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.

Chitosan rate of uptake in HEK293 cells is influenced by soluble versus microparticle state and enhanced by serum-induced cell metabolism and lactate-based media acidification.

UNLABELLED: Chitosan is a biocompatible polysaccharide composed of glucosamine and N-acetylglucosamine. The polymer has a unique behavior of fluctuating between soluble chains at pH 6 and insoluble microparticles at pH 7. The purpose of this study was to test the hypothesis that chitosan structure, solubility state, and serum influence the rate of cell uptake. Chitosans with 80% and 95% degree of deacetylation (medium and low viscosity) were tagged with rhodamine and analyzed for particle size, media solubility, and uptake by HEK293 epithelial cells using live confocal microscopy and flow cytometry. In media pH 7.4 with or without 10% serum, chitosans fully precipitated into 0.5 to 1.4 µm diameter microparticles with a slight negative charge. During 24 h of culture in serum-free medium, chitosan particles remained extracellular. In cultures with serum, particles were taken up into intracellular vesicles in a serum dose-dependent manner. Opsonization of chitosan with serum, or replacement of serum by epidermal growth factor (EGF) failed to mediate serum-free chitosan particle uptake. Serum stimulated cells to acidify the media, partly by lactate generation. Media acidified to pH 6.5 by 7 mM lactate maintained 50% of chitosan in the soluble fraction, and led to minor uniform serum-free uptake in small vesicles. CONCLUSION: Media acidification mediates minor in vitro uptake of non-biofouled soluble chitosan chains, while serum-biofouled insoluble chitosan microparticles require sustained serum exposure to generate energy required for macropinocytosis.

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.

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.

Chitosan inhibits platelet-mediated clot retraction, increases platelet-derived growth factor release, and increases residence time and bioactivity of platelet-rich plasma in vivo.

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.

High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation.

Chitosan is a biodegradable natural polysaccharide that has shown potential for gene delivery, although the ideal molecular weight (MW) and degree of deacetylation (DDA) for this application have not been elucidated. To examine the influence of these parameters on gene transfer, we produced chitosans with different DDAs (98%, 92%, 80% and 72%) and depolymerized them with nitrous acid to obtain different MWs (150, 80, 40 and 10 kDa). We produced 64 formulations of chitosan/pDNA complexes (16 chitosans, 2 amine-to-phosphate (N:P) ratios of 5:1 and 10:1 and 2 transfection media pH of 6.5 and 7.1), characterized them for size and surface charge, and tested them for gene transfection in HEK 293 cells in vitro. Several formulations produced high levels of transgene expression while two conditions, 92-10-5 and 80-10-10 [DDA-MW-N:P ratio] at pH 6.5, showed equivalence to our best positive control. The results also revealed an important coupling between DDA and MW of chitosan in determining transgene expression. Maximum expression was obtained with a certain combination of DDA and MW that depended on N:P ratio and the pH, but similar expression levels could be achieved by simultaneously lowering MW and increasing DDA or lowering DDA and increasing MW, suggesting a predominant role of particle stability, through co-operative electrostatic binding, in determining transfection efficiency.

Osteochondral Biopsy Analysis Demonstrates That BST-CarGel Treatment Improves Structural and Cellular Characteristics of Cartilage Repair Tissue Compared With Microfracture.

OBJECTIVE: The efficacy and safety of BST-CarGel, a chitosan-based medical device for cartilage repair, was compared with microfracture alone at 1 year during a multicenter randomized controlled trial (RCT) in the knee. The quality of repair tissue of osteochondral biopsies collected from a subset of patients was compared using blinded histological assessments. METHODS: The international RCT evaluated repair tissue quantity and quality by 3-dimensional quantitative magnetic resonance imaging as co-primary endpoints at 12 months. At an average of 13 months posttreatment, 21/41 BST-CarGel and 17/39 microfracture patients underwent elective second look arthroscopies as a tertiary endpoint, during which ICRS (International Cartilage Repair Society) macroscopic scoring was carried out, and osteochondral biopsies were collected. Stained histological sections were evaluated by blinded readers using ICRS I and II histological scoring systems. Collagen organization was evaluated using a polarized light microscopy score. RESULTS: BST-CarGel treatment resulted in significantly better ICRS macroscopic scores (P = 0.0002) compared with microfracture alone, indicating better filling, integration, and tissue appearance. Histologically, BST-CarGel resulted in a significant improvement of structural parameters-Surface Architecture (P = 0.007) and Surface/Superficial Assessment (P = 0.042)-as well as cellular parameters-Cell Viability (P = 0.006) and Cell Distribution (P = 0.032). No histological parameters were significantly better for the microfracture group. BST-CarGel treatment also resulted in a more organized repair tissue with collagen stratification more similar to native hyaline cartilage, as measured by polarized light microscopy scoring (P = 0.0003). CONCLUSION: Multiple and independent analyses in this biopsy substudy demonstrated that BST-CarGel treatment results in improved structural and cellular characteristics of repair tissue at 1 year posttreatment compared with microfracture alone, supporting previously reported results by quantitative magnetic resonance imaging.

