Electrical Stimulation of Denervated Rat Skeletal Muscle Retards Capillary and Muscle Loss in Early Stages of Disuse Atrophy

The purpose of the present study is to investigate the effects of low-frequency electrical muscle stimulation (ES) on the decrease in muscle mass, fiber size, capillary supply, and matrix metalloproteinase (MMP) immunoreactivity in the early stages of denervation-induced limb disuse. Direct ES was performed on the tibialis anterior muscle following denervation in seven-week-old male rats. The rats were divided into the following groups: control (CON), denervation (DN), and denervation with direct ES (DN + ES). Direct ES was performed at an intensity of 16 mA and a frequency of 10 Hz for 30 min per day, six days a week, for one week. We performed immunohistochemical staining to determine the expression of dystrophin, CD34, and MMP-2 in transverse sections of TA muscles. The weight, myofiber cross-sectional area (FCSA), and capillary-to-fiber (C/F) ratio of the tibialis anterior (TA) muscle were significantly reduced in the DN group compared to the control and DN + ES groups. The MMP-2 positive area was significantly greater in DN and DN + ES groups compared to the control group. These findings suggest beneficial effects of direct ES in reducing muscle atrophy and capillary regression without increasing MMP-2 immunoreactivity in the early stages of DN-induced muscle disuse in rat hind limbs.

Introduction
Limb disuse due to denervation (DN) causes musculoskeletal atrophy accompanied by a reduction in muscle tissue capillary supply. In particular, steep decreases in muscle mass and force generated by the muscle are observed in earlystage disuse atrophy. Significant decreases in muscle mass and fiber size occur within one week of denervation, and by two weeks the tibialis anterior (TA) muscle atrophies to about 50% of the control [1][2][3]. Muscle fiber atrophy is accompanied by increased capillary density, reduced capillary-tofiber (C/F) ratio, and decreased capillary diameter after hind limb unloading and denervation [4][5][6]. Capillaries supply muscle fibers with oxygen and nutrients and remove waste metabolic products. Capillary density is also reduced because less blood flow is required to meet the metabolic demands in the atrophic muscle. Therefore, therapeutic approaches that enhance blood flow induced by muscle contractions might ameliorate muscle atrophy and capillary loss during the early stages of DN-induced disuse.
Electrical muscle stimulation (ES) has been utilized as a therapeutic intervention and a functional substitute for voluntary muscle contraction in patients with spinal cord injury (SCI) [7] and long-standing denervation. Some clinical studies have found that direct ES to denervated muscles increases muscle mass and average fiber diameter [8,9]. Moreover, the early application of ES treatment following orthopedic surgery may effectively attenuate loss of muscle strength and improve functional performance [10]. In contrast, other preclinical studies have demonstrated no beneficial effects of chronaxie-based ES (frequency: 20 Hz; pulse duration: twice the chronaxie value; 3 s on, 6 s off) on nerve crush-induced muscle fiber atrophy and muscle excitability [11]. In fact, chronaxie-based ES has been reported to induce muscle fibrosis and atrophy in denervated muscles [12]. Furthermore, although high-intensity (16 mA) ES was effective in retarding denervated muscle atrophy, it may have adverse effects on the regeneration of nerve terminals in the neuromuscular junction and/or the membrane systems involved in excitation-contraction coupling [13]. The effects of ES on muscle atrophy appear to be influenced by the type of disuse model used, as well as the nature of the experimental ES regimen, which can vary in the intensity, frequency, and number of contractions [14][15][16].
In recent years, it has been reported that matrix metalloproteinases (MMPs) play an important role in the homeostasis and maintenance of myofiber functional integrity; MMPs break down the extracellular matrix (ECM) and regulate skeletal muscle cell migration, differentiation, and regeneration [17]. MMPs are also essential for the angiogenic response to physical activity, which is a complex process thought to be regulated by a balance of proangiogenic and antiangiogenic factors [18]. At the initial stage of capillary sprout formation, the continuous basement membrane, which is composed of type IV collagen and laminin-rich connective tissue membrane underlying endothelial cell layers, must first be dissolved by proteinases before the migration and proliferation of distinct endothelial cell populations occur. MMP-2 (66 kDa, type IV collagenase, gelatinase A) activity is important in the early stages of endothelial cell morphogenesis/capillary formation, as MMP-2 and MMP-9 are responsible for the fragmentation of basement membrane type IV collagen 9 [19,20]. Furthermore, based on inhibition studies of MMP-2, ablation of MMP-2 may impair not only angiogenesis, but also the growth of regenerated muscle fibers via decreased VEGF and nNOS expression [21]. Therefore, MMP-2 may also play a significant role in myogenesis and muscle regeneration [22,23].
The activity of MMP-2 has been reported to increase following DN and limb immobilization [24,25] or muscle stretching in the denervated muscle [26]. Similarly, chronic ES and exercise training elevate MMP-2 expression to various levels depending on intensity [27,28]. On the other hand, other studies show that ES treatment (20 Hz for 3 s followed by 6 s of rest, repeated for 20 maximal contractions, applied every 48 h for 28 days) does not alter MMP-2 activity [25], indicating that discrepancies might occur among interventions with different conditions. To our knowledge, the histomorphological effects of low-frequency ES on muscle fibers, capillaries, and MMP-2 immunoreactivity in early-stage DNinduced hind limb disuse have yet to be studied, although some studies have demonstrated that even low-frequency ES (10 Hz, incomplete tetanus) reduces DN-induced muscle loss [13,16]. Thus, it is important to explore the potential use of low-frequency ES (e.g., 10 Hz) to maintain both muscle mass and capillaries. We hypothesize that low-frequency ES reduces muscle atrophy and capillary loss in early stages of DN-induced limb disuse.
The purpose of the present study is to investigate the histomorphometric effects of low-frequency ES on the decrease in muscle mass, fiber size, capillary density, and MMP immunoreactivity in the early stages of DN-induced limb disuse.

Materials and Methods
Twenty-two male Fischer 344 rats (CLEA, Tokyo, Japan) were individually housed in standard cages at constant temperature (23 ± 2 ∘ C), humidity (55% ± 5%), and 12 h light-dark cycles. Rats were provided with CE-2 rodent chow (CLEA) and water ad libitum. Seven-week-old rats were randomly assigned to one of the following groups: age-matched controls (CON, = 8), denervation with direct ES (DN + ES, = 8), or denervation without ES (DN, = 6). The sample size ( = 6-7/group) was calculated with reference to FCSA data from previous studies from our laboratory, using the following formula [6,[29][30][31]: where is the ratio of the larger group to the smaller group, is the standard deviation, Δ is the effect size, = 0.05, = 0.2 (for 80% power with 95% confidence), 훼/2 = 1.96, and All animal manipulations and protocols were carried out in accordance with the guidelines presented in the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences, published by the Physiological Society of Japan. All procedures were approved by the Animal Committee of the National Institute of Fitness and Sports and the Animal Committee of Niigata University of Health and Welfare.
The rats in the DN and DN + ES groups were anesthetized with an intraperitoneal injection (50 mg/kg) of sodium pentobarbital. An incision was made on the skin covering the left buttock and the sciatic nerve was exposed and separated from the surrounding tissue. The sciatic nerve was frozen for 5 s using a 5 mm diameter stainless steel rod cooled in liquid nitrogen [13,16,[32][33][34][35]. This freezing procedure uniformly damages nerve fibers, similar to nerve crushing, cutting, or transection with a suture [13,32,34,35]. Evoked electromyography was performed with ES at a location proximal to the freezing site of the sciatic nerve to confirm the efficacy of TA muscle denervation [36].
On the day after surgery, the left TA muscles of the DN + ES rats under isoflurane inhalation anesthesia (2%) were percutaneously electrically stimulated. The stimulation protocol was delivered as previously described [16,37]. Bipolar silver surface electrodes (3 mm diameter) were briefly attached to the shaved anterior surface of the left leg. ES was performed using electrostimulators with an isolator (SEM-4201, Nihon Kohden, Tokyo, Japan) for 30 minutes a day, six days a week, for one week at an intensity of 16 mA, a frequency of 10 Hz, and a pulse width of 250 s. The ES regimen comprised 2 s of stimulation followed by 6 s of rest. Muscle contractions induced by ES at 10 Hz are characterized by minimal summation during twitch contraction [16]. The ES regimen used in the previous study (10 Hz for 2 s followed by 6 s of rest, repeated for 225 cycles over 30 min) caused as many as 4500 instances (10 × 2 × 225) of muscle contraction, mechanical stimuli, or both. One week after denervation, rats in the control, DN, and DN + ES groups were anesthetized with Table 1: Body weight (BW), muscle weight relative to BW, myofiber cross-sectional area (FCSA), capillary-to-fiber (C/F) ratio, capillary density (CD), and MMP-2 positive area in the TA muscles. sodium pentobarbital (50 mg/kg body weight). TA, extensor digitorum (EDL), and soleus (Sol) muscles were extracted and weighed. Left TA muscle samples were mounted on a piece of cork with OCT compound and frozen in isopentane cooled in liquid nitrogen for histological analysis. Samples were stored at −80 ∘ C until use. The TA muscle samples were cut into 10 m cross sections with a cryostat (CM3050S, Leica, Germany) at −20 ∘ C and mounted on silanized slides for immunohistochemical and haematoxylin and eosin staining. After fixing with ice-cold 4% paraformaldehyde for 15 min, sections were blocked at room temperature for 1 h with 10% normal goat serum (NGS) and 1% Triton X-100 in PBS and then washed twice in PBS for 5 min. Next, sections were incubated in 5% NGS and 0.3% Triton X-100 in PBS for 16-20 h at 4 ∘ C with a primary antibody against dystrophin as a plasma membrane marker (1 : 500 dilution, Abcam, Tokyo, Japan), CD34 as a marker for endothelial cells (1 : 250 dilution, Abcam, Tokyo, Japan), and MMP-2 (1 : 2000 dilution, Abcam, Tokyo, Japan). The sections were washed several times with PBS, incubated with Alexa Fluor 488 or 568 conjugated secondary antibody (1 : 500 dilution, Abcam, Tokyo, Japan) in 5% NGS and 0.1% Triton X-100 PBS for 1 h at room temperature, and then mounted with Vectashield mounting medium. Images of TA muscle sections were obtained using a fluorescent light microscope (BX60; Olympus, Tokyo, Japan) and a CCD camera (DP72; Olympus, Tokyo, Japan). Digital images at 200x magnification were used to determine the cross-sectional area (CSA) of muscle fibers and the capillary number in each TA muscle. The FCSA of at least 75 fibers in each muscle were measured using Image-Pro Premier software (Media Cybernetics, Rockville, MD, USA). The total number of transverse capillaries and fibers in four random 154 × 205 m fields was manually counted to determine the capillary density (CD) and the C/F ratio [6,38]. The MMP-2 positive areas were measured within the area of interest (386 × 386 m) in each TA muscle.
Data are presented as mean ± SD. Data sets were analyzed using one-way analysis of variance (ANOVA) followed by either the Bonferroni post hoc test or the Kruskal-Wallis test followed by Steel-Dwass multiple comparison tests, depending on the normality of the data distribution [39]. values less than 0.05 were considered significant.

