Chitosan shows effective nucleic acid delivery. To understand the influence of chitosan’s molecular weight, dose, payload, and hyaluronic acid coating on in vivo toxicity, immune stimulation, biodistribution and efficacy, precisely characterized chitosans were formulated with unmodified or chemically modified siRNA to control for innate immune stimulation. The hemocompatibility, cytokine induction, hematological and serological responses were assessed. Body weight, clinical signs, in vivo biodistribution and functional target knockdown were monitored. Hemolysis was found to be dose- and MW-dependent with the HA coating abrogating hemolysis. Compared to cationic lipid nanoparticles, uncoated and HA-coated chitosan nanoparticles did not induce immune stimulation or hematologic toxicity. Liver and kidney biomarkers remained unchanged with chitosan formulations, while high doses of cationic lipid nanoparticles led to increased transaminase levels and a decrease in body weight. Uncoated and HA-coated nanoparticles accumulated in kidneys with functional knockdown for uncoated chitosan formulations reaching 60%, suggesting potential applications in the treatment of kidney diseases.

With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena. To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents. To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration. To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B. Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics. The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.

Surgical reattachment of torn rotator cuff tendons can lead to satisfactory clinical outcome but failures remain common. Ortho-R product is a freeze-dried formulation of chitosan (CS) that is solub...

A novel approach for stimulating articular cartilage repair was developed and evaluated in skeletally aged Arcott sheep with signs of early osteoarthritis. Freeze-dried (FD) chitosan formulations were optimized to produce ultraporous cylinders that slowly rehydrate and disperse into bioactive chitosan microparticles in coagulating blood plasma. FD-chitosan implants (80% Degree of Deacetylation, 85 kDa) were produced at 3 doses (initial concentrations of 5, 10, 20 mg/mL, pH 2.5). Full-thickness cartilage defects were created bilaterally in medial femoral condyles of 8-9 year-old sheep (N = 12), microdrilled with 11 holes, then in one knee per sheep, one implant cylinder was inserted into each bleeding drill hole. At 1 day (N = 2), 3 months (N = 5) and 9 months (N = 5) post-operative, repair tissues were analyzed macroscopically and by micro -computed tomography, histology, biochemistry, and mechanics. Chitosan microparticles were detected in day 1 subchondral blood clots and mostly cleared at 3 months. At 3 months, microdrill holes were 2 -fold larger, filled with angiogenic granulation tissue, callus, and woven bone, with more chondroinduction in treated versus control drill holes (p = 0.021). At 9 months, biomaterial treatment enhanced bone plate repair and stimulated 68% cartilage resurfacing vs 53% for drill-only controls (p = 0.047). Both treated and control cartilage repair tissues had lower glycosaminoglycan content than intact cartilage and were thinner, stiffer, and more permeable. Upon indentation, hyaline-like repair cartilage showed poroelastic behavior. This study showed that FD-chitosan can be locally delivered to incorporate chitosan microparticles into subchondral bone blood clots and exert anabolic therapeutic effects on articular cartilage resurfacing in aged sheep knees.

Bone-marrow stimulation (BMS) improves knee-joint function but elicits incomplete repair. Liquid chitosan (CS)-glycerol phosphate/blood clots have been shown to improve BMS-based cartilage repair. Platelet-rich-plasma (PRP)-a rich source of growth factors and cytokines-improves recruitment and chondrogenic potential of subchondral mesenchymal stem cells. We hypothesised that repair response in a rabbit chronic-defect model will improve when freeze-dried CS/PRP is used to augment BMS. Bilateral trochlear defects created in New Zealand white rabbits were allowed to progress to a chronic stage over 4 weeks. Chronic defects were debrided and treated by BMS in second surgery, then augmented with PRP (BMS + PRP) or freeze-dried CS/PRP implants (BMS + CS/PRP). The quality of 8-week repair tissue was assessed by macroscopic, histological, and micro computed tomography (Micro-CT) analysis. ICRS macroscopic scores indicated fibrocartilaginous or fibrous repair in control defects that were improved in the BMS + CS/PRP group. An overall improvement in repair in BMS + CS/PRP group was further confirmed by higher O'Driscoll scores, %Saf-O and %Coll-II values. Micro-CT analysis of subchondral bone indicated ongoing remodelling with repair still underway. Quality and quantity of cartilage repair was improved when freeze-dried CS/PRP implants were used to augment BMS in a chronic defect model.

