detect cartilage damage in vivo. Cartilage damage and OA progression were detected by the use of fluorescence-imaging (IVIS) and histopathology. Histopathology analyses showed that mild osteoarthritic changes had occurred. This corresponded with a higher fluorescence on IVIS imaging due to more nanosome binding. These results suggest that theragnostic nanosomes may be useful for detection of early PTOA as well as for targeted delivery of interventional agents. FROM THE CLINICAL EDITOR: With the aging population, osteoarthritis now poses a significant problem worldwide. Early detection may help slow the progression of the disease. In this study, the authors described the use of fluorescent nanosomes to detect early cartilage damage in a mouse model of osteoarthritis. This detection method may also prove to be useful for targeted delivery of drugs in the future.

Bone morphology in 46 BXD recombinant inbred strains and femur-tibia correlation.

We examined the bone properties of BXD recombinant inbred (RI) mice by analyzing femur and tibia and compared their phenotypes of different compartments. 46 BXD RI mouse strains were analyzed including progenitor C57BL/6J (n = 16) and DBA/2J (n = 15) and two first filial generations (D2B6F1 and B6D2F1). Strain differences were observed in bone quality and structural properties (P < 0.05) in each bone profile (whole bone, cortical bone, or trabecular bone). It is well known that skeletal phenotypes are largely affected by genetic determinants and genders, such as bone mineral density (BMD). While genetics and gender appear expectedly as the major determinants of bone mass and structure, significant correlations were also observed between femur and tibia. More importantly, positive and negative femur-tibia associations indicated that genetic makeup had an influence on skeletal integrity. We conclude that (a) femur-tibia association in bone morphological properties significantly varies from strain to strain, which may be caused by genetic differences among strains, and (b) strainwise variations were seen in bone mass, bone morphology, and bone microarchitecture along with bone structural property.

Genomic locus on chromosome 1 regulates susceptibility to spontaneous arthritis in mice deficiency of IL-1RA.

BACKGROUND: To understand the role of genetic factors on chromosome 1 in the regulation of spontaneous arthritis in mice deficient in IL-1 receptor antagonist protein (IL_1RA), we previously used speed congenic breeding to transfer the QTL region from DBA/1(-/-) mice that are resistant to spontaneous arthritis into BALB/c(-/-) mice which are susceptible. We were able to establish two congenic strains which exhibited a delayed onset and reduced severity of disease. In this study, we asked a different set of questions. How will the QTL region from BALB/c(-/-) interact with the rest of the genome in the DBA/1(-/-) background? Will the DBA/1(-/-) mice become susceptible to spontaneous arthritis if the QTL genomic region on chromosome 1 was replaced with the genomic fragment of the same region from BALB/c(-/-)? We conducted the congenic breeding with the similar procedure as that of congenic strains with BALB/c(-/-) background. RESULT: Instead of BALB/c(-/-), DBA/1(-/-) was used as the recurrent parent while BALB/c(-/-) was used as the donor parent. By the 6(th) generation we determined that all of the chromosomes in the progeny were of DBA/1(-/-) origin with the exception of the QTL portion of chromosome 1 which is heterozygous of BALB/c(-/-) and DBA/1(-/-) origin. We then intercrossed selected mice to produce homozygous strains containing the homozygous genomic region of BALB/c(-/-) on chromosome 1, while the rest of genome are homozygous DBA/1(-/-). This strain was observed for the development of spontaneous arthritis. Up to 9 weeks of age, both congenic strain and DBA/1(-/-) did not develop arthritis. However, after 9 weeks, the congenic strain started to exhibit signs of arthritis, while the DBA/1(-/-) remained free from disease. CONCLUSION: The result indicates a strong influence of genetic factor(s) on the QTL of chromosome 1 on the susceptibility to spontaneous arthritis. Identification of genetic factors within this QTL region in the future will significantly enhance our understanding of molecular mechanism of spontaneous arthritis.

Theranostic immunoliposomes for osteoarthritis.