Results
Body weight did not significantly differ between the groups (Table 1), though TA muscle weight significantly decreased relative to body weight after denervation ( < 0.05). Relative TA muscle weight was significantly higher in the DN + ES group compared to the DN group ( < 0.05). Relative EDL muscle weight was significantly lower ( < 0.05) in the DN and DN + ES groups than in the control group, but no significant difference was observed between the DN and DN + ES groups (Table 1). Figure 1 shows representative images of TA muscle FCSA in the control, DN, and DN + ES groups. The CSA of TA muscle fibers were significantly smaller in the DN group than in the control and DN + ES groups ( < 0.05, Table 1).
We examined the expression and localization of CD34, a capillary endothelial marker, and dystrophin, a sarcolemmal membrane marker (Figures 1(a)-1(c)). Immunohistochemical staining revealed that CD34-positive capillaries were observed around the dystrophin-positive myofibers. The C/F ratios in the control, DN, and DN + ES groups were 1.22 ± 0.33, 0.73 ± 0.20, and 1.17 ± 0.26, respectively. The C/F ratio was significantly lower ( < 0.05) in the DN than in the control and DN + ES groups (Table 1). No significant differences were observed in capillary density between groups. MMP-2 was immunolocalized at the periphery of myofibers (Figure 2), and the MMP-2 positive area was significantly greater ( < 0.05) in the DN and the DN + ES groups compared to the control group (Table 1).

Discussion
The current study presents new data on the effects of lowfrequency ES in early stages of disuse atrophy on denervated rat muscles. The results show that ES reduced the decrease in muscle weight, FCSA, and C/F ratio in early-stage DNinduced muscle disuse. The MMP-2 positive area increased in denervated TA muscle, whereas it remained unchanged in denervated TA muscle that was treated daily with ES.
Our results show that EDL and Sol muscle weight were lower in the DN and DN + ES groups than in the control group, and there was no difference between the DN and DN + ES groups. The EDL lies in a deeper layer than the TA, but both muscles are innervated by the tibial nerve, which is a branch of the sciatic nerve. Thus, the present data suggest that successful sciatic nerve denervation had no differential effect on the DN and the DN + ES groups. In general, relatively higher intensities or frequencies of ES are likely to result in greater effects on muscle atrophy and capillary regression in early-stage DN-induced disuse. Muscle stimulation with higher-frequency ES regimens (20-100 Hz) tends to show beneficial effects of reducing the disuse-induced decreases in muscle mass, FCSA, and capillary supply [15,25,[40][41][42]. However, a low-frequency ES regimen (10 Hz, 8 and 16 mA, 30 min/day, for 3 weeks) in stimulated TA muscle retarded the atrophy of denervated muscle [13]. Using the same ES conditions (10 Hz, 16 mA, 30 min/day, for one week), the present study demonstrated that daily ES treatment reduced the DN-induced decrease in FCSA. Therefore, low-frequency ES treatment might have a beneficial effect on muscle disuse atrophy. Direct ES has been reported to increase the C/F ratio in intact fast-type dominant muscle [42,43]. Previous studies that compared the effects of different ES frequencies on resting blood flow and capillary density reported that optimal ES frequency for angiogenesis lies in the range of 10-40 Hz [44]. Moreover, previous reports describe a significant increase in C/F ratio after only four to seven days of low-frequency ES (10 Hz) in intact TA and EDL muscles [45,46]. Our data showed that the C/F ratios in the DN + ES groups were significantly greater than in the DN group, but not different from the control group. This suggests that daily low-frequency ES treatment effectively ameliorates muscle capillary regression in early-stage DN-induced disuse. However, our data also revealed that capillary density was unchanged following DN and ES treatment. In the present study, two-dimensional capillary density was determined by counting the total number of capillaries in a muscle section and was expressed as the number of capillaries per CSA unit. The measure of capillary density is likely subject to muscle atrophy or hypertrophy in fiber size if the C/F ratio is stable. Hudlicka et al. suggested that muscle fiber hypertrophy is always associated with reduced capillary density [47]. However, there are discrepancies among previous reports; some found that muscle fiber atrophy is accompanied by increased capillary density two and eight weeks after denervation [5], whereas others report a decrease [48]. Nevertheless, longterm denervation results in reduced capillary density and C/F ratio [49]. Some reports have also established that inactivity affects capillary density in slow and fast muscles differently [5,50]. Thus, the incongruent results regarding capillary density might be a result of differences in the duration of disuse, degree of muscle atrophy (fiber size), and muscle type.
A potential explanation for the effects of ES on capillary proliferation involves vascular endothelial growth factor (VEGF), which is known to be an important physiological regulator of angiogenesis. Several lines of evidence suggest that VEGF may also play a vasculoprotective role [51,52]. In animal studies, ES-induced muscle contraction or mechanical stress facilitates cytokine (myokine) production [53] and increases blood flow and capillary vascularity [54,55]. Mechanical stress, such as shear stress and cyclic mechanical stretching, is known to induce VEGF expression [56,57]. In fact, continuous low-voltage ES in a hind limb ischemia model of rats for five days has been reported to increase VEGF production [58]. Therefore, the enhanced blood flow and cyclic stretching generated by ES-induced muscle contractions may stimulate VEGF production and lead to angiogenesis in the ES muscles.
Furthermore, MMPs play an important role in the homeostasis and maintenance of myofiber functional integrity and regulate skeletal muscle regeneration and angiogenesis by degrading ECM components [17,18]. Capillary sprout formation is initiated by fragmentation of the capillary basal lamina and the subsequent migration and proliferation of distinct endothelial cell populations in response to angiogenic stimuli [19,59]. Various blood and connective tissue cells (e.g., macrophages, monocytes, and endothelial cells) secrete MMP-2 [60], and MMP-2 is involved in the degradation of type IV collagen, laminin, elastin, fibronectin, and many other ECM proteins [61]. Interestingly, an ablation study of MMP-2 reported downregulation of VEGF in the MMP-2 knockout mouse [62], and MMP-2 ablation is known to impair the growth of regenerated muscle fibers and reduce angiogenesis via decreased VEGF, Flt-1, and nNOS expression. Thus, MMP-2 may influence angiogenesis and muscle regeneration via upregulation of VEGF and its receptor Flt-1 and may also affect angiogenesis through regulation of nNOS-derived NO [21]. Our data revealed that the DN and DN + ES groups had a greater MMP-2 positive area compared to the control group, but no difference was observed between the DN and DN + ES groups. Previous studies report increased MMP-2 activity following DN and limb immobilization [24,25] and found that MMP-2 expression is elevated to different extents depending on the intensity of chronic ES [27,28]. On the other hand, low-intensity ES (20 Hz for 3 s followed by 6 s of rest, repeated for 20 maximal contractions, applied every 48 h for 28 days) did not alter MMP-2 activity [25]. Some discrepancies might exist among interventions with different conditions. A previous study reported that exercise-induced expression of MMP-2 is intensity dependent; high-intensity endurance exercise at approximately 70% of maximum oxygen consumption (VO 2 max) increases MMP-2 expression, whereas low-intensity endurance exercise (approximately 50% VO 2 max) did not alter MMP-2 expression in skeletal muscles [27]. Although we did not determine VO 2 during daily ES treatment, our ES regimen caused approximately 25% of maximal contraction force in the TA muscle [16]. Therefore, we may not have detected de novo alteration of MMP-2 in the DN + ES rats because of the low-intensity intervention.
The present findings should be interpreted in the context of their potential limitations. First, we used young, growing rats that were undergoing skeletal remodeling and thus needed the appropriate baseline controls. Although there was no inhibition of longitudinal bone growth of the tibiae in the young DN rats over time [63], young animals usually have greater potential for musculoskeletal recovery after limb disuse [64,65]. Our results might quantitatively differ if skeletally mature rats were used with a DN-induced disuse model. Second, our methodology precluded analysis of the activity of tissue inhibitors of metalloproteinases (TIMPs) and other pro-and antiangiogenic factors. A zymography analysis would be ideal to evaluate MMP-2 and TIMP-1 activity, and analysis of inhibitors of metalloproteinases will be the focus of future experiments. Muscle atrophy is accompanied by changes in both MMPs and TIMPs, and the ratio between these metalloproteinases may be important in determining myoblast migration and differentiation [61,66]. Furthermore, MMPs and TIMPs play a significant role in regulating angiogenesis, which is the formation of new blood vessels [19]. For example, TIMP-2 plays an important role in ECM turnover by mediating the activity of MMPs, including MMP-2 [60]. Therefore, data on the balance between MMP-2 and TIMP-2 may provide useful information for evaluating the effects of low-frequency ES on reducing early-stage muscle atrophy and capillary regression in denervated muscles. Further studies using adult rats and other parameters and methodical techniques are needed for a more complete understanding of the relationship between ES-induced muscle contractions and capillary and muscle loss in the early stages of DN-induced muscle disuse.
In conclusion, this is the first histomorphological study to our knowledge that assesses the influence of low-frequency ES on MMP-2 immunoreactivity, fiber atrophy, and capillary regression in denervated muscle in early stages of disuse. Our results showed that FCSA and C/F ratios decreased with increased MMP-2 immunoreactivity following DN and that daily low-frequency ES treatment on denervated muscle reduced the decrease in FCSA and C/F ratios but did not alter higher levels of MMP-2 produced by DN-induced disuse. These findings suggest that direct ES has beneficial effects on early-stage DN-induced muscle disuse in the rat hind limb by ameliorating muscle atrophy and capillary regression without inducing a further increase in MMP-2 immunoreactivity. The use of direct ES to generate muscle contractions for the rehabilitation of skeletal muscles is well established, and the influence of different ES regimens on the balance of pro-and antiangiogenic factors is of great interest for future investigations as it may contribute to the understanding of the mechanisms responsible for the beneficial effects of ES.

