Non-invasive electroarthrography measures cartilage in live horses and correlates to direct measurements of cartilage streaming potentials in weight bearing regions of equine metacarpophalangeal joints.

OBJECTIVE: To perform non-invasive Electroarthrography (EAG) on live horses and establish relationships between EAG and direct measurements of cartilage streaming potentials in weight bearing areas of the equine metacarpophalangeal joint. DESIGN: EAG was performed bilaterally on the metacarpophalangeal joints of live horses (n = 3). Separate experiments used metacarpophalangeal joint explants (n = 11) to measure EAG obtained during simulated loading followed by direct measurements of cartilage streaming potentials on joint surfaces using the Arthro-BST probe. Joints were assigned to relatively normal (n = 5) and mildly degraded (n = 6) groups based on histological scoring of Safranin-O/Fast Green stained sections. RESULTS: EAG, involving application of electrodes to skin surrounding the joint and repeated weight shifting, was well-tolerated in live horses. One pair of distal forelimbs were available for analogous ex vivo EAG testing and measurements were strongly correlated to in vivo EAG measurements obtained on the same joints (r = 0.804, p = 0.016, n = 8). Both indirect (EAG) and direct (Arthro-BST) measurements of cartilage streaming potentials distinguished between normal and mildly degraded cartilage with statistically significant differences at 5 of 6 and 4 of 6 electrodes during simulated standing and walking, respectively. Strong and moderate correlations for weight bearing regions on the dorsal phalanx and central metacarpus were detected during both standing and walking. At the metacarpus/sesamoid interface a moderate correlation occurred during walking. CONCLUSION: Non-invasive EAG was used successfully in a clinical scenario and correlated to direct measurements of streaming potentials in weight bearing cartilage. These data support the potential of EAG to contribute to the diagnosis and treatment of degenerative joint diseases.

siRNA Delivery with Chitosan: Influence of Chitosan Molecular Weight, Degree of Deacetylation, and Amine to Phosphate Ratio on in Vitro Silencing Efficiency, Hemocompatibility, Biodistribution, and in Vivo Efficacy.

Chitosan (CS) shows in vitro and in vivo efficacy for siRNA delivery but with contradictory findings for incompletely characterized systems. For understanding which parameters produce effective delivery, a library of precisely characterized chitosans was produced at different degrees of deacetylation (DDAs) and average molecular weights (Mn). Encapsulation and transfection efficiencies were characterized in vitro. Formulations were selected to examine the influence of Mn and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection. Hemocompatibility and in vivo biodistribution were then investigated for different Mn, N:P ratios, and doses. Nanoparticle uptake and gene silencing correlated with increased surface charge, which was obtained at high DDA and high Mn. A minimum polymer length of ∼60-70 monomers (∼10 kDa) was required for stability and knockdown. In vitro knockdown was equivalent to lipid control with no metabolic or genotoxicity. An inhibitory effect of serum on biological performance was dependent on DDA, Mn, and N:P. In vivo biodistribution in mice show accumulation of nanoparticles in kidney with 40-50% functional knockdown.

Rotator cuff repair: a review of surgical techniques, animal models, and new technologies under development.

Rotator cuff tears are the most common musculoskeletal injury occurring in the shoulder. Current surgical repair fails to heal in 20% to 95% of patients, depending on age, size of the tear, smoking, time of repair, tendon quality, muscle quality, healing response, and surgical treatments. These problems are worsened by the limited healing potential of injured tendons attributed to the presence of degenerative changes and relatively poor vascularity of the cuff tendons. Development of new techniques to treat rotator cuff tears requires testing in animal models to assess safety and efficacy before clinical testing. Hence, it is important to evaluate appropriate animal models for rotator cuff research with degeneration of tendons, muscular atrophy, and fatty infiltration similar to humans. This report reviews current clinical treatments and preclinical approaches for rotator cuff tear repair. The review will focus on current clinical surgical treatments, new repair strategies under clinical and preclinical development, and will also describe different animal models available for rotator cuff research. These findings and future directions for rotator cuff tear repair will be discussed.