Rotator cuff tears result in shoulder pain, stiffness, weakness and loss of motion. After surgical repair, high failure rates have been reported based on objective imaging and it is recognized that current surgical treatments need improvement. The aim of the study was to assess whether implants composed of freeze-dried chitosan (CS) solubilized in autologous platelet-rich plasma (PRP) can improve rotator cuff repair in a rabbit model. Complete tears were created bilaterally in the supraspinatus tendon of New Zealand White rabbits ( n = 4 in a pilot feasibility study followed by n = 13 in a larger efficacy study), which were repaired using transosseous suturing. On the treated side, CS-PRP implants were injected into the transosseous tunnels and the tendon itself, and healing was assessed histologically at time points ranging from one day to two months post-surgery. CS-PRP implants were resident within transosseous tunnels and adhered to tendon surfaces at one day post-surgery and induced recruitment of polymorphonuclear cells from 1 to 14 days. CS-PRP implants improved attachment of the supraspinatus tendon to the humeral head through increased bone remodelling at the greater tuberosity and also inhibited heterotopic ossification of the supraspinatus tendon at two months. In addition, the implants did not induce any detectable deleterious effects. This preliminary study provides the first evidence that CS-PRP implants could be effective in improving rotator cuff tendon attachment in a small animal model.

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Chitosan (CS)/siRNA polyplexes have great therapeutic potential for treating multiple diseases by gene silencing. However, clinical application of this technology requires the development of concentrated, hemocompatible, pH neutral formulations for safe and efficient administration. In this study we evaluate physicochemical properties of chitosan polyplexes in various buffers at increasing ionic strengths, to identify conditions for freeze-drying and rehydration at higher doses of uncoated or hyaluronic acid (HA)-coated polyplexes while maintaining physiological compatibility. Optimized formulations are used to evaluate the impact of the siRNA/oligonucleotide sequence on polyplex physicochemical properties, and to measure their in vitro silencing efficiency, cytotoxicity, and hemocompatibility. Specific oligonucleotide sequences influence polyplex physical properties at low N:P ratios, as well as their stability during freeze-drying. Nanoparticles display greater stability for oligodeoxynucleotides ODN vs siRNA; AT-rich vs GC-rich; and overhangs vs blunt ends. Using this knowledge, various CS/siRNA polyplexes are prepared with and without HA coating, freeze-dried and rehydrated at increased concentrations using reduced rehydration volumes. These polyplexes are non-cytotoxic and preserve silencing activity even after rehydration to 20-fold their initial concentration, while HA-coated polyplexes at pH∼7 also displayed increased hemocompatibility. These concentrated formulations represent a critical step towards clinical development of chitosan-based oligonucleotide intravenous delivery systems.

Menisci display exquisitely complex structure and play an essential weight-bearing role in the knee joint. A torn meniscus is one of the most common knee injuries which can result in pain and mechanical abnormalities. Tear location is one aspect which determines the endogenous healing response; tears that occur in the peripheral densely vascularized zone of the meniscus have the potential to heal while the healing capacity is more limited in the less vascularized inner zones. Meniscectomy was once widely performed, but led to poor radiographic and patient-reported mid- and long-term outcomes. After the advent of arthroscopy, orthopaedic opinion in the 1980s has been swaying toward salvaging or repairing the torn meniscus tissue to prevent osteoarthritis rather than performing meniscectomy. Meniscus repair in young active individuals has been shown to be effective, reproducible, and reliable if indications are met; however, only a small proportion of all tears are considered repairable with available technologies. Biological augmentation techniques and meniscus tissue engineering strategies are being devised to enhance the likelihood and rate of healing in meniscus repair. Preclinical and clinical studies have shown that introduction of cellular elements of the blood, bone marrow, and related growth factors have the potential to enhance meniscus repair. This article reviews the current state of clinical management of meniscus tears (primary repair) as well as augmentation techniques to improve healing by meniscus wrapping with extracellular matrix materials, trephination, synovial rasping and abrasion, fibrin/blood clot placement, and platelet-rich plasma injections. In addition, the rationale for using polymer/autologous blood component implants to improve meniscus repair will be discussed.