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy:
Biology
Engineering
Materials Science
Medicine

text:
Poly(ethylmethacrylate-co-diethylaminoethyl acrylate) coating improves endothelial re-population, bio-mechanical and anti-thrombogenic properties of decellularized carotid arteries for blood vessel replacement

Decellularized vascular scaffolds are promising materials for vessel replacements. However, despite the natural origin of decellularized vessels, issues such as biomechanical incompatibility, immunogenicity risks and the hazards of thrombus formation, still need to be addressed. In this study, we coated decellularized vessels obtained from porcine carotid arteries with poly (ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) with the purpose of improving endothelial coverage and minimizing platelet attachment while enhancing the mechanical properties of the decellularized vascular scaffolds. The polymer facilitated binding of endothelial cells (ECs) with high affinity and also induced endothelial cell capillary tube formation. In addition, platelets showed reduced adhesion on the polymer under flow conditions. Moreover, the coating of the decellularized arteries improved biomechanical properties by increasing its tensile strength and load. In addition, after 5 days in culture, ECs seeded on the luminal surface of 8g7-coated decellularized arteries showed good regeneration of the endothelium. Overall, this study shows that polymer coating of decellularized vessels provides a new strategy to improve re-endothelialization of vascular grafts, maintaining or enhancing mechanical properties while reducing the risk of thrombogenesis. These results could have potential applications in improving tissue-engineered vascular grafts for cardiovascular therapies with small caliber vessels.

Cardiovascular diseases, including coronary artery and peripheral vascular disorders, affect a large amount of the population worldwide each year, and as such vascular grafts for by-pass procedures or replacement of damaged vessels are in high demand in vascular surgery. Currently, vascular grafts involve the use of autologous vessels or synthetic inserts. Despite autografts being the gold standard replacement option with long-term patency rates, their availability is limited due to pre-existing damage or their use in previous procedures and the requirement of an additional clinical procedure. Alternative sources for vascular grafts are synthetic grafts made of Dacron ® and expanded polytetrafluoroethylene 1 . Synthetic grafts have been used successfully for large caliber vessel reconstructions; however, they are not suitable for the replacement of vessels smaller than 6 mm in diameter mainly due to the risk of thrombus formation and neointimal hyperplasia which could lead to lumen occlusion 1,2 .
To address these limitations, novel tissue engineering strategies have emerged to develop alternative conduits for clinical applications 3 . Biocompatible vascular scaffolds are designed to resemble native tissue as well as their functional and biomechanical properties. In this sense, decellularization of naturally available biomaterials has been identified as a promising approach for vascular replacement of small-diameter vascular vessels 4 . Tissue decellularization consists of removing all cellular components leaving behind the extracellular matrix (ECM) along with the main structural components of a blood vessel, such as collagen and elastin, which will then act as a natural scaffold with mechanical properties close to those of native tissue 4 . Moreover, decellularized matrices are less immunogenic than native tissue in as much as most of the antigenic cellular components are removed resulting in more favorable scaffolds for transplantations 5 .
Consequently, decellularized allogeneic and xenogeneic ECM scaffolds are now being investigated for the reconstruction of a wide range of specialized tissues with potential therapeutic applications including their use for cardiovascular conduits [6][7][8] . However, some studies have shown thrombus formation after acellular graft implantation partly attributable to the lack of a viable vascular endothelium 9,10 . In fact, when a vascular lesion is produced, platelets migrate to the affected area causing thrombus formation 11 . Moreover, the decellularization process leads to the exposition of highly thrombogenic collagen on the vascular wall and platelet adhesion. Thus, strategies to enhance adhesion of endothelial cells (ECs) or endothelial progenitor cells (EPCs) improving the rapid endothelialization of vascular scaffolds while reducing platelet adhesion are key for vascular tissue engineering 6,12 .
Poly(ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) is a biocompatible polymer that has been described to enhance viability, attachment and function of progenitor and mature ECs. Moreover, the coating of intra-vascular devices with this biopolymer has been shown to facilitate the attachment of ECs while minimizing platelet binding 13 .
In the present study, we demonstrated better endothelial coverage, reduced risk of thrombosis and comparable biomechanical properties of 8g7-coated decellularized arteries compared to native ones. The adhesion and growth of human umbilical vein endothelial cells (HUVECs) on the uncoated and polymer-coated luminal surface of acellular carotid arteries was examined through a variety of techniques, including scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Reduced platelet aggregation to the polymer was established under physiological shear flow through perfusion of whole blood in a parallel-plate flow chamber. In addition, the biomechanics of native acellular and polymer coated decellularized arteries were compared with the native arteries. This study highlights the potential of combining synthetic polymer coatings with natural acellular vessels to develop novel tissue-engineered grafts for vascular repair.

Results
Capillary-like tube formation assay on biopolymer coated surfaces. The tube formation assay was used to investigate the capacity of 8g7 polymer to induce Angiogenesis in vitro 14 . HUVECs were seeded onto 8g7 polymer-coated glass coverslips as well as onto uncoated glass coverslips and 12-well plate coated with matrigel ( Fig. 1). HUVECs were able to attach and grow on all surfaces (Fig. 1A). After 4 h, HUVECs cultured on both 8g7 polymer and matrigel formed capillary-like tubular structures, while uncoated glass coverslips did not (Fig. 1A). The number of capillary-like structures formed was counted at different areas of surfaces. No significant differences were found between number of capillary-like tubes formed by cells grown onto, 8g7 polymer or matrigel (Fig. 1B), indicating that HUVECs maintain their functional ability on both. The average number of cells that form a branch of the capillary-like structure when cells grown on polymer coated coverslips was 8 ± 0.29 with a mean number of branching points of 3.6 while an a average of 12 ± 0.3 cells were found to form tubes when cells were grown on matrigel with a mean number of branching points of 3.9 (P = 0.068) (Fig. 1C). Furthermore, the viability of HUVECs cultured onto polymer coated coverslips was not compromised as no dead cells were observed (data not shown).

Platelet adhesion test on polymer-coated coverslips.
To determine whether platelets adhere onto the 8g7 polymer under flow conditions, citrated whole blood was perfused over polymer-coated and uncoated coverslips in a parallel plate chamber. The percentage of surface covered with platelets was determined following a shear rate of 600 s −1 , 800 s −1 and 1200 s −1 for 4 min ( Fig. 2A). Whilst no differences were seen under moderately low shear stress conditions (600 s −1 ) between uncoated and 8g7 polymer-coated coverslips, a significantly reduced number of platelets adhered to 8g7 polymer-coated coverslips under conditions of increased shear rate (800 s −1 and 1200 s −1 ) (Fig. 2B).
Characterization of decellularized arteries. Histological analyses were performed to demonstrate the efficacy of the decellularization process in native carotid arteries. H&E staining showed complete removal of cellular components after the decellularization procedure (Fig. 3C) when compared with native arteries (Fig. 3A,B). In addition, the ECM of decellularized arteries remained intact with the characteristic concentric layers of elastin and collagen (stained pink) (Fig. 3C). Complete removal of vascular cells was confirmed by Masson's trichrome staining. Native tissue showed high density of cells embedded in collagen fibers (stained turquoise) (Fig. 3D,E), while no cells were found in the decellularized arteries, which displayed a well preserved ECM of collagen and elastin fibers that maintained the same circumferential orientation of the native tissue (Fig. 3F). DAPI staining was used to further confirm the removal of all cells in the vessel walls. Native arteries stained with DAPI showed cell nuclei at the intimal and medial layer (Fig. 3G), while no nuclei were detected in decellularized arteries (Fig. 3H). SEM analyses of the luminal surface of decellularized arteries confirmed the removal of cellular debris (Fig. 3J). Also, the basic extracellular microstructure of decellularized arteries did not appear altered (Fig. 3J) compared to native ones (Fig. 3I).
Charaterization of polymer coating and mechanical testing. The chemical structure of the 8g7 (poly(ethyl methacrylate-co-diethylaminoethyl acrylate)) polymer containing tertiary amine is shown in Fig. 4A. The polymer was coated on glass coverslips and the thickness of the polymer coating was determined by SEM Scientific RepoRts | 7: 407 | DOI:10.1038/s41598-017-00294-6 ( Fig. 4B) to be 1.78 ± 0.51 μm. In addition, polymer coated decellularized arteries were analyzed by TEM and the thickness of the polymer layer on luminal side was found to be 0.63 ± 0.2 μm (Fig. 4C). The mechanical testing revealed significant differences in burst pressure between the native arteries (1330 ± 135 mbar) and decellularized arteries (1115 ± 108 mbar) with the polymer-coated decellularized arteries (1153 ± 138 mbar) showing marginal improvement (Fig. 4). The polymer-coated arteries were able to withstand a maximum load of 19.2 ± 12.4 N with an ultimate tensile strength for circumferential stress of 4.6 ± 0.4 MPa, which resembled the mechanical properties of native vessels (maximum load = 18.5 ± 1.8 N and ultimate circumferential tensile strength = 3.9 ± 0.9 MPa). However, decellularized arteries showed a significantly lower maximum load (15.2 ± 1.7 N) and ultimate tensile strength (2.8 ± 0.4 MPa) (Fig. 4).
Biopolymer coated vessels support HUVECs growth. We studied the ability of 8g7 to support ECs growth in order to reconstitute the endothelial layer of the decellularized vessels. The coating protocol was optimized to ensure coating of the entire intimal layer of the decellularized vessel using an ad hoc system ( Figure S2). HUVECs were used to repopulate the endothelium of the decellularized arteries. Cells were seeded over the entire internal layer of decellularized and polymer-coated decellularized arteries and histological analyses were performed to study cell attachment. DAPI staining of vessel cross-sections showed cell nuclei aligned over the lumen surfaces of both uncoated (Fig. 5A) and polymer-coated recellularized arteries (Fig. 5D), indicating that cells attaching to the polymer formed an outline similar to native arteries (Fig. 3G).
Further staining with H&E and Masson's Trichrome staining confirmed these results ( Fig. 5B,C,E,F) and indicated that the biopolymer supported the formation of a more homogeneous interconnected cell layer (Fig. 5E,F).  Confocal microscopy analyses showed a confluent monolayer of ECs in the section of the vessel lumen. Cell tracker green staining evidenced live cells forming a homogenous carpet on the internal surface of both uncoated ( Fig. 5G) and polymer-coated recellularized arteries (Fig. 5H). HUVECs adhered tightly to the inner layer of decellularized arteries surface and were able to grow and migrate into the pores created by the removal of the cells after the decellularization process ( Fig. 5G,H). The intimal layer of polymer-coated recellularized arteries appeared regenerated with cells arranged following a wave-like alignment of the luminal fibers (Fig. 5H). Moreover, we found a significant increase in the number of cells attached to the polymer-coated decellularized arteries when compared with the decellularized arteries ( Fig. 5I).
Ultrastructural analyses. Scanning electron microscopy was performed on artery samples ( Fig. 6A-F) with SEM images of a decellularized artery without polymer coating and a polymer-coated decellularized artery showing homogeneous and uniform coating. A suitable endothelial surface was achieved on non polymer-coated (Fig. 6B,C) and polymer-coated (Fig. 6E,F) arteries after recelluarization (5 days culture with HUVECs) with most of the seeded HUVECs distributed on the intima layer, forming a cell mono-layer. The polymer-coated recellularized arteries (Fig. 6E,F) showed a more compact cellular distribution when compared to uncoated ones (Fig. 6B,C). In fact, for the 8g7 coated decellularized arteries, no gap between cells could be found while the uncoated recellularized arteries showed clear gaps between cells (Fig. 6C, white arrows) indicating a discontinuous cell re-endothelialization. These observations demonstrate that 8g7 increases cell attachment and improves cell coverage of the luminal surface while maintaining the cobblestone morphology of the cells.
To confirm these results, TEM analyses of both, native and polymer-coated recellularized arteries sections (cut perpendicular to the arteries) were performed. Micrographs showed the attachment of seeded cells on the intimal surface of the arteries (Fig. 6G,H). Caveolae, a typical feature associated to HUVECs were observed (Fig. 6H). Furthermore, the polymer seemed to facilitate the formation of a continuous epithelium, because specific cell unions, such as cell junctions (white asterisk) and cell adherence to the ECM (white arrows) were observed (Fig. 6H).