Combined analysis of polycation/ODN polyplexes by analytical ultracentrifugation and dynamic light scattering reveals their size, refractive index increment, stoichiometry, porosity, and molecular weight.

Analytical ultracentrifugation (AUC) and dynamic light scattering (DLS) were combined to characterize polyplexes formed with 10 kDa chitosan or 10 kDa PEI and oligodeoxynucleotides (ODN). Combined analysis revealed that both polyplexes were highly porous (over 80%) and that their weight average hydrodynamic diameters were of 46 and 55 nm for chitosan/ODN and PEI/ODN complexes, respectively. Transformation of the sedimentation coefficient distribution to a size and molecular weight distribution gave an average molecular weight of 19 and 29 MDa for chitosan and PEI polyplexes, respectively. Data from AUC also allowed for the calculation of the actual dn/dc and N/P ratios of each polyplex. Additional data from scanning electron microscopy and static light scattering confirmed the conclusions that were initially derived from AUC and DLS, thus validating that the combination of AUC and DLS is a powerful approach to characterize polyplexes in terms of refractive index increment, size, and molecular weight distributions, as well as porosity.

Ionization behavior of chitosan and chitosan-DNA polyplexes indicate that chitosan has a similar capability to induce a proton-sponge effect as PEI.

Polycations having a high buffering capacity in the endosomal pH range, such as polyethylenimine (PEI), are known to be efficient at delivering nucleic acids by overcoming lysosomal sequestration possibly through the proton sponge effect, although other mechanisms such as membrane disruption arising from an interaction between the polycation and the endosome/lysosome membrane, have been proposed. Chitosan is an efficient delivery vehicle for nucleic acids, yet its buffering capacity has been thought to be significantly lower than that of PEI, suggesting that the molecular mechanism responsible for endolysosomal escape was not proton sponge based. However, previous comparisons of PEI and chitosan buffering capacity were performed on a mass concentration basis instead of a charge concentration basis, the latter being the most relevant comparison basis because polycation-DNA complexes form at ratios of charge groups (amine to phosphate), rather than according to mass. We hypothesized that chitosan has a high buffering capacity when compared to PEI on a molar basis and could therefore possibly mediate endolysosomal release through the proton sponge effect. In this study, we examined the ionization behavior of chitosan and chitosan-DNA complexes and compared to that of PEI and polylysine on a charge concentration basis. A mean field theory based on the use of the Poisson-Boltzmann equation and an Ising model were also applied to model ionization behavior of chitosan and PEI, respectively. We found that chitosan has a higher buffering capacity than PEI in the endolysosomal pH range, while the formation of chitosan-DNA complexes reduces chitosan buffering capacity because of the negative electrostatic environment of nucleic acids that facilitates chitosan ionization. These data suggest that chitosans have a similar capacity as PEI to mediate endosomal escape through the proton sponge effect, possibly in a manner which depends on the presence of excess chitosan.

The TGF-ß co-receptor, CD109, promotes internalization and degradation of TGF-ß receptors.

Transforming growth factor-ß (TGF-ß) is implicated in numerous pathological disorders, including cancer and mediates a broad range of biological responses by signaling through the type I and II TGF-ß receptors. Internalization of these receptors via the clathrin-coated pits pathway facilitates SMAD-mediated signaling, whereas internalization via the caveolae pathway is associated with receptor degradation. Thus, molecules that modulate receptor endocytosis are likely to play a critical role in regulating TGF-ß action. We previously identified CD109, a GPI-anchored protein, as a TGF-ß co-receptor and a negative regulator of TGF-ß signaling. Here, we demonstrate that CD109 associates with caveolin-1, a major component of the caveolae. Moreover, CD109 increases binding of TGF-ß to its receptors and enhances their internalization via the caveolae. In addition, CD109 promotes localization of the TGF-ß receptors into the caveolar compartment in the presence of ligand and facilitates TGF-ß-receptor degradation. Thus, CD109 regulates TGF-ß receptor endocytosis and degradation to inhibit TGF-ß signaling. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