Powered by TCPDF (www.tcpdf.org) This material is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form. You must obtain permission for any other use. Electronic or print copies may not be offered, whether for sale or otherwise to anyone who is not an authorised user. Kestilä, I.; Thevenot, J.; Finnilä, M. A.; Karhula, S. S.; Hadjab, I.; Kauppinen, S.; Garon, M.; Quenneville, E.; Haapea, M.; Rieppo, L.; Pritzker, K. P.; Buschmann, M. D.; Nieminen, Heikki; Saarakkala, Seppo

The stability of DNA/chitosan complexes upon exposure to hyaluronic acid, chondroitin sulfate, and heparin, was assessed by fluorescence spectroscopy to quantify DNA release. Only the highly charged heparin was found to release DNA from the complexes. Complex stability upon exposure to heparin increased with the degree of deacetylation and molecular weight of chitosan and with the ratio of chitosan amino groups to DNA phosphate groups (N/P ratio) in the complexes. Isothermal titration microcalorimetry revealed that among polyanions tested, only heparin has a binding affinity to chitosan approaching that of DNA and can therefore release DNA from the complexes. These results also indicate that anionic com-

Platelet-rich plasma (PRP) has been used to treat different orthopedic conditions, however, the clinical benefits of using PRP remain uncertain. Chitosan (CS)-PRP implants have been shown to improve meniscus, rotator cuff and cartilage repair in pre-clinical models. The purpose of this current study was to investigate in vitro and in vivo mechanisms of action of CS-PRP implants. Freeze-dried formulations containing 1% (w/v) CS (80% degree of deacetylation and number average molar mass 38 kDa), 1% (w/v) trehalose as a lyoprotectant and 42.2 mM calcium chloride as a clot activator were solubilized in PRP. Gravimetric measurements and molecular/cellular imaging studies revealed that clot retraction is inhibited in CS-PRP hybrid clots through physical coating of platelets, blood cells and fibrin strands by chitosan, which interferes with platelet aggregation and platelet-mediated clot retraction. Flow cytometry and ELISA assays revealed that platelets are activated and granules secreted in CS-PRP hybrid clots and that cumulative release of platelet-derived growth factor (PDGF-AB) and epidermal growth factor is higher from CS-PRP hybrid clots compared to PRP clots in vitro. Finally, CS-PRP implants resided for up to 6 weeks in a subcutaneous implantation model and induced cell recruitment and granulation tissue synthesis, confirming greater residency and bioactivity compared to PRP in vivo.