Discussion
In vascular tissue engineering, replacement grafts should recreate the conditions for cellular and tissue organization. Decellularized vessels could provide a promising alternative to synthetic vascular grafts that present disappointing failure rates when replacing small diameter vessels 2, 15 . Moreover, the poor health of patients with  vascular diseases restricts the possibility of obtaining homograft tissues. Thus, the use of decellularized xenografts may be a feasible alternative with potential unlimited availability 16 . In fact, xenogeneic ECM has already been used successfully as a scaffold to replace/repair numerous tissues and organs in both preclinical animal studies and human clinical applications 7 . However, the outcomes of the few clinical studies have raised controversies due to poor decellularization, fibrosis and a strong inflammatory response elicited by the xenogeneic collagen matrix 17,18 . Moreover, it has been shown that the decellularized porcine matrix acts as a platelet-activating surface. Platelet adhesion and aggregate formation was shown to be favored only on the surface of decellularized heart valve matrix or partially denuded matrix whereas after seeding with ECs no platelet activation was detected 19 . Therefore, avoiding the direct exposition of the acellular surface to the blood and a healthy confluent endothelial layer seem to be critical requirements to circumvent adhesion and aggregation of platelets and, thereby, graft rejection and thrombosis. Consequently, our study aimed to develop decellularized artery scaffolds that provide a suitable microenvironment to support ECs expansion while diminishing platelet adherence capabilities.
Many strategies have been developed to repopulate vascular grafts and establish a complete endothelium including among others the coating of decellularized heart vessels with substrates such as fibronectin 20 , heparin and vascular endothelial growth factor (VEGF) [21][22][23][24] . However, synthetic polymer coatings are more suitable for clinical applications as they are well defined materials, can be synthesized reproducibly and according to GMP standards. In a previous study polyurethane and polyacrylate libraries were screened using polymer microarray technology 13 , to identify synthetic biopolymers with essential attributes to support adhesion and expansion of both progenitor and mature ECs while minimizing platelet attachment. The best selected polymer (8g7) demonstrated comparable levels of binding for both EPC and mature ECs and an equivalent high expression of endothelial surface markers when compared to long-established substrates for in vitro culture conditions i.e., collagen type I and polystyrene 13 .
Based on that previous work, this study was designed to combine the properties of ECM-based natural grafts and 8g7 biopolymer to generate a tissue-engineered vascular scaffold that supports re-endothelialization and reduced platelet aggregation. First, the adhesion, growth and capillary tube formation abilities of ECs seeded on 8g7 were demonstrated. ECs grown on polymer-coated coverslips showed their functional capacity to induce endothelial tube-like structures similar to matrigel. Despite extensive use of matrigel for the attachment and differentiation of ECs, based on its ability to provide a suitable microenvironment to promote angiogenesis and endothelial proliferation 25 , it is derived from mouse sarcomas and hence unsuitable for clinical applications due to the risk of pathogen transmission and immunogenicity. From a clinical point of view, the growth of ECs needs a fully defined microenvironment which could be provided by chemically defined synthetic polymers such as 8g7. We hypothesize that a balance between the mechanical properties of the polymer and the growth factors of the culture medium may regulate the formation of tubule-like structures. In fact, the characteristics of the substrate have been shown before to direct cytoskeleton organization and to affect cell adhesion and disposition 26 . Other factors such as the substrate stiffness have been shown to influence the formation of tubule-like structures 27 with softer substrates showing better capillary network, indicating that the mechanical characteristic of the substrate have an impact on endothelisation 28,29 .
Previous studies have demonstrated the efficacy of other substrates to reduce platelet attachment 30 , although results are interesting, it is relevant to note that those studies were performed under static parameters. Here, we established the ability of 8g7 to reduce platelet aggregation under flow conditions using a dynamic method that mimics physiological flow. As platelet adhesion and aggregation mechanisms are shear stress dependent 31 , in vitro flow devices, such as parallel-plate flow chambers that mimic the in vivo conditions, are widely used for the analysis of platelet function in whole blood over the range of physiological shear stresses 32 . Such devices can also be used to evaluate thrombogenic potential of platelet over coated surfaces 33,34 . Interestingly, perfusion of whole blood in a parallel-plate flow chamber demonstrated that 8g7-coated coverslips exhibit reduced platelet adhesion and aggregation in increasing shear rates in comparison to the uncoated coverslips. This significant decrease in platelet attachment confirms the anti-thrombotic properties of 8g7 under a pseudo-physiological flow. The adhesion of cells to a biomaterial surface has been proposed to be a two-step process, in which the first step of 'passive adhesion' is controlled by physicochemical interactions (such as hydrophobicity and surface charge) and the second step of 'active adhesion' achieved due to the interactions of surface proteins on the cell and substrate 35,29 . Though physicochemical interactions such as hydrophilicity can play an important role in cellular attachment, as seen in temperature-modulated poly(N-isopropyl acrylamide) gels, the detachment temperature (and hence the level of hydrophilicity) is known to be dependent on the cell type (e.g., hepatocytes or endothelial cells) 35 , thus highlighting a much more complex association between cell binding and substrate hydration. Similarly, the influence of electrostatic charges on cell-binding is not fully understood. Moreover, different proteins have varying affinity to a polymer surface; and the interaction of proteins attached to the substrate and the receptor proteins on the cell surface has an important role in cell adhesion. It has been shown that platelet adhesion is not only related to the hydrophilicity of the substrate and the amount of plasma proteins absorbed but also on their conformation. Hence two surfaces poly(hydroxyethyl methacrylate) and poly(2-methoxyethylacrylate), both allow low protein binding, however, poly(2-methoxyethyl acrylate) that preserves the native conformation of adsorbed proteins displays remarkably lower platelet binding 36 .
Among the several protocols described for decellularization of vessels, various studies have shown that enzyme-based decellularization techniques lead to the destruction of the ECM 37 . However, in the protocol used in this study, previously reported by Seridan et al. 38 , trypsin treatment was applied for a short period of time followed by treatment with Triton X-100, which effectively removed the cellular material leaving an intact ECM structure formed mainly by collagen and elastin proteins as shown by histological and SEM analyses. These results are also similar to other studies which used Triton X-100 as a detergent for artery decellularization and showed a well preserved ECM 39,40 . Though the decellularization procedure did not cause extensive damage to the extracellular matrix of the arteries, it resulted in poor mechanical properties, in agreement with previous studies 41,42 . In the current work, mechanical tests revealed that uncoated decellularized arteries ruptured at a lower pressure than native arteries, as previously reported 43 and coating with the 8g7 polymer enhanced the mechanical properties of the decellularized vessels. Combination of decellularized vessels with polymers such as poly ε-caprolactone (PCL) 44 and poly(D,L-lactide) (PDLLA) 45 has been reported to improve the biomechanical properties of the graft. Due to their diverse mechanical properties, a wide range of biocompatible polyacrylates has found varied applications in biomedicine ranging from poly(methyl methacrylate) (PMMA) used in artificial teeth and bone cements to polyhydroxyethyl methacrylate used in soft contact lenses, drug delivery systems and dressings [46][47][48][49][50] . Vascular grafts made of polyacrylates are also commercially available 51 . In this work we have demonstrated that the mechanical properties of decellularised arteries improved significantly when coated with poly(ethyl methacrylate-co-diethylaminoethyl acrylate). In general, poly(ethyl methacrylate) based polymers have Young's modulus lower than 'hard' polyacrylates (such as PMMA) and higher than 'soft' polyacrylates (such as poly(ethyleneglycol methacrylate) 52 .
In spite of the excellent physical and mechanical properties of promising synthetic polymers like poly(vinyl alcohol), in vitro and in vivo studies have failed to show any significant endothelial cell attachment on the grafts luminal surfaces 53 . Moreover, in vitro experiments with grafts made of synthetic materials currently in clinical use showed that ECs produced isolated clumps and appeared as small spheroids with single point contact 54 . Synthetic vascular grafts coated with either antibodies or integrin-binding peptides have been developed for increasing cell adhesion to the inner lumen directly from the circulating blood stream 12, 55-57 . However, nonspecific adhesion of non-EPC remains major concern as this can lead to problems such as restenosis, inflammation or hyperplasia 58 . Thus, physical and biochemical surface modifications have been seen to be necessary to improve the cell attachment on these synthetic polymers 53,59,60 . From a clinical point of view, the growth of ECs needs a fully defined microenvironment which could be provided by chemically defined synthetic polymers such as 8g7.
Polymer 8g7 provides a superior substrate for attachment and proliferation of ECs where cells appeared homogenously distributed and exhibit the typical cobblestone monolayer morphology associated with high levels of attachment and enhanced cellular proliferation 54 . Moreover, decellularized tissue-engineered arteries can be stored and thus made readily available for patients similar to synthetic graft materials. Emerging approaches have started to engineer coated grafts by combining biological tissue and synthetic polymers. Recently, Gong et al. fabricated a hybrid tissue engineered vascular graft by the application of an electrospun nano-polycaprolactone coating on the outside of a decellularized rat aorta, which improved the biomechanics of the decellularized vessel. However, they also needed to incorporate exogenous heparin to modify the acellular vascular intimal surfaces 44 . In another study, the anticoagulant lepirudin was added to a Poly-(D,L-lactide) coating applied in decellularized arteries. Although it showed a reduction in the thrombogenicity and in the ECM's rate of degeneration, no evidence of recellularization was documented after in vivo implantation of the graft, suggesting that polymer coating might result in inhibiting cellular repopulation 45 .
Here, we demonstrated for the first time that the endothelium of decellularized arteries is regenerated by coating with a synthetic polymer with lower affinity for platelets while showing improved mechanical properties.
These results suggest that polymer coating of decellularized arteries may improve the in vivo blood compatibility of decellularized vessels and encourage future implantation studies to evaluate its patency, resistance to haemodynamic forces preventing thromboembolic events necessary for the long-term performance of small diameter vessels.