Optimal processing method to obtain four-color confocal fluorescent images of the cytoskeleton and nucleus in three-dimensional chondrocyte cultures.

Tissue engineering of articular cartilage requires accurate imaging of the chondrocyte cytoskeleton. Past studies have applied various fixation and permeabilization protocols without optimization of parameters. In this study, we have examined procedures using glutaraldehyde and paraformaldehyde as fixatives and Triton X-100 and Octyl-POE as permeabilizing detergents. A four-color fluorescence confocal method was developed to simultaneously image actin, tubulin, vimentin, and the nucleus. We found optimal preservation and morphology of the chondrocyte cytoskeleton after simultaneous fixation and permeabilization with glutaraldehyde and Triton X-100. These images displayed less cellular shrinkage and higher-resolution filamentous structures than with paraformaldehyde or when permeabilization followed fixation.

Aged bovine chondrocytes display a diminished capacity to produce a collagen-rich, mechanically functional cartilage extracellular matrix.

Most fundamental studies in cartilage tissue engineering investigate the ability of chondrocytes from young animals to produce cartilaginous matrix under various conditions, while current clinical applications such as autologous chondrocyte implantation, use chondrocytes from donors that are decades past skeletal maturity. Previous investigations have suggested that several characteristics of primary chondrocytes are age-dependent but none have quantified cell proliferation, proteoglycan synthesis and accumulation, collagen synthesis and accumulation, compressive and tensile mechanical properties in order to examine the effects of donor age on all of these parameters. We enzymatically isolated primary bovine chondrocytes from fetal, young and aged animals and cultured these cells in agarose gels to assess the above-mentioned properties. We found that fetal and young (but still skeletally mature i.e. 18-month-old bovine) chondrocytes behaved similarly, while aged chondrocytes (5- to 7-year-old bovine) displayed diminished proliferation ( approximately 2x less), a slightly reduced proteoglycan accumulation per cell ( approximately 20%), and significantly less collagen accumulation per cell ( approximately 55%) compared to the younger cells. Histological observations and mechanical properties supported these findings, where a particularly significant reduction in tensile stiffness produced by aged chondrocytes compared to younger cells was observed. Our findings suggest that donor age is an important factor in determining the outcome and potential success when tissue-engineered cartilage is produced from articular chondrocytes. More specifically, primary chondrocytes from aged donors may not possess sufficient capacity to produce the extracellular matrix that is required for a mechanically resilient tissue.

Chondroinduction Is the Main Cartilage Repair Response to Microfracture and Microfracture With BST-CarGel: Results as Shown by ICRS-II Histological Scoring and a Novel Zonal Collagen Type Scoring Method of Human Clinical Biopsy Specimens.