Purpose: Calcified cartilage (CC) has an important role in solute transportation and biomechanics between the subchondral bone and articular cartilage. The morphological changes of CC, including thickening of CC, tidemark duplication and tidemark roughness, have all been associated with cartilage degeneration during osteoarthritis (OA). Current methods to visualize these changes are mainly based on the use of 2D histological sections. Micro-computed tomography (μCT) is a volumetric imaging technique useful for characterizing calcified tissues. Here we present a novel method to volumetrically analyze the roughness of tidemark and the vessel perforations through CC from μCT image stacks. Furthermore, we investigated these changes at different stages of OA. Methods: Samples were harvested from six patients (age 49–67) undergoing total knee replacement surgery and two asymptomatic cadavers (age 26 and 49). Osteochondral cores (n = 15, Ø = 4mm) were drilled from the weight bearing area of lateral tibial plateaus. Samples were fixed in buffered 4% formaldehyde and subsequently imaged with μCT (Skyscan 1272, Brüker microCT, Kontich, Belgium: voltage 50 kV, current 200 μA, exposure 3200 ms, frame averaging 3, projection images 1200, isotropic voxel size 2.8 μm). Projections were reconstructed with Nrecon software (v1.6.9.8). After μCT imaging, samples were subjected to conventional histological sectioning and stained with safranin O for histopathological OARSI grading. Furthermore, the number of tidemarks (TM.N) and CC thickness (CC.Th) were analyzed from the stained histological sections. The μCT data analysis was conducted with Matlab software (v8.5), except for calcified tissue and vessel segmentations that were done with a custom-made C++ algorithm and CTAn software (v1.14.4.1), respectively. Volumes of interest (VOIs) of 600×400×Z (Z proportional to CC thickness) were fitted in the center of the CC surface. The calculated parameters were: 1) vessel area fraction (VAF, ratio of flat vessel surface area to VOI surface area [600x400]), 2) number of vessels per mm2 (NV/A), and 3) tidemark roughness (TMR, mean ratio of tidemark length vs. fitted line along the tidemark from all slices). Furthermore, a local binary pattern (LBP) -based analysis was applied to the segmented tidemark in 3D. Briefly, the LBP method defines a specific pattern for each studied voxel based on its neighborhood, as a measure of local volumetric orientation. From the LBP analysis, the amount of different patterns (ADP) as well as the entropy of patterns (EP) were calculated to describe the variance in local CC surface orientation. Figure 1 shows the full analysis protocol. Spearman's correlations were calculated to associate analyzed features with OARSI grading and TM.N. Pearson's correlations were used in all other comparisons. Results: Volumetric representation of two VOIs are shown in Figure 2. VAF correlated with ADP (rp = 0.833, p < 0.0001), and TMR (rp = 0.923, p < 0.0001). EP showed a similar trend with VAF, although not statistically significant (rp = 0.369, p = 0.176). NV/A correlated with ADP (rp = 0.633, p = 0.011), CC.Th (rp = −0.615, p = 0.015) and the TM.N (rs = −0.531, p = 0.042). OARSI grade correlated with EP (rs = −0.578, p = 0.024) and ADP (rs = −0.600, p = 0.018). Scatter plots from μCT volume analyses are shown in Figure 3. Conclusions: Tidemark roughness and vessel perforations through CC were analyzed volumetrically from μCT images using the novel method. The parameters describing local morphology of the tidemark were significantly associated with OA progression. In contrast, no association between the vessel perforations through CC and OA progression was found. On the other hand, increase in vessel perforations was associated with a thinner CC and fewer tidemark duplications. Increased vessel perforations were also linked to the increase in the tidemark roughness and complexity, and to some extent with the randomness of the tidemark morphology. These results suggest an interaction between CC internal and external structure and vessel perforations through CC. Furthermore, these findings support that OA progression may change the local morphology of the tidemark. This volumetric analysis method provides means for further investigation of the tidemark morphology and the structural changes of CC with OA progression.Figure 2: 3D visualization of the 600×400×Z VOIs used in the analyzes. Vessels that perforate to the tidemark surface are shown in red and the calcified tissue is shown in cyan.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3: Scatter plots of Vessel Area Fraction (VAF) and Number of vessels per mm2 (NV/A) against Tidemark roughness (TMR), Entropy of patterns (EP) and Amount of different patterns (ADP). Colors indicate different OARSI grade groups. Positive trend is observed between vessel perforation and parameters that define roughness (TMR) and local orientation variances (ADP, EP) of the tidemark. ADP is the amount of different patterns per average surface area evaluated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Chitosan (CS)-based polyplexes are efficient non-viral gene delivery systems that are most commonly prepared by manual mixing. However, manual mixing is not only poorly controlled but also restricted to relatively small preparation volumes, limiting clinical applications. In order to overcome these drawbacks and to produce clinical quantities of CS-based polyplexes, a fully automated in-line mixing platform was developed for production of large batches of small-size and homogeneous CS-based polyplexes. Operational conditions to produce small-sized homogeneous polyplexes were identified. Increasing mixing concentrations of CS and nucleic acid was directly associated with an increase in size and polydispersity of both CS/pDNA and CS/siRNA polyplexes. We also found that although the speed of mixing has a negligible impact on the properties of CS/pDNA polyplexes, the size and polydispersity of CS/siRNA polyplexes are strongly influenced by the mixing speed: the higher the speed, the smaller the size and polydispersity. While in-line and manual CS/pDNA polyplexes had similar size and PDI, CS/siRNA polyplexes were smaller and more homogenous when prepared in-line in the non-laminar flow regime compared to manual method. Finally, we found that in-line mixed CS/siRNA polyplexes have equivalent or higher silencing efficiency of ApoB in HepG2 cells, compared to manually prepared polyplexes.