Conclusions
Our study demonstrates a novel method of coating decellularized vessels with poly(e thylmethacrylate-co-diethylaminoethyl acrylate). This material is biocompatible and readily synthesized. The polymer coating enhances attachment of ECs promoting endothelium regeneration, while it is able to reduce platelet attachment and improve the mechanical properties of decellularized vessels. This work suggests that the combination of the natural vascular architecture of decellularized arteries with the properties of 8g7 appear to be ideal for the development of chimeric tissue engineered vessels aiming to promote endothelialization and prevent thrombosis. These novel mixed grafts could help to overcome the current compliance mismatch between synthetic grafts and natural arteries.

Materials and Methods
Polymer synthesis. All chemicals were purchased from Sigma Aldrich (Gillingham, UK) and used as supplied without purification. Protocols are detailed in the supplementary section (Tables S1 and S2 and Figure S1). All methods and experimental protocols were approved and carried out in accordance to the guidelines and regulations of the University of Granada.
Polymer coating of coverslips. Polymers were spin-coated onto glass coverslips. Two sizes of glass coverslips were used, circular (19 mm diameter) and square (18 × 18 mm) (Menzel-Gläser, Braunschweig, Germany). Polymer solutions in THF (2% w/v) were spin-coated at 2000 rpm for 10 seconds using a spin coater (6708D, Speedline Technologies). The coated surfaces were dried in a convection oven at 40 °C overnight and sterilized using UV light prior to cell culture.
Capillary-like tube formation assay on polymer-coated coverslips and matrigel. HUVEC cell line was obtained from American Type Culture Collection (ATCC ® CRL-1730 ™ ) and cultured following ATCC recommendations. HUVECs were cultured in complete EGM-2 growth medium (Lonza, Basel, Switzerland) under standard cell culture conditions of 37 °C and 5% CO 2 . Cells were used between passages 4 and 5. Endothelial cells grown on matrices such as matrigel are able to migrate and align forming capillary-like or cord-like structures 61,62 . Here we have assessed if the 8g7 polymer can mimic matrigel's properties and induced the HUVECs cells to form the described tubule-like structures. To do so, HUVECs were seeded and incubated on polymer coated and uncoated coverslips (30000 cells/well) at 37 °C and 5% CO 2 in 12-well plates. In parallel, HUVECs were cultured in matrigel (BD Biosciences, San Jose, CA) coated 12-well plates as a control. After 4 h, cells were imaged (Nikon Eclipse Ti-U, USA) and the number of capillary-like structures was measured. Each cord portion between the ramifications was considered as one capillary unit 14,61 . Mean ± standard deviation (SD) values were obtained by evaluating four representative areas of each culture from three independent experiments and carried out by two observers. Additionally, the average number of cells that form each branch of the tubule-like and the mean number of branching points (the nodes where branches meet 63 ) per each capillary-like structure formed were quantified from four random fields at 10x magnification and obtained from three independent experiments using ImageJ ™ program 62 . Platelet adhesion on the polymer under flow conditions. Platelet adhesion on uncoated and polymer-coated coverslips was assessed using a parallel-plate perfusion chamber 64,65 . Blood from healthy human donors was drawn by venipuncture and processed immediately after collection 13 . Informed patient consent was obtained for all samples used in this study and samples were collected in accordance with the Research Ethics Committees of the Hospital Clínic of Barcelona (CEIC 2009/4700), and University of Granada. Two coverslips were individually inserted into the separate receptacles of the perfusion chamber and citrate-anticoagulated blood was recirculated at shear rates of 600 s −1 , 800 s −1 , and 1200 s −1 for 5 min. The coverslips were then rinsed with 0.15 M phosphate buffered saline (PBS), fixed with 0.5% glutaraldehyde in 0.15 M PBS at 4 °C for 24 h and stained with 0.02% toluidine blue for morphometric evaluation. The perfused surface was imaged using a video camera (Leica) fitted to a light microscope (Polyvar, Reichert-Jung). Two independent experiment were performed (n = 2) and percentage of the area covered by platelets of 3 different areas per coverslip was obtained using ImageJ ™ program, by comparing the area of platelet coverage to the area of the image.
Tissue harvest. Porcine carotid arteries were obtained from a slaughterhouse and transported to the laboratory stored in cold PBS with 1% penicillin/streptomycin. Immediately after arrival, carotid arteries were cleaned of excess of connective and adventitial tissues and rinsed in sterile PBS. Common carotid arteries 3-4 mm in diameter were cut into approximately 3-5 cm long segments ( Figure S2), frozen in PBS at −80 °C and defrosted just before use.
Decellularization process. The decellularization process of carotid tissue samples was performed as previously described using enzymatic digestion and detergent extraction 38 . Carotid arteries were immersed in de-ionized water for 24 h at 4 °C followed by treatment with 0.05% Trypsin with 0.02% EDTA (Sigma Aldrich, St. Louis, MO, USA) for 1 h at 37 °C. After a short rinse in PBS to remove excess trypsin, the samples were treated with a solution of 2% Triton X-100 and 0.8% ammonium hydroxide (Sigma) in de-ionized water for 72 h at 4 °C changing the solution every 24 h. After the decellularization process, samples were washed in de-ionized water three times, for 24 h each, to remove chemical residues. All steps were carried out under continuous shaking except trypsin incubation.
Polymer-coating of decellularized arteries. After cannulation with a three-way stopcock, (see Supplementary Data, Figure S2), 300 μL of the 8g7 polymer solution (2.0% w/v in 25% acetic acid) was injected into the decellularized arteries and incubated for 30 min at 4 °C under gentle shaking. Decellularized arteries were also injected with 25% acetic acid solution as a control. Coated and uncoated decellularized carotid arteries were dried 2h at room temperature in a fume hood, washed twice with PBS and irradiated with UV light for 30 min before use.
Mechanical testing. Samples were divided into three groups: (i) native arteries, (ii) decellularized arteries and (iii) polymer-coated decellularized arteries. The biomechanical properties of all the groups (n = 3, in each group) were determined by conducting uniaxial tensile tests using an ad hoc and lab-made device ( Figure S3). The controlled static load by pumping water (using a peristaltic pump) at 8 grams increments (with a pause of 20 seconds after each increment), while keeping the vessels continuously hydrated. The strain was measured by image correlation techniques from a high-resolution video. The video was triggered at the beginning of pumping and a custom image capture software was used to image at a rate of 1 frame/increment. This allowed to track the motion of the speckle pattern applied on the tissue, and their large displacements were calculated by analyzing the recorded images ( Figure S4) as previously reported 38,66 .
Ultimate tensile strength, load and burst pressure were determined. For ultimate tensile strength and load, ring sections were cut from each sample, mounted on parallel aligned custom-made holders and loaded until failure. The burst pressure was calculated from their failure load where radial stress was calculated and converted to millibars taking into account load, mean radius and ultimate circumferential tensile strength 67 . Diameter, width, thickness and length of each sample used for the calculations were measured using a high-precision slide caliper.
Arteries recellularization. Both, 8g7 polymer coated and uncoated decellularized carotid arteries were pre-incubated overnight in complete EGM-2 medium (Lonza, Basel, Switzerland) cannulated with a three-way stopcock. The excess media was removed and HUVECs (at passage 4, 1 × 10 6 cells/mL) were seeded onto the luminal surface of the arteries (length, 15 mm; inner diameter, 4 mm) (n = 3). Vessels were rotated 90° around its longitudinal axis every 30 min until all surfaces had been exposed to cells. After 360° rotation (2 h), cannulated arteries were immersed in media and the graft was incubated for another 24 h to ensure complete cells attachment. After 24 h and every 2 days thereafter, medium was changed. Non-seeded processed arteries were maintained in media and served as control samples. Cells were maintained in culture for 5 days before being collected for analysis.
Histological and immunofluorescence analyses. Samples were divided into five groups: (i) native arteries, (ii) decellularized arteries, (iii) polymer-coated decellularized arteries, (iv) non polymer-coated recellularized arteries and (v) polymer-coated recellularized arteries. Ring segments of 5 mm thickness were taken from each sample group for histological analyses. Samples were fixed in 4% paraformaldehyde, embedded in paraffin in an automatic tissue processor (TP1020, Leica, Germany), cut into 5 μm sections and stained with Hematoxylin and Eosin (H&E) and Masson's Trichrome as previously reported 68 . The number of cells on decellularized arteries and polymer-coated decellularized was quantified from four representative histological staining images at 40x magnification from three independent experiments using ImageJ ™ program. For 4′, 6-diamidino-2-phenylindole (DAPI, Sigma) staining, slides were deparaffinized, rehydrated and stained with mounting medium (Vectashield, Burlingame, CA, USA) containing DAPI. Bright field and fluorescent (DAPI) images were acquired with an inverted microscope (Nikon H550s, USA).
Laser confocal microscopy. Samples of uncoated and polymer-coated recellularized arteries were washed twice with PBS and labeled with the fluorescent dye, CellTracker Green/CMFDA (Invitrogen Inc., Carlsbad, CA,