Although there have been substantial advancements in the treatment of inflammatory arthritis, treatments for osteoarthritis (OA) have lagged and currently are primarily palliative until joints become totally dysfunctional and prosthetic replacement is needed. One obstacle for developing a preventive therapy for OA is the lack of good tools for efficiently diagnosing the disease and monitoring its progression during the early stages when the effect of therapeutic drugs or biologics is most likely to be effective. We have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA. These immunoliposomes bind to damaged but not normal cartilage. Utilizing these reagents, we can quantitate exposure of type II collagen during cartilage degradation in individual joints in vivo in a guinea pig. Immunoliposomes could be used to determine the effectiveness of therapeutic interventions in small animals as well as vehicles for localized drug delivery to OA chondrocytes. FROM THE CLINICAL EDITOR: This team of authors have developed near infrared immunoliposomes conjugated with type II collagen antibody for diagnosis and treatment of early OA, with promising results demonstrated in a guinea pig model.

A programmable arthritis-specific receptor for guided articular cartilage regenerative medicine.

Objective: Investigational cell therapies have been developed as disease-modifying agents for the treatment of osteoarthritis (OA), including those that inducibly respond to inflammatory factors driving OA progression. However, dysregulated inflammatory cascades do not specifically signify the presence of OA. Here, we deploy a synthetic receptor platform that regulates cell behaviors in an arthritis-specific fashion to confine transgene expression to sites characterized by cartilage degeneration. Methods: An scFv specific for type II collagen (CII) was used to produce a synthetic Notch (synNotch) receptor that enables "CII-synNotch" mesenchymal stromal cells (MSCs) to recognize CII fibers exposed in damaged cartilage. Engineered cell activation by both CII-treated culture surfaces and on primary tissue samples was measured via inducible reporter transgene expression. TGFß3-expressing cells were assessed for cartilage anabolic gene expression via qRT-PCR. In a co-culture with CII-synNotch MSCs engineered to express IL-1Ra, ATDC5 chondrocytes were stimulated with IL-1α, and inflammatory responses of ATDC5s were profiled via qRT-PCR and an NF-κB reporter assay. Results: CII-synNotch MSCs are highly responsive to CII, displaying activation ranges over 40-fold in response to physiologic CII inputs. CII-synNotch cells exhibit the capacity to distinguish between healthy and damaged cartilage tissue and constrain transgene expression to regions of exposed CII fibers. Receptor-regulated TGFß3 expression resulted in upregulation of Acan and Col2a1 in MSCs, and inducible IL-1Ra expression by engineered CII-synNotch MSCs reduced pro-inflammatory gene expression in chondrocytes. Conclusion: This work demonstrates proof-of-concept that the synNotch platform guides MSCs for spatially regulated, disease-dependent delivery of OA-relevant biologic drugs.

Synergistic effect of combined growth factors in porcine intervertebral disc degeneration.

Although intervertebral disc (IVD) degeneration is one of most common causes of morbidity, its etiology remains unclear. In healthy discs, the rates of synthesis and breakdown of the extracelluar matrix (ECM) are in equilibrium because of intricate regulation by growth factors and catabolic cytokines. Important among these physiologic growth factors are transforming growth factor-ß (TGF-ß1) and bone morphogenetic protein-2 (BMP-2). Disc degeneration is thought to be associated with a loss of this homeostasis between proteoglycan (PG) synthesis and cytokine-induced degradation leading to up-regulation of matrix metalloproteinases (MMP) families and down-regulation of extracelluar matrix production. Several strategies using biological agents have been attempted to manage IVD degeneration, improving the function and anabolic capabilities of IVD cells and inhibiting matrix degradation. The purpose of this study is to compare the effects of the anabolic cytokines BMP-2 and TGF-ß1 with those of the catabolic cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) on porcine annulus fibrosus (AF). The results of this study show that the application of pro-inflammatory cytokines like tumor necrosis factor-α and interleukin-1ß to normal annulus fibrosus cells leads to a significant increase in tissue levels of the degradative protease MMP-1. Treatment with a combination of minimum doses of both BMP-2 and TGF-ß1 caused a greater decrease in MMP-1 and increase in aggrecan than either cytokine alone, suggesting a synergistic effect of the combined cytokines.

Construction of a tissue-engineered annulus fibrosus.