Objective. Bone marrow stimulation procedures initiate repair by fracturing or drilling subchondral bone at base of cartilaginous defect. Earlier studies have shown that defect location and animal age affect cartilage repair outcome, suggesting a strong influence of structural and biological characteristics of subchondral bone. Here, we analyzed comprehensive biological characteristics of bone marrow progenitor cells (BMPCs) in subchondral bone of young and old rabbit condyle and trochlea. We tested the hypothesis that in vitro biological properties of BMPCs are influenced by location, age of donor and method of their isolation. Design. In vitro biological properties, including cell yield, colony-forming unit fibroblasts (CFU-f), surface marker expression, and differentiation potential were determined. Comparisons were carried out between trochlea versus condyle and epiphyseal versus metaphyseal bone using old (N = 5) and young animal knees (N = 8) to generate collagenase and explant-derived BMPC cultures. Results. CFU-f, cell yield, expression of stem cell markers, and osteogenic differentiation were significantly superior for younger animals. Trochlear subchondral bone yielded the most progenitors with the highest clonogenic potential and cartilaginous matrix expression. Trochlear collagenase-derived BMPCs had higher clonogenic capacity than explant-derived ones. Epiphyseal cells generated a larger chondrogenic pellet mass than metaphyseal-derived BMPCs. All older pellet cultures and one non-responder young rabbit failed to accumulate glycosaminoglycans (GAGs). Conclusion. Taken together, these results suggest that properties intrinsic to subchondral progenitors could significantly influence cartilage repair potential, and could partly explain variability in cartilage repair outcomes using same cartilage repair approach.

Nanoparticle toxicity represents a major hurdle limiting clinical translation with both cationic lipids and polymers that can produce preclinical toxicity and serious adverse events in clinical trials. Advancements in cationic lipid head groups have improved the therapeutic window but clinical application still requires the administration of prophylactic anti-inflammatory steroids. Chitosans, a family of natural polycationic and bio-degradable polymers have shown in vitro and in vivo efficacy for nucleic acid delivery. However, the effect of chitosan molecular weight (Mn), dose and payload type–ie unmodified versus modified siRNA–on cytokine induction, hematological and serological responses, body weight and clinical signs have not been investigated nor reported following intravenous (IV) administration in mice. To understand the influence of Mn, dose, payload and hyaluronic acid (HA) coating, on in vivo toxicity, immune stimulation, biodistribution and efficacy, precisely characterized low (10 kDa) and high (120 kDa) Mn chitosans with a fixed degree of deacetylation of 92% were produced and formulated with unmodified (immune stimulating) vs chemically modified (non-immune stimulating) siRNA, and tested for hemocompatibility as per ASTM standards for dose selection and IV administration to mice. Cytokine induction (IL-1β, IL-6, TNF-α, IFN-γ and KC), hematological (Hb, Hematocrit, platelets,…) and serological (ALT, AST, BUN, Cr,...) responses were assessed at 4 (Cytokine) and 24 hours (Serology) post-administration. HA was used to coat nanoparticle to improve hemocompatibility. Body weight and clinical signs were monitored following …