CD109-mediated degradation of TGF-ß receptors and inhibition of TGF-ß responses involve regulation of SMAD7 and Smurf2 localization and function.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-ß signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-ß signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-ß co-receptors can modulate TGF-ß signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-ß co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-ß signaling. Despite CD109's potential relevance as a regulator of TGF-ß action in vivo, the mechanisms by which CD109 regulates TGF-ß signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-ß signaling by promoting TGF-ß receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-ß receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-ß signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.

Intracellular trafficking and decondensation kinetics of chitosan-pDNA polyplexes.

The transfection efficiency (TE) of chitosan-plasmid DNA (pDNA) polyplexes can be critically modulated by the polymer degree of deacetylation (DDA) and molecular weight (MW). This study was performed to test the hypothesis that the TE dependence on chitosan MW and DDA is related to the polyplex stability, hence their intracellular decondensation/unpacking kinetics. Major barriers to nonviral gene transfer were studied by image-based quantification. Although uptake increased with increased DDA, it did not appear to be a structure-dependent process affecting TE, nor was nuclear entry. Colocalization analysis showed that all chitosans trafficked through lysosomes with similar kinetics. Fluorescent resonant energy transfer (FRET) analysis revealed a distinct relationship between TE and polyplex dissociation rate. The most efficient chitosans showed an intermediate stability and a kinetics of dissociation, which occurred in synchrony with lysosomal escape. In contrast, a rapid dissociation before lysosomal escape was found for the inefficient low DDA chitosan whereas the highly stable and inefficient complex formed by a high MW and high DDA chitosan did not dissociate even after 24 hours. This study identified that the kinetics of decondensation in relation to lysosomal escape was a most critical structure-dependent process affecting the TE of chitosan polyplexes.

Nanomaterial Delivery Systems for mRNA Vaccines.

The recent success of mRNA vaccines in SARS-CoV-2 clinical trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after intramuscular injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clinical trials. The review concludes with an analysis of the determinants of the performance of lipid nanoparticles in mRNA vaccines.

Quality of Cartilage Repair from Marrow Stimulation Correlates with Cell Number, Clonogenic, Chondrogenic, and Matrix Production Potential of Underlying Bone Marrow Stromal Cells in a Rabbit Model.

OBJECTIVE: Previous studies have shown that intrinsic behavior of subchondral bone marrow stem cells (BMSCs) is influenced by donors and locations. To understand the variability in cartilage repair outcomes following bone marrow stimulation, we tested the hypothesis that in vivo cartilage repair correlates with in vitro biological properties of BMSCs using a rabbit model. METHODS: Full-thickness cartilage defects were created in the trochlea and condyle in one knee of skeletally mature New Zealand White rabbits (n = 8) followed by microdrilling. Three-week repair tissues were analyzed by macroscopic International Cartilage Repair Society (ICRS) scores, O'Driscoll histological scores, and Safranin-O (Saf-O) and type-II collagen (Coll-II) % stain. BMSCs isolated from contralateral knees were assessed for cell yield, surface marker expression, CFU-f, %Saf-O, and %Coll-II in pellet culture followed by correlation analyses with the above cartilage repair responses. RESULTS: In vivo cartilage repair scores showed strong, positive correlation with cell number, clonogenic, chondrogenic, and matrix production (Coll-II, GAG) potential of in vitro TGF-ßIII stimulated BMSC cultures. Trochlear repair showed clear evidence of donor dependency and strong correlation was observed for interdonor variation in repair and the above in vitro properties of trochlear BMSCs. Correlation analyses indicated that donor- and location-dependent variability observed in cartilage repair can be attributed to variation in the properties of BMSCs in underlying subchondral bone. CONCLUSION: Variation in cell number, clonogenic, chondrogenic, and matrix production potential of BMSCs correlated with repair response observed in vivo and appear to be responsible for interanimal variability as well as location-dependent repair.