The intervertebral disc is composed of load-bearing fibrocartilage that may be subjected to compressive forces up to 10 times the body weight. The multilaminated outer layer, the annulus fibrosus (AF), is vulnerable to damage and its regenerative potential is limited, sometimes leading to nuclear herniation. Scaffold-based tissue engineering of AF using stem cell technology has enabled the development of bi-laminate constructs after 10 weeks of culture. It is difficult to know if these constructs are limited by the differentiation state of the stem cells or the culture system. In this study, we have characterized an expandable scaffold-free neoconstruct using autologous AF cells. The construct was prepared from pellet cultures derived from monolayer cultures of AF cells from mature pigs that became embedded in their own extracellular matrix. The pellet cultures were incubated for 24 h in a standardized conical tube and then carefully transferred intact to a culture flask and incubated for 21 days to allow continued matrix synthesis. Cell viability was maintained above 90% throughout the culture period. The engineered scaffold-free construct was compared with the native AF tissue by characterization of gene expression of representative markers, histological architecture, and biochemical composition. The morphological and biochemical characteristics of the cultured disc construct are very similar to that of native AF. The cell number per gram of construct was equal to that of native AF. Expression of aggrecan was elevated in the engineered construct compared with RNA extracted from the AF. The glycosaminoglycan content in the engineered construct showed no significant difference to that from native construct. These data indicate that scaffold-free tissue constructs prepared from AF cells using a pellet-culture format may be useful for in vitro expansion for transplantation into damaged discs.

Albumin-binding RNAi Conjugate for Carrier Free Treatment of Arthritis.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin 'hitchhiking' for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary: Lipophilic siRNA conjugates optimized for albumin binding and "hitchhiking" can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.

Characterization of Non-Invasively Induced Post-Traumatic Osteoarthritis in Mice.

The pathophysiology of post-traumatic arthritis (PTOA) is not fully understood. This study used non-invasive repetitive mechanical loading (ML) mouse models to study biochemical, biomechanical, and pain-related behavioral changes induced in mice. Mouse models reflected the effects of the early stages of PTOA in humans. For the PTOA model, cyclic comprehensive loading (9N) was applied to each mouse's left knee joint. ML-induced biochemical and molecular changes were analyzed after loading completion. Cartilage samples were examined using gene expression analysis. Tissue sections were used in subsequent OA severity scoring. Biomechanical features and pain-related behavior were studied after 24 h and three weeks post-ML sessions to examine the development of PTOA. The loaded left knee joint showed a greater ROS/RNS signal than the right knee, which was not loaded. There was a significant increase in cartilage damage and MMP activity in the mechanically loaded joints relative to non-loaded control knee joints. Similarly, we found a difference in the viscoelastic tangent, which highlights significant changes in mechanical properties. Biochemical analyses revealed significant increases in total NO, caspase-3 activity, H2O2, and PGE2 levels. Gene expression analysis highlighted increased catabolism (MMP-13, IL-1ß, TNF-α) with a concomitant decrease in anabolism (ACAN, COL2A1). Histopathology scores clearly indicated increases in OA progression and synovitis. The gait pattern was significantly altered, suggesting signs of joint damage. This study showed that biomechanical, biochemical, and behavioral characteristics of the murine PTOA groups are significantly different from the control group. These results confirm that the current mouse model can be considered for translational PTOA studies.

Aging affects response to cyclic tensile stretch: paradigm for intervertebral disc degeneration.

Much evidence supports a fundamental role for mechanical forces in modulating differentiation, homeostasis, and remodelling of musculoskeletal cells. Little is known, however, regarding mechanobiology and gene expression of intervertebral disc (IVD) cells from older individuals. To characterise the effect of mechanical stimulation on cells from older discs, an in vitro study of IVD cells harvested from different aged pigs was conducted to measure extracellular matrix (ECM) gene expression in response to cyclic tensile stress (CTS). Gene expression of annulus fibrosus (AF) cells from IVDs of mature and older pigs was quantified for the predominant ECM genes; type I collagen, type II collagen and aggrecan, and matrix metalloproteinase 1 (MMP-1), a collagenase that degrades fibrillar collagens. AF cells cultured on flexible-bottom plates were stretched 10 % at 0.5 Hz frequency. After 24 h, gene expression was assayed using reverse transcriptase polymerase chain reaction (RT-PCR). Basal mRNA levels without stretching for type II collagen and aggrecan were lower in older annular cells whereas MMP-1 levels were higher compared to mature cells. Following CTS, an adaptive response was elicited in annular cells from both age groups. ECM protein genes were upregulated, whereas MMP-1 was downregulated. The magnitude of response was significantly greater in older cells as compared to mature cells. These data suggest that the cells from the AF of older animals manifest lower basal levels of mRNA for type II collagen and aggrecan and higher levels of MMP-1 possibly due to decreased tensile stress experienced in vivo and is not the result of reduced capacity for response.