Purpose: The collagen network has a unique organization in articular cartilage. The most common ways to characterize the collagen network orientation are polarized light microscopy (PLM) and electron microscopy techniques. However, these techniques are limited to the study of thin sections or tissue surfaces. Here, we present an analysis method for micro-computed tomography (micro-CT) data to evaluate the microstructural orientation of dehydrated articular cartilage samples in 3D using structure tensor analysis. Methods: Cylindrical osteochondral samples (n = 6, diameter 4 mm) were prepared from tibial plateaus of human cadavers (N = 3) and of patients who underwent total knee replacement surgery (N = 3). The samples were split in half, and then fixed in formaldehyde. One half was subjected for standard histological sectioning protocol, while the second half was dehydrated in ascending ethanol series and treated with hexamethyldisilazane (HMDS). Subsequently, the second half was dried in room temperature overnight and imaged using desktop μCT (SkyScan 1272, Bruker microCT, Kontich, Belgium; 40kV, 250 μA, 3600 projections, 5 frames/projection, 1815 ms/frame, isotropic voxel size 1.6 μm, no additional filtration). The data was reconstructed using NRecon software (v 1.6.10.4). Structure tensor analysis was applied to the micro-CT data (volume-of-interest: 500 μm x 500 μm x cartilage depth) in 2D (i.e. x-z plane) and in 3D to determine the extracellular matrix orientation in articular cartilage. Eigen analysis was applied to the structure tensors in each voxel, and the eigen-vector with the smallest eigenvalue (i.e. the smallest gradient) was set as the direction of the extracellular matrix. Depth-dependent elevation angle (elevation from the x-y plane) profiles were calculated by averaging the elevation angles of the direction vectors in x-y plane. The histological sections (thickness 5 μm) were imaged with PLM (Abrio PLM system, CRi, Inc., Woburn, MA, USA) to obtain a reference for the collagen network orientation. The micro-CT-based and PLM depth-dependent profiles were rescaled to 200 pixels and compared to each other with Pearson's correlation analysis. Data analyses were conducted using MATLAB (v 8.5, Natick, MA, USA). Results: Figure 1 shows representative images of single micro-CT slice, and the elevation angles obtained using structure tensor methods and PLM. When the structure tensor analysis was conducted in 2D, the average elevation angle in the surface layer (1–10% of the thickness) was 41 ± 4 degrees (with respect to the cartilage surface) and gradually changed to 61 ± 5 degrees in the deep layer (40–100% of the thickness) (Figure 2A). The mean correlation (± standard deviation) between the depth-dependent profiles obtained using 2D structure tensor analysis and PLM was r = 0.82 ± 0.08. The elevation angles obtained using 3D structure tensor analysis are presented in Figure 2B. The average elevation angle obtained using 3D structure tensor analysis was 29 ± 4 degrees at the surface layer and gradually changed to 47 ± 3 degrees in the deep layer. The mean correlation between the depth-dependent profiles obtained using 3D structure tensor analysis and PLM was r = 0.87 ± 0.05. Conclusions: The natural contrast of dehydrated articular cartilage obtained using micro-CT imaging was utilized to reveal 3D structural information of extracellular matrix. The presented micro-CT analysis approach is fully based on image texture analysis, which evaluates the orientations of structures from the grayscale differences between the neighboring voxels. On the other hand, PLM gives direct information on the collagen network orientation. Therefore, it is not surprising that the techniques are not in absolute agreement. Nevertheless, the shapes of the depth-dependent elevation angle profiles obtained using micro-CT analysis and PLM are very similar, as indicated by the high correlation coefficients (r > 0.8) between the profiles of adjacent blocks. The main advantage of the presented method over PLM is its ability to study larger tissue volumes in 3D, while PLM is limited to the study of thin tissue sections in 2D and collagen orientation only in that plane. Based on the findings of this study, we suggest that micro-CT imaging of HMDS-dehydrated articular cartilage samples can be used to obtain information on the orientation of extracellular matrix.Figure 2. Depth-dependent average elevation angles as a function of normalized cartilage thickness obtained using PLM in 2D (red) and using structure tensor analysis A) in 2D (black) and B) in 3D (black). The shaded areas represent the standard deviations in the sample set.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Chitosan (CS) end-group conjugation methods are rarely reported in the literature, mainly since the CS terminal aldehyde moiety produced by nitrous acid depolymerization is only present in trace amounts in its reactive form. In a previous study, our group proposed an intermolecular thioacetylation process that allowed terminal conjugation of thiol-reactive species to chitosan with 50% efficiency. However, this reaction is incompatible with acid-labile substituents and the conversion efficiency of CS end-groups could be limited by the size of the thiol-reactive species engaged in the reaction, mainly by steric hindrance since two substituents are required to obtain the stabilized thioacetal derivative. In the present study, we developed a novel CS end-group thioacetylation approach relying on a new regioselective linker that bears three thiol moieties. This trivalent linker, referred to as triskelion here, was specifically designed for activation of the CS 2,5-anhydro-D-mannose (M-Unit) end-group and consists of a thiolhook for efficient aldehyde conjugation through an intramolecular reaction and a thiol-tail that remains available for subsequent end-group functionalization with any thiol-reactive species. The chemical synthesis of this linker provided the desired material with high yields over three steps. The in situ intramolecular thioacetylation process between the triskelion linker and 2,5-anhydro-D-mannose (MUnit, monomeric) was assessed by semi-quantitative LC-MS studies, revealing that the corresponding intramolecular thioacetal largely predominated ( 90%). This regioselective derivatization was also performed onto M-Unit CS aldehydes and the desired CS-b-triskelion conjugates were obtained with functionalization degrees over 85%, as confirmed by NMR spectroscopy (H and DOSY). As a final assessment of the CS-b-triskelion thiol-tail reactivity, these conjugates were successfully engaged with thiol-reactive magnetic beads into disulfide bond displacement with 50% efficiency. The proposed CS terminal activation with the triskelion linker opens new perspectives for biomedical applications, especially brush-like surface modifications and other copolymer formation through disulfide linkages or Michael-type additions.