Non-invasive Electroarthrography Measures Load-Induced Cartilage Streaming Potentials via Electrodes Placed on Skin Surrounding an Articular Joint.

OBJECTIVE: We aimed to demonstrate that electroarthrography (EAG) measures streaming potentials originating in the cartilage extracellular matrix during load bearing through electrodes adhered to skin surrounding an articular joint. DESIGN: Equine metacarpophalangeal joints were subjected to simulated physiological loads while (1) replacing synovial fluid with immersion buffers of different electrolyte concentrations and (2) directly degrading cartilage with trypsin. RESULTS: An inverse relationship between ionic strength and EAG coefficient was detected. Compared to native synovial fluid, EAG coefficients increased (P < 0.05) for 5 of 6 electrodes immersed in 0.1X phosphate-buffered saline (PBS) (0.014 M NaCl), decreased (P < 0.05) for 4 of 6 electrodes in 1X PBS (0.14 M NaCl), and decreased (P < 0.05) for all 6 electrodes in 10X PBS (1.4 M NaCl). This relationship corresponds to similar studies where streaming potentials were directly measured on cartilage. EAG coefficients, obtained after trypsin degradation, were reduced (P < 0.05) in 6 of 8, and 7 of 8 electrodes, during simulated standing and walking, respectively. Trypsin degradation was confirmed by direct cartilage assessments. Streaming potentials, measured by directly contacting cartilage, indicated lower cartilage stiffness (P < 10-5). Unconfined compression data revealed reduced Em, representing proteoglycan matrix stiffness (P = 0.005), no change in Ef, representing collagen network stiffness (P = 0.15), and no change in permeability (P = 0.24). Trypsin depleted proteoglycan as observed by both dimethylmethylene blue assay (P = 0.0005) and safranin-O stained histological sections. CONCLUSION: These data show that non-invasive EAG detects streaming potentials produced by cartilage during joint compression and has potential to become a diagnostic tool capable of detecting early cartilage degeneration.

Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration.

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.

Complete physicochemical characterization of DNA/chitosan complexes by multiple detection using asymmetrical flow field-flow fractionation.

Asymmetrical flow field-flow fractionation (AF4) coupled with UV-vis spectrophotometry, multiangle light scattering (MALS), and dynamic light scattering (DLS) detection was used to analyze dispersions of DNA/rhodamine B labeled chitosan (Ch-rho) complexes frequently used as gene delivery vectors. The method yielded, in a single experiment, important characteristics of the complexes, such as their hydrodynamic radius, size distribution, conformation, composition, and the amount of free Ch-rho in the dispersions. Samples for analysis were obtained by varying experimental parameters known to influence the transfection efficiency of DNA/chitosan complexes, including the DNA concentration at mixing (82-164 µg/mL), the ratio of chitosan amino groups to DNA phosphate groups (3 ≤ N/P ratio ≤ 15), the chitosan molecular weight (10-76 kDa), and its degree of deacetylation. In all preparations, DNA/Ch-rho complexes had hydrodynamic radii ranging from 15 to 160 nm. Both the DNA concentration and the Ch-rho molecular weight influence the size distribution of the complexes: a greater fraction of large particles was detected in dispersions prepared with the most concentrated DNA solution or the Ch-rho of highest molar mass. All dispersions contained free Ch-rho in solution. The free Ch-rho content ranged from 53 to 92% of the total Ch-rho concentration in dispersions prepared with N/P ratios from 3 to 15, respectively, implying that the N/P ratio of the complexes ranged from 1.3 to 1.6 in all samples. The accuracy of the free Ch-rho determination by AF4/UV-vis/MALS+DLS was confirmed by an independent method involving (1) ultracentrifugation of the dispersions prepared with unlabeled chitosan and (2) analysis of the supernatant by the Orange II dye depletion method. This study demonstrates the ability of AF4/UV-vis/MALS+DLS to provide a complete physicochemical characterization of DNA/polycation complexes used in nonviral gene delivery.