Autoimmunity due to molecular mimicry as a cause of neurological disease.

One hypothesis that couples infection with autoimmune disease is molecular mimicry. Molecular mimicry is characterized by an immune response to an environmental agent that cross-reacts with a host antigen, resulting in disease. This hypothesis has been implicated in the pathogenesis of diabetes, lupus and multiple sclerosis (MS). There is limited direct evidence linking causative agents with pathogenic immune reactions in these diseases. Our study establishes a clear link between viral infection, autoimmunity and neurological disease in humans. As a model for molecular mimicry, we studied patients with human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a disease that can be indistinguishable from MS (refs. 5,6,7). HAM/TSP patients develop antibodies to neurons. We hypothesized these antibodies would identify a central nervous system (CNS) autoantigen. Immunoglobulin G isolated from HAM/TSP patients identified heterogeneous nuclear ribonuclear protein-A1 (hnRNP-A1) as the autoantigen. Antibodies to hnRNP-A1 cross-reacted with HTLV-1-tax, the immune response to which is associated with HAM/TSP (refs. 5,9). Immunoglobulin G specifically stained human Betz cells, whose axons are preferentially damaged. Infusion of autoantibodies in brain sections inhibited neuronal firing, indicative of their pathogenic nature. These data demonstrate the importance of molecular mimicry between an infecting agent and hnRNP-A1 in autoimmune disease of the CNS.

Recent Advances in Clinical Translation of Intra-Articular Osteoarthritis Drug Delivery Systems.

Osteoarthritis (OA) is a degenerative disease of the joints and a leading cause of physical disability in adults. Intra-articular (IA) therapy is a popular treatment strategy for localized, single-joint OA; however, small-molecule drugs such as corticosteroids do not provide prolonged relief. One possible reason for their lack of efficacy is high clearance rates from the joint through constant lymphatic drainage of the synovial tissues and synovial fluid and also by their exchange via the synovial vasculature. Advanced drug delivery strategies for extended release of therapeutic agents in the joint space is a promising approach to improve outcomes for OA patients. Broadly, the basic principle behind this strategy is to encapsulate therapeutic agents in a polymeric drug delivery system (DDS) for diffusion- and/or degradation-controlled release, whereby degradation can occur by hydrolysis or tied to relevant microenvironmental cues such as pH, reactive oxygen species (ROS), and protease activity. In this review, we highlight the development of clinically tested IA therapies for OA and highlight recent systems which have been investigated preclinically. DDS strategies including hydrogels, liposomes, polymeric microparticles (MPs) and nanoparticles (NPs), drug conjugates, and combination systems are introduced and evaluated for clinical translational potential.

Top-Down Fabricated microPlates for Prolonged, Intra-articular Matrix Metalloproteinase 13 siRNA Nanocarrier Delivery to Reduce Post-traumatic Osteoarthritis.

Post-traumatic osteoarthritis (PTOA) associated with joint injury triggers a degenerative cycle of matrix destruction and inflammatory signaling, leading to pain and loss of function. Here, prolonged RNA interference (RNAi) of matrix metalloproteinase 13 (MMP13) is tested as a PTOA disease modifying therapy. MMP13 is upregulated in PTOA and degrades the key cartilage structural protein type II collagen. Short interfering RNA (siRNA) loaded nanoparticles (siNPs) were encapsulated in shape-defined poly(lactic-co-glycolic acid) (PLGA) based microPlates (µPLs) to formulate siNP-µPLs that maintained siNPs in the joint significantly longer than delivery of free siNPs. Treatment with siNP-µPLs against MMP13 (siMMP13-µPLs) in a mechanical load-induced mouse model of PTOA maintained potent (65-75%) MMP13 gene expression knockdown and reduced MMP13 protein production in joint tissues throughout a 28-day study. MMP13 silencing reduced PTOA articular cartilage degradation/fibrillation, meniscal deterioration, synovial hyperplasia, osteophytes, and pro-inflammatory gene expression, supporting the therapeutic potential of long-lasting siMMP13-µPL therapy for PTOA.