Objective: To evaluate cross-correlations of ex vivo electromechanical properties with cartilage and subchondral bone plate thickness, as well as their sensitivity and specificity regarding early cartilage degeneration in human tibial plateau. Method: Six pairs of tibial plateaus were assessed ex vivo using an electromechanical probe (Arthro-BST) which measures a quantitative parameter (QP) reflecting articular cartilage compression-induced streaming potentials. Cartilage thickness was then measured with an automated thickness mapping technique using Mach-1 multiaxial mechanical tester. Subsequently, a visual assessment was performed by an experienced orthopedic surgeon using the International Cartilage Repair Society (ICRS) grading system. Each tibial plateau was finally evaluated with mCT scanner to determine the subchondral-bone plate thickness over the entire surface. Results: Cross-correlations between assessments decreased with increasing degeneration level. Moreover, electromechanical QP and subchondral-bone plate thickness increased strongly with ICRS grade (p = 0.86 and p = 0.54 respectively), while cartilage thickness slightly increased (p = 0.27). Sensitivity and specificity analysis revealed that the electromechanical QP is the most performant to distinguish between different early degeneration stages, followed by subchondral-bone plate thickness and then cartilage thickness. Lastly, effect sizes of cartilage and subchondral-bone properties were established to evaluate whether cartilage or bone showed the most noticeable changes between normal (ICRS 0) and each early degenerative stage. Thus, the effect sizes of cartilage electromechanical QP were almost twice those of the subchondral-bone plate thickness, indicating greater sensitivity of electromechanical measurements to detect early osteoarthritis. Conclusion: The potential of electromechanical properties for the diagnosis of early human cartilage degeneration was highlighted and supported by cartilage thickness and mCT assessments. (c) 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.