s2fieldsofstudy: Biology Medicine

text:
Collagen Autoantibodies and Their Relationship to CCP Antibodies and Rheumatoid Factor in the Progression of Early Rheumatoid Arthritis

Serum autoantibodies to cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) are important markers for diagnosis and prognosis of rheumatoid arthritis (RA), but their autoantigens are not cartilage-specific. Autoantibodies to joint-specific type II collagen (CII) also occur in RA, and monoclonal antibodies of similar specificity induce collagen antibody-induced arthritis in animals, but their role in RA is uncertain. We utilized an enzyme-linked immunosorbent assay (ELISA) with the CB10 peptide of CII to compare the frequency of autoantibodies with those of anti-CCP and RF in stored sera from a prospective study of 82 patients with early RA to examine the outcome, defined as remission (n = 23), persisting non-erosive arthritis (n = 27), or erosions (n = 32). Initial frequencies of anti-CB10, anti-CCP and RF were 76%, 54%, and 57% in RA, and 4%, 0%, and 9% in 136 controls. The frequency of anti-CB10 was unrelated to outcome, but anti-CCP and RF increased with increasing severity, and the number of autoantibodies mirrored the severity. We suggest RA is an immune complex-mediated arthritis in which the three antibodies interact, with anti-CII inducing localized cartilage damage and inflammation resulting in citrullination of joint proteins, neoepitope formation, and a strong anti-CCP response in genetically-susceptible subjects, all amplified and modified by RF.

Introduction
Rheumatoid arthritis is a chronic inflammatory arthritis of unknown cause. Dating back to the late 1940s when rheumatoid factor (RF) was first detected in patients with rheumatoid arthritis, circumstantial evidence suggested the perpetrator was autoimmune. However, precise early diagnosis is difficult, and clinical diagnosis is made by rheumatologists based on exclusion of other possible diagnoses and consideration of the fulfilment of criteria proposed by the American Rheumatism Association (ARA) in 1954 and revised in subsequent years [1][2][3][4]. These criteria have defined the disease more precisely and attracted widespread research on the clinical, histologic, immunologic, pharmacologic, and genetic aspects of RA, as well as addressing controversial issues regarding the understanding of the disease. The findings have largely substantiated the view that the mechanism underlying RA is autoimmune, but the identity of the joint-specific autoantigen involved in the failure of immunological tolerance is uncertain.
RF as a diagnostic marker of RA may precede clinical symptoms by many years [5] and is associated with more severe disease and poor prognosis. Evidence of synthesis of RF within the joint, helical CB10 fragment of the native CII molecule to detect antibodies to CII [51]. CB10 is one of the two major peptides derived by digestion of CII with cyanogen bromide that contain important arthritogenic epitopes in animal studies, and represents 30% of intact CII. Using this CB10 ELISA, we detected autoantibodies in 84 (88%) of 96 patients with RA compared with 24% using intact CII. In contrast, anti-CB10 was present in only 4 of 33 (12%) patients with psoriatic arthritis, 2 of 34 (10%) with osteoarthritis, and three of 93 (3%) blood donors [51]. CB11, the second peptide that contains major arthritogenic epitopes in animal studies was also highly reactive, but the disease specificity of the response was reduced.
In that study, the patients had established RA, and were attending the Rheumatology Clinic of the Royal Melbourne Hospital, a tertiary referral centre. All patients fulfilled the 1987 ARA criteria; 78% had active disease, and the median duration of arthritis was 13 years. The aim of the present study was to determine whether anti-CB10 was present in the serum of patients with early RA, and to compare the frequency of anti-CB10, anti-CCP and RF, using stored sera from a community-based prospective study of early inflammatory arthritis from first referral to a specialist rheumatologist. Sera were tested from 82 patients for whom the final diagnosis was early RA to examine the associations of these three autoantibodies with the outcome: remission, persisting non-erosive arthritis, or development of erosions ( Table 1). The study was carried out before the development of biologic therapies [52] and before anti-CCP was included among the American College of Reumatology (formerly ARA) criteria.

Frequency of Autoantibodies to CB10 in Established RA
Receiver operating characteristic (ROC) curve analysis was performed to establish a cut-off for the anti-CB10 ELISA, using sera from 52 patients from a cross-sectional study of long-standing erosive RA and 136 control sera. The area under the ROC curve was 0.738 (95% CI 0.669 to 0.799). Taking a level of specificity of 95%, a value greater than 48 units was considered positive; under these conditions, 34 (65%) of the RA sera were positive, and six of 136 controls (4%, 3/63 blood donors, 1/31 systemic lupus erythematosus (SLE), 2/42 other unspecified patients).

Autoantibodies to CB10 in Early RA
Antibodies to CB10 were compared in the initial serum sample from 82 sera from patients with early arthritis, and the 136 control sera (Figure 1). Using the previously established value of 48 units to define the upper limit of normal, 62 (76%) of the patients were positive compared with 65% of the patients with long-standing RA. The frequency of anti-CB10 at presentation was not related to the progress of the arthritis, and most patients had anti-CB10, although the frequency was highest in those who attained remission (87% remission; 67% persisting; 75% erosive) ( Figure 2, Table 2). There was no association between the presence of anti-CB10, and either one or two copies of the SE. patients with long-standing RA. The frequency of anti-CB10 at presentation was not related to the progress of the arthritis, and most patients had anti-CB10, although the frequency was highest in those who attained remission (87% remission; 67% persisting; 75% erosive) ( Figure 2, Table 2). There was no association between the presence of anti-CB10, and either one or two copies of the SE.    patients with long-standing RA. The frequency of anti-CB10 at presentation was not related to the progress of the arthritis, and most patients had anti-CB10, although the frequency was highest in those who attained remission (87% remission; 67% persisting; 75% erosive) ( Figure 2, Table 2). There was no association between the presence of anti-CB10, and either one or two copies of the SE.

Anti-CCP in Early Inflammatory Arthritis
For anti-CCP, 44 (54%) of the patients with early arthritis had antibodies, compared with 36 (69%) of the patients with long-standing RA. The frequency of anti-CCP at disease onset was increased among those patients who developed erosive arthritis when compared with those with persisting non-erosive arthritis and was lowest in those who attained remission (remission 22%, persisting non-erosive 52%, erosive 78%), but the levels of anti-CCP among positive sera were similar in each group. Thus, the median level and range of positive anti-CCP among five patients who remitted was 835 (300-2000), compared with 516 (60-3100) for 14 patients with persisting arthritis and 814 (35-3420) for 25 patients with erosive arthritis. Levels of anti-CCP were not related to length of time before the development of erosions.
Among the total group of 69 patients for whom HLA typing was available, there was a significant association between the SE, and anti-CCP, and 26 of 40 patients (65%) with the SE had anti-CCP, compared with 10 of 29 (34%) without (χ 2 = 6.27, p = 0.0122), consistent with the reported linkage between the SE and enhanced antigen presentation of citrullinated peptides [31]. However, the 10 patients with anti-CCP but without the SE were all DR2 (n = 7) and /or DR7 (n = 4), and seven of these had erosive arthritis. High levels of anti-CCP have been associated with HLA DRB1*15 (previously recognised with HLA DRB1*16 as DR2) in patients with RA who lack the SE [53].

Rheumatoid Factor in Early Inflammatory Arthritis
RF was present in the initial serum from 47 (57%) of the patients with early arthritis, compared with 67% of patients with long-standing arthritis. Like anti-CCP, RF occurred most frequently in erosive arthritis, and least in those patients who attained remission (remitting 30%, persisting non-erosive 67%; erosive 69%).

Predictive Value of Autoantibodies
Despite the increased frequency of both anti-CCP2 and RF with more severe disease, none of the autoantibodies alone was sufficient to predict the course of the disease. Combinations of antibodies at presentation were more informative, and generally, the more autoantibodies that were detected, the poorer the outcome for the patient ( Figure 3A). Six patients had no detectable antibodies, and 27 had a single antibody, of whom 21 had anti-CB10, two, both with erosive arthritis, had anti-CCP and 4 patients had RF ( Figure 3B). Of the 33 patients who had no autoantibodies or only one of the three, 16 (48%) attained remission, and eight (24%) developed erosive arthritis, whereas of 29 patients who had three autoantibodies, three (10%) attained remission, and 16 (55%) developed erosive arthritis.