One-step analysis of DNA/chitosan complexes by field-flow fractionation reveals particle size and free chitosan content.

The composition of samples obtained upon complexation of DNA with chitosan was analyzed by asymmetrical flow field flow fractionation (AF4) with online UV-visible, multiangle light scattering (MALS), and dynamic light scattering (DLS) detectors. A chitosan labeled with rhodamine B to facilitate UV-vis detection of the polycation was complexed with DNA under conditions commonly used for transfection (chitosan glucosamine to DNA phosphate molar ratio of 5). AF4 analysis revealed that 73% of the chitosan-rhodamine remained free in the dispersion and that the DNA/chitosan complexes had a broad size distribution ranging from 20 to 160 nm in hydrodynamic radius. The accuracy of the data was assessed by comparison with data from batch-mode DLS and scanning electron microscopy. The AF4 combined with DLS allowed the characterization of small particles that were not detected by conventional batch-mode DLS. The AF4 analysis will prove to be an important tool in the field of gene therapy because it readily provides, in a single measurement, three important physicochemical parameters of the complexes: the amount of unbound polycation, the hydrodynamic size of the complexes, and their size distribution.

Young adult chondrocytes proliferate rapidly and produce a cartilaginous tissue at the gel-media interface in agarose cultures.

Primary chondrocytes cultured in agarose can escape the gel, accumulate at the interface between agarose and the culture medium, and form an outgrowing tissue. These outgrowths can appear as voluminous cartilage-like nodules that have never been previously investigated. In the present study, bovine articular chondrocytes from three age groups (fetal, young adult, aged) were seeded and cultured in agarose to test the hypothesis that hyaline-like cartilage outgrowths develop at the interface by appositional growth, in an age-dependant manner. Macroscopic appearance, cell content, cell division, cytoskeletal morphology, and extracellular matrix (ECM) composition were analyzed. Fetal chondrocytes produced a fibrous interfacial tissue while aged chondrocytes produced ECM-poor cell clusters. In contrast young adult chondrocytes produced large cartilaginous outgrowths, rich in proteoglycan and collagen II, where cells in the central region displayed a chondrocyte morphology. Cell proliferation was confined to the peripheral edge of these outgrowths, where elongated cell morphology, cell-cell contacts, and cell extensions toward the culture medium were seen. Thus these voluminous cartilaginous outgrowths formed in an appositional growth process and only for donor chondrocytes from young adult animals. This system offers an interesting ability to proliferate chondrocytes in a manner that results in a chondrocyte morphology and a cartilaginous ECM in central regions of the outgrowing tissue. It also provides an in vitro model system to study neocartilage appositional growth.

Effects of refrigeration and freezing on the electromechanical and biomechanical properties of articular cartilage.

In vitro electromechanical and biomechanical testing of articular cartilage provide critical information about the structure and function of this tissue. Difficulties obtaining fresh tissue and lengthy experimental testing procedures often necessitate a storage protocol, which may adversely affect the functional properties of cartilage. The effects of storage at either 4°C for periods of 6 days and 12 days, or during a single freeze-thaw cycle at -20°C were examined in young bovine cartilage. Non-destructive electromechanical measurements and unconfined compression testing on 3 mm diameter disks were used to assess cartilage properties, including the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Cartilage disks were also examined histologically. Compared with controls, significant decreases in SPI (to 32.3±5.5% of control values, p<0.001), Ef (to 31.3±41.3% [corrected] of control values, p=0.046), Em (to 6.4±8.5% of control values, p<0.0001), and an increase in k (to 2676.7±2562.0% of control values, p=0.004) were observed at day 12 of refrigeration at 4°C, but no significant changes were detected at day 6. A trend toward detecting a decrease in SPI (to 94.2±6.2% of control values, p=0.083) was identified following a single freeze-thaw cycle, but no detectable changes were observed for any biomechanical parameters. All numbers are mean±95% confidence interval. These results indicate that fresh cartilage can be stored in a humid chamber at 4°C for a maximum of 6 days with no detrimental effects to cartilage electromechanical and biomechanical properties, while one freeze-thaw cycle produces minimal deterioration of biomechanical and electromechanical properties. A comparison to literature suggested that particular attention should be paid to the manner in which specimens are thawed after freezing, specifically by minimizing thawing time at higher temperatures.