Sequential Changes in Autoantibodies
Levels of autoantibodies were measured in sequential sera from 20 patients, of whom five attained remission, six had persisting non-erosive arthritis, and nine had erosive arthritis. Although the levels of anti-CB10 fluctuated markedly in some patients but remained steady in others, overall there was a trend to lower levels of anti-CB10 as the disease progressed, and whereas 18 of the 20 (90%) patients had antibodies initially, only 11 (55%) had antibodies in the final sample (χ 2 = 4.20, p = 0.04). Complete loss of anti-CB10 was particularly associated with progression to erosive arthritis, and of the nine patients with erosive arthritis who were examined sequentially, six became negative for anti-CB10 at the end of the study, and two patients were negative throughout.
In contrast to the decrease in anti-CB10 reactivity seen in the patients followed sequentially, levels of anti-CCP among the 10 patients who had anti-CCP initially (four persisting non-erosive, six

Sequential Changes in Autoantibodies
Levels of autoantibodies were measured in sequential sera from 20 patients, of whom five attained remission, six had persisting non-erosive arthritis, and nine had erosive arthritis. Although the levels of anti-CB10 fluctuated markedly in some patients but remained steady in others, overall there was a trend to lower levels of anti-CB10 as the disease progressed, and whereas 18 of the 20 (90%) patients had antibodies initially, only 11 (55%) had antibodies in the final sample (χ 2 = 4.20, p = 0.04). Complete loss of anti-CB10 was particularly associated with progression to erosive arthritis, and of the nine patients with erosive arthritis who were examined sequentially, six became negative for anti-CB10 at the end of the study, and two patients were negative throughout.
In contrast to the decrease in anti-CB10 reactivity seen in the patients followed sequentially, levels of anti-CCP among the 10 patients who had anti-CCP initially (four persisting non-erosive, six erosive), remained stable whether low level (<100), mid-level (>100 < 1000), or high level (>1000), and none lost reactivity. Four patients developed anti-CCP during the follow-up. In each case the patients had anti-CB10 initially. One patient who developed erosive arthritis had anti-CCP (800 units) when tested at one year. Both tests were still positive at four years, although the levels of anti-CB10 were falling, and both tests were negative by eight years. A second patient with persisting arthritis had a single high anti-CCP (550 units) at five years; the test was again negative at six years. Two further patients, one of whom remitted and one who developed erosive arthritis, had transient low levels of anti-CCP (<70 units), although both were strongly positive for anti-CB10.
Among the 20 patients for whom sequential sera were tested, 11 patients had RF initially (five persisting non-erosive, six erosive). There was no consistent pattern of changes observed in these patients. Three of these patients recorded at least one negative test, and the test became positive in one patient.

Discussion
RA is a disease of variable onset and expression that reflects its multigenic origin [54]. It has been considered to be an immune complex disease, based on the local production of autoantibodies, historically rheumatoid factor, with immune complexes and a low level of complement within the joint [6,11]. For many years type II collagen has been proposed as the elusive joint-specific autoantigen [55], but studies have been limited by the lack of reproducible and reliable assays for antibodies to CII. Using an assay to detect antibodies to the CB10 peptide of CII, we reported antibodies in the sera of 88% of patients with long-standing severe RA [51]. In the present study, we have examined the potential role of autoantibodies in the development of RA and the clinical course in 82 subjects with early inflammatory arthritis, most fulfilling the 1987 criteria [1] and excluding other inflammatory arthritis, prior to the availability of biologic therapies. These subjects were followed prospectively over a period of up to 10 years, employing assays that were comparable in sensitivity and specificity in established RA, to compare the presence of three autoantibody populations, anti-CB10, anti-CCP and RF in stored sera taken at the onset of symptoms. The outcome was categorised as attainment of remission, persistence of arthritis, or development of erosions. No similar study in early RA has been reported, and although the number of subjects studied was small, current emphasis on the importance of early treatment and the advent of new biologics would now make such a study difficult to perform.
Of the three autoantibodies, anti-CB10, used as a measure of anti-CII, was detected in 76% of patients overall, including 87% of 23 patients who attained remission, 12 of whom had only anti-CB10, and was the most abundant of any of the three autoantibodies. Levels of anti-CB10 were higher in the patients with early RA than in patients with established disease and in the 20 patients for whom sequential samples were available levels decreased with time. Similar falls in levels of circulating anti-CII have been observed by others [33][34][35]56] and a possible explanation for this is that serum antibodies to CII are progressively removed from the circulation and sequestered within the cartilage as damage to the matrix and proteoglycan loss exposes more CII to complex with the antibody. Elution of anti-CII from cartilage of subjects with RA [57] lends support to this notion. These results are consistent with a role for CII as the elusive joint-specific autoantigen in RA, but the frequency of anti-CII in patients who remitted suggests that this antibody alone is insufficient to promote severe arthritis.
In contrast to anti-CB10, anti-CCP occurred in only 54% of the patients overall but was strongly associated with the development of erosions, being present in 78% of those who developed erosive arthritis. Anti-CCP production was linked to the SE, but not all patients who developed erosions had the SE and the development of erosions were more strongly associated with anti-CCP than with the SE, possibly as a result of an additional HLA-association with DR15 [53]. Anti-CCP represents reactivity with a wide range of citrullinated antigens, few of which may be expressed in the joint, and in contrast to the changes in levels of anti-CB10, levels of anti-CCP in serum showed very little change.
The third autoantibody, RF, was present in 57% of patients at onset, and in 69% of those who developed erosive arthritis. Unlike anti-CCP, serum levels of RF varied inconsistently amongst those who were followed sequentially. Possibly, it plays a role in immunoregulation by cross-linking Fc-regions of antibodies, increasing the net avidity and specificity of antibodies of low affinity, forming immune complexes and activating complement. Accordingly, it could play an essential role in removing pathogens [13]. RF has been particularly associated with antibody responses to repetitive, highly-ordered patterns of epitopes, such as those that occur in collagen [58].
Taken together, all of these autoantibodies could contribute to immune complex formation. Once on-going inflammation is established, the specificity of the initiating autoantibodies (anti-CII) may become an ongoing but minor component of the flagrant inflammatory response as RF and antibodies to various citrullinated antigens become the driving force for the feedback loop involving inflammation, cytokine production, matrix damage, and chondrocyte activation. In the present study, the presence of all three autoantibodies, anti-CB10, anti-CCP, and RF, was a predictor of outcome: 13% in those who attained remission, 37% in those with persistent disease, and 50% in those with erosions. For each of these antibody systems, the autoantibodies would contribute directly to immune complex mediated damage according to their class and subclass by cellular interactions involving binding to Fc-receptors, and complement activation.
This view of RA as an immune complex-mediated disease of articular joints has been supported by the development of models of antibody induced arthritis, both CAIA, induced using mAb to CII [48], and also K/BxN serum-transfer arthritis (K/BxN STA) [59], induced using mAb derived from mice of the K/BxN transgenic strain that spontaneously develop severe arthritis. In the K/BxN STA model autoantibodies are to glucose-6-phosphate isomerase, a ubiquitously expressed intracellular enzyme that is expressed on the cartilage surface in mice. In both of these models, despite the substantial differences in the inductive phase of the arthritis there are common pathways of expression and similar outcomes in which the inflammatory response is driven by autoantibodies that form immune complexes within the joint, activation of innate immune cells, such as neutrophils and macrophages, and immune mediators, including cytokines, chemokines, complement, Toll-like receptors, Fc receptors, and integrins. In both models, the arthritis exhibits leukocyte invasion, synovitis, pannus formation, and cartilage and bone destruction similar to that seen in RA [48,55,59]. These symptoms are entirely antibody-initiated, representing the effector arm of an immune response primarily driven by Fc-mediated interactions.
The repetitive sequence of epitopes on the CII molecule of the cartilage favours formation of multimeric immune complexes and activation of the complement system. This attracts a cellular infiltrate resulting in the release of cytokines, chemokines, and various enzymes which contribute to inflammation and further damage to cartilage and surrounding tissue. Particularly relevant is the incursion of polymorphonuclear leucocytes, monocytes, and macrophages that generate calcium-dependent peptidylarginine deiminase enzymes [16,60] with the capacity to citrullinate arginine molecules on the autoantigenic motifs of CII [49], as well as a range of peptides that are part of the joint structure, but are not specific to joints. These citrullinated neoepitopes result in T cell responses under the influence of the SE [31], stimulating localised ACPA production within the joint [61] and the development of antibodies that are primarily determined by the presence of the SE rather than the disease itself [62]. However, a response to a neoepitope seen as a "foreign" antigen may well be poorly regulated, with enhanced potential to drive local immune complex formation. It then becomes a marker of severe disease [63] as confirmed by our finding of 78% of subjects who developed erosions.
In both CAIA and K/BxN STA, combinations of mAb reactive with different epitopes on the antigen more readily induce arthritis than any single mAb alone [64,65] and this may merely represent enhanced ability to form mAb/Ag multimers, with increased FcR and complement activation.
However, although these diseases can be considered as induced by immune complexes, in which Fab-specificity of the antibody is secondary to immune activation by Fc-mediated effects, further levels of complexity are contributed by the specific Fab interactions of epitopes recognised and this complexity may be what is seen in the polyclonal response in RA. In CAIA, both arthritogenic and protective mAbs have been identified, and epitope analysis has identified regions of the CII molecule where mAb binding causes direct cartilage damage [44,46,[66][67][68][69][70].
Articular cartilage has an unusual structure, as it is a relatively acellular tissue in which a small number of chondrocytes are surrounded by an abundant extracellular matrix. It comprises a network of fibrils of CII and other less abundant collagens, in which are entrapped negatively-charged glycosaminoglycans and proteoglycans, particularly hyaluronan and aggrecan, and other minor, but important, components that interact to maintain the integrity of the matrix and allow frictionless articular movement. The chondrocytes are entirely responsible for the formation and synthesis of the matrix. In cartilage explant cultures, in the absence of inflammatory mediators, several arthritogenic mAbs to CII cause marked proteoglycan loss from the cartilage surface where the antibody penetrates, with progressive denaturation and damage to the collagen matrix [46,69,71].The same arthritogenic mAbs also affect synthesis of new matrix by chondrocytes in vitro, with effects both on cell morphology and on collagen synthesis that differ according to the specificity of the mAb [46,70]. These changes are Fab-mediated, do not require cells other than chondrocytes, and are much more striking in cartilage where the chondrocytes have been killed by freeze-thawing [68]. It is likely that mAb derived from mice with CIA interfere with interactions within the cartilage, either between collagen and chondrocytes, or collagen and other matrix components. In adult cartilage, chondrocytes are relatively inert, and matrix turnover occurs only slowly, but the balance between damage and repair in culture is suggestive of the relapsing-remitting progress that is often seen in human RA. Moreover, as chondrocyte activity and matrix synthesis decreases with age, an imbalance between damage and repair as a result of antibody binding is consistent with the increased incidence of RA with age. Similar studies have not been performed with ACPA or RF, but the arthritogenic anti-CII mAb react with surface-exposed epitopes on CII that contain arginine, and equivalent ACPA mAb reactive with the citrullinated epitopes have been derived that enhance the development of arthritis [50,72], and may well have similar disruptive structural effects.
Although this is the only study we are aware of that has examined the three autoantibody populations in early RA a major limitation of this study, and of others, is that we have measured antibodies in serum, not synovial fluid. It would be both impractical and unethical to attempt to replicate such a prospective study using synovial fluid, but we are mindful that the site of inflammation is within the joint. Synovial fluid and serum represent two different compartments, with limited exchange between them and levels of IgG, IgM, or IgA in normal synovial fluids are a fraction of the levels in serum [73,74]. As each of the autoantibodies we have studied has been shown to be produced locally within the joint and is more abundant at the site of inflammation and damage, measurements of serum autoantibodies may be misleading and represent only a 'spill-over' from the site of the lesion in the joint. Despite this reservation, the measurement of the triad of autoantibodies-anti-CB10, anti-CCP and RF-has provided insight into the outcome of RA in subjects diagnosed with early inflammatory arthritis, and in nominating CII as the putative autoantigen implicated in the failure of immunological tolerance in RA, we have outlined the key steps in the evolution of the efferent arm of the autoantibody response.
Finally, an important aspect of this study is the observation that anti-CB10 were present very early in the course of disease in these patients. The diagnosis of early inflammatory arthritis as RA was a clinical diagnosis made in the course of treatment by a specialist rheumatologist (AS) after exclusion of other potential diagnoses. Not all patients fulfilled the ARA criteria for RA when first seen, noting that nodules and erosions are late sequelae, and four criteria require symptoms to be present for six weeks, whereas some patients in each group were first seen and commenced disease-modifying drugs within that period. We have rarely detected anti-CB10 in other diseases associated with arthritis, including osteoarthritis and psoriatic arthritis [51], or in the present study, in patients with SLE. The increasing emphasis of the importance of early treatment for RA, and the recognition that ARA criteria are not helpful for diagnosis in the first 12 months of the disease [75] suggest that the detection of autoantibodies to CII has important implications, clinically. The presence of these autoantibodies in early arthritis points to an autoimmune reaction affecting articular joints and should alert the clinician to the likelihood of a developing rheumatoid arthritis. Tests, including those determining the profile of autoantibodies, can then be undertaken to establish diagnosis, and implement treatment appropriate to the findings and the follow-up required.