Efficiency of Chitosan/Hyaluronan-Based mRNA Delivery Systems In Vitro: Influence of Composition and Structure.

Messenger RNA (mRNA)-containing nanoparticles were produced by electrostatic complexation with a library of pharmaceutical grade chitosans with different degrees of deacetylation and hyaluronic acids (HAs) (native vs. sulfated). Polymer length (Mn), HA degree of sulfation (DS), and amine to phosphate to carboxyl + sulfate (from HA) ratio (N:P:C) were controlled. In vitro transfections were performed in the presence/absence of trehalose and at different pH. Particle size and ζ-potential were correlated with transfection efficiency. Polymer length and charge densities (degree of deacetylation, degree of sulfation) of both HA and chitosan had a direct influence on transfection efficiency through modulation of avidity to mRNA. N:P:C ratio, trehalose, mixing concentration, and nucleic acid dose influenced transfection efficiency with optimized formulations reaching ∼60%-65% transfection efficiency relative to commercially available lipid control with no apparent toxicity for transfection at slightly acidic pH 6.5.

New insights into chitosan-DNA interactions using isothermal titration microcalorimetry.

The interaction of chitosan with plasmid DNA was investigated as a function of pH, buffer composition, degree of deacetylation (DDA), and molecular weight (M(n)) of chitosan, using isothermal titration microcalorimetry (ITC). The Single Set of Identical Sites model was used to obtain the enthalpy of interaction, the binding constant, and the stoichiometry of binding. The chitosan-DNA interaction was shown to be coupled with proton transfer from the buffer to chitosan, as revealed by the dependence of the measured heat release on the ionization enthalpy of the buffer. The measured enthalpy of binding was almost entirely due to proton transfer, because it was accounted for by the enthalpy of ionization of the buffer and of chitosan once the number of protons transferred was calculated. This proton transfer during binding resulted in the protonation of an additional 17, 37, and 58% of total glucosamine units at pH 5.5, 6.5, and 7.4, respectively. The strong polyanionic nature of DNA facilitates the ionization of glucosamines of chitosan upon complexation and is responsible for proton transfer. Interestingly, using the chitosan-DNA stoichiometry provided by ITC and the calculated degree of ionization of chitosan in the complex, the charge ratio of protonated amines to negative phosphate groups in the complex was nearly constant at 0.50-0.75 after saturation and was independent of the pH, buffer type and chitosan molecular characteristics. The chitosan-DNA binding constant was in the range of 10(9)-10(10) M(-1). The binding constant was pH-dependent and was greater at lower pH due to increased electrostatic attraction to DNA when chitosan is highly charged. Furthermore, the DDA and molecular weight of chitosan exerted a great influence on binding affinity which increased by almost an order of magnitude with an increase of the latter from 7 to 153 kDa. The binding affinity did not change significantly with DDA from 72 to 80% when the M(n) was kept constant near 80 kDa, but it increased substantially with DDA from 80 to 93% to reach a value similar to that obtained with chitosan of M(n) = 153 kDa and 80% DDA. These results provide insight into the previously reported dependence of the transfection efficiency of DNA/chitosan complexes on chitosan DDA and molecular weight, where complex stability and chitosan-DNA binding strength play a critical role.

Injectable freeze-dried chitosan-platelet-rich-plasma implants improve marrow-stimulated cartilage repair in a chronic-defect rabbit model.

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.

Freeze-dried chitosan-platelet-rich plasma implants improve supraspinatus tendon attachment in a transosseous rotator cuff repair model in the rabbit.

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.