Patients
Serum samples were tested from 82 patients diagnosed with early RA who were being followed as part of a prospective study of recent-onset inflammatory arthritis [52]. The patients were from the rheumatology community-based private practice of one of the authors (AS) who performed all assessments. The diagnosis was based on the clinical assessment of the rheumatologist and patients with evidence of other recognized inflammatory arthritis, such as psoriatic arthritis, crystal arthritis, or other connective tissue diseases, were excluded. Inclusion diagnosis were two or more swollen and painful joints present for at least two weeks. Patients were not required to fulfil 1987 ARA criteria [1]. Median duration of arthritis at baseline was three months (range: 1 week-8 months) [51]. As part of this study, clinical assessments included the Ritchie articular index for tender joints [76], the total number of swollen joints, 1987 ARA criteria fulfilled, ESR, and presence of erosions in radiographs of the hands and feet assessed based on a Larsen score of 2 or more [77] (Table 1). Positivity for the DRB1 shared epitope (SE) that has been linked to the pathogenesis of RA was assessed by DRB1 genotyping performed as a two-step process using PCR amplification followed by hybridization with sequence-specific oligonucleotides [52]. The initial serum samples tested were obtained within eight months of the onset of symptoms (median: 3 months; range: 2 weeks-8 months). All patients were followed prospectively for at least two years (median: 4.5 years; range: 2-10 years); 32 (40%) developed erosive arthritis, usually within three years, and 21 (26%), including two with erosions, attained remission that was defined as no evidence of clinical synovitis and no requirement for disease-modifying drugs or corticosteroids for 12 months [78]. Details of the groups of patients are shown in Table 1. To examine changes in antibody levels, 123 additional sera were tested from 20 of the patients with early RA, with a median follow-up of five years (range: 2-8 years).
For comparison, sera from 52 patients with long-standing RA, according to the 1987 criteria of the American College of Rheumatology (ACR) were included to determine the frequency of autoantibodies in established disease. These patients had a median age of onset of 45.5 years (range: 25-74 years), and duration of 15 years (range: 2-61 years). They were included as part of a cross-sectional study of rheumatoid arthritis for whom clinical and genetic data have previously been reported [79,80]. All had erosions at the time of testing. One hundred thirty-six additional controls included 63 sera from blood donors attending the Red Cross Blood Bank in Melbourne, and 73 sera obtained under code from pathology laboratories. Of these, 31 were from patients with systemic lupus erythematosus, all of whom had antibodies to ds-DNA, and 42 were a random selection of sera discarded by the hospital biochemistry laboratory and, for these sera, no clinical details were available. Sera from the patients with RA were collected over a 10-year period, and stored at −20 • C for later testing. All tests were performed at the same time, and sequential sera from a single patient were tested in the same assay.
All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and was approved by the Royal Melbourne Hospital Board of Medical Research and Ethics Committee of the Royal Melbourne Hospital. The Monash University Human Research Ethics Committee (MUHREC) approved the use of the stored sera under the provisions of "Ethics Approval for the Use of Stored Human Tissue Samples".

Preparation of CB10
Bovine type II collagen was prepared from cartilage of the nasal septum by pepsin digestion and differential salt precipitation as described previously [81], and digested with cyanogen bromide [82]. CB10 was purified using cation exchange chromatography at 42 • C on carboxymethyl cellulose (CM-cellulose, Whatman CM-62) equilibrated in 0.02 M sodium citrate, and elution with a 0.01 M-0.16 M linear salt gradient. To further purify CB10 from CB11, CB8 and CB9,7 fractions were pooled, desalted and lyophilised, and applied to a P-30 column (100-200 mesh, exclusion limit 40 kD, globular proteins. Biorad, Hercules, CA, USA) equilibrated in 0.1 M acetic acid. To determine the purity of the peptides, fractions were run on 15% tricine gels and stained with 0.2% (w/v) Coomassie blue. Purified CB10 was renatured by stepwise cooling from 20 • C to 0.2 • C [83].

Detection of Antibodies to CB10 by ELISA
Antibodies to CB10 were measured using a previously developed ELISA in which intact CII was replaced with the CB10 peptide. Microtiter plates were coated overnight at 4 • C with 100 µL of 20 µg/mL CB10 in PBS pH 7.4, and blocked for 2 h with 200 µL of 1% skimmed milk in PBS (SMPBS) containing 0.05% Tween 20. Sera were tested in duplicate at a dilution of 1:200 in the blocking buffer, using a 2 h incubation. Antibodies were detected using horseradish peroxidase-conjugated (HRP) sheep anti-human IgG (Amrad, Hawthorn, Australia) diluted 1:2000 in SMPBS without Tween 20. As CB10 is unstable above 20 • C, all incubations to this point were performed at 4 • C, using cold buffers. Plates were equilibrated to room temperature for 10 min, and colour development was carried out at room temperature using 100 µL 3,3 , 5,5 tetramethylbenzidine liquid substrate for ELISA (Sigma-Aldrich, Sydney, Australia); absorbance was read at 655 nm at exactly 10 min. The monoclonal antibody M2139 that reacts with the J1 conformational epitope in CB10 [44], was included to confirm that the CB10 retained its correct conformation. It was defined to contain 100 units of reactivity, and used as a standard with titration from 1:200 on every plate.

Measurement of Autoantibodies to Citrullinated Protein Antigens
ACPA were measured using the commercial anti-CCP2 assay from Euro-Diagnostica that is based on a panel of cyclic citrullinated peptides, according to the manufacturer's instructions, with a manufacturer-derived cut-off for positivity of 25.

Measurement of RF
The presence and titer of rheumatoid factor was measured by external laboratories as part of the initial clinical assessment of the patients, but stored sera were retested for this study using a Roche turbidimetric assay for RF according to the manufacturer's instructions, with a manufacturer-derived cut-off for positivity of 14.

Statistical Analysis
MedCalc statistical software [84] was used for derivation and analysis of receiver operating characteristic curves (ROC curves) for the autoantibodies.

Conclusions
RA is a common chronic inflammatory disease of articular joints that often leads to joint destruction. The cause is unknown, but is best explained by autoimmunity, mainly based on the presence of autoantibodies and compelling evidence from experimental animal models. However, in autoimmunity the induction and effector mechanisms presently lack transparency and, in RA, the autoantigen implicated in the failure of immunological tolerance is still in dispute. In the present study we propose the arthritogenic response commences with an autoimmune response to CII and this results in immune complex formation due to autoantibody binding to repeated epitopes on the large triple helical molecule of CII on the articular joint surface. This destabilizes cartilage, attracts cells associated with inflammation, and results in an independent neoimmune response to citrullinated proteins that include not only CII, but a number of ubiquitous proteins that are under the influence of the SE. Formation of immune complexes result in the development of RF, which may either potentiate the immune response or have a role in clearance. This sequence of events is supported by our finding of a high frequency of anti-CB10 representative of anti-CII in subjects with early inflammatory arthritis, the persistence of anti-CCP as severity increases, and the presence of "the full-house" of autoantibodies-anti-CB10, anti-CCP, and RF-in the subjects with severe disease.