BACKGROUND: Current cartilage repair histological scoring systems are unable to explain the relationship between collagen type II deposition and overall repair quality. PURPOSE/HYPOTHESIS: The purpose of this study was to develop a novel zonal collagen type (ZCT) 5-point scoring system to measure chondroinduction in human clinical biopsy specimens collected after marrow stimulation. The hypothesis was that the ZCT scores would correlate with the International Cartilage Repair Society-II (ICRS-II) overall histological repair assessment score and glycosaminoglycan (GAG) content. STUDY DESIGN: Descriptive laboratory study. METHODS: After optimizing safranin O staining for GAG and immunostaining for human collagen type II and type I (Col2 and Col1, respectively), serial sections from clinical osteochondral repair biopsy specimens (13 months after microfracture or microfracture with BST-CarGel; n = 39 patients) were stained and 3 blinded readers performed histomorphometry for percentage of staining, ICRS-II histological scoring, polarized light microscopy (PLM) scoring, and 5-point ZCT scoring based on tidemark morphology, zonal distribution of Col2 and Col1, and Col1 percentage stain. Because 1 biopsy specimen was missing bone, 38 biopsy specimens were evaluated for ICRS-II, PLM, and ZCT scores. RESULTS: Chondroinduction was identified in 21 biopsy specimens as a Col2 matrix fused to bone that spanned the deep-middle-superficial zones ("full-thickness hyaline repair"), deep-middle zones, or deep zone ("stalled hyaline") that was covered with a variable-thickness Col1-positive matrix, and was scored, respectively, as ZCT = 1 (n = 4 biopsy specimens), ZCT = 2 (n = 6) and ZCT = 3 (n = 11). Other biopsy specimens (n = 17) were fibrocartilage (n = 9; ZCT = 4), fibrous tissue (n = 4, ZCT = 5), or non-marrow derived (n = 4; ZCT = 0). Non-marrow derived tissue had a mean mature tidemark score of 84 out of 100 versus a regenerating tidemark score of 24 for all other biopsy specimens (P = .005). Both "stalled hyaline" repair and fibrocartilage had the same mean Col2 percentage stain; however, fibrocartilage was distinguished by heavy Col1 deposits in the deep zone, a 2-fold higher mean Col1 percentage stain (P = .001), and lower surface integrity (P = .03). ZCT scores correlated with GAG content and the ICRS-II overall assessment score, especially when combined with the PLM score for collagen organization (R = 0.82). Histological scores of the deep zone strongly predicted the ICRS-II overall assessment score (R = 0.99). CONCLUSION: The ICRS-II overall repair assessment score and GAG content correlated with the extent of Col2 deposition free of fibrosis in the deep/middle zone rather than bulk accumulation of Col2. CLINICAL RELEVANCE: Biopsy tissue from the BST-CarGel randomized clinical trial (microfracture without and with BST-CarGel, as treatment groups were not unblinded) showed regenerated tissue consistent with a chondroinduction mechanism in at least half of the treated lesions.

Effect of corneal hydration on the quality of the femtosecond laser anterior lamellar cut.

The goal of this study was to assess the effect of corneal hydration on the quality of the femtosecond laser (FSL) anterior lamellar cut. The Visumax FSL was used to dissect an 8-mm-diameter corneal flap in 22 eye bank corneas showing various levels of hydration. The intended ablation depth was 220 µm in all eyes, which corresponded to the maximal depth available with this laser. After the cut, the achieved ablation depth was measured using optical coherence tomography images, flap separability was assessed by measuring the mean force generated to detach the flap, and stromal bed roughness was assessed by measuring the Haralick contrast level on the 1000× scanning electron microscopy images of the ablated surfaces. The preoperative central corneal thickness ranged from 547 to 1104 µm (mean ± SEM: 833 ± 30 µm). A negative correlation was found between the level of corneal hydration and the ablation depth measured in the mid-peripheral cornea (r =  -0.626, p = 0.003), the ablation being more superficial in more edematous corneas. The Haralick contrast also tended to increase as a function of corneal hydration (r = 0.416, p = 0.061), suggesting that laser ablation in edematous corneas results in rougher stromal surfaces. These results support the hypothesis that the quality of the FSL lamellar cut decreases as the level of corneal hydration increases. Although FSL is still considered in the field as the tool of the future for corneal dissection, a better understanding of the limits of this tool will be needed before it can replace manual or automated stromal dissection techniques in hydrated corneas.

Microdrilled cartilage defects treated with thrombin-solidified chitosan/blood implant regenerate a more hyaline, stable, and structurally integrated osteochondral unit compared to drilled controls.

This study analyzed the long-term cartilage and subchondral bone repair of microdrilled defects treated with chitosan glycerol-phosphate/blood implant, using thrombin (Factor IIa) to accelerate in situ solidification. We also evaluated the cartilage repair response to six smaller microdrill holes compared with two larger holes. Bilateral knee trochlear cartilage defects were created in n=8 skeletally mature rabbits, drilled with six proximal 0.5 mm and two distal 0.9 mm holes, then covered with in situ-solidified IIa-implants (treated) or with IIa-alone (control). After 6.5 months of repair, cartilage repair tissues were analyzed by histological scoring and histomorphometry for hyaline matrix characteristics and osseous integration. Subchondral repair bone was analyzed by 3D microcomputed tomography and compared to acute defects (n=6) and intact trochlea (n=8). Implant-treated cartilage repair tissues had higher structural integrity through the entire defect (p=0.02), twofold higher percent staining for glycosaminoglycan (p=0.0004), and ~24% more collagen type II staining over the smaller drill holes (p=0.008) compared with controls. Otherwise, hole diameter had no specific effect on cartilage repair. The subchondral bone plate was partially restored in treated and control defects but less dense than intact trochlea, with evidence of incomplete regeneration of the calcified cartilage layer. More residual drill holes (p=0.054) were detected in control versus treated defects, and control defects with more than 40% residual holes presented abnormally thicker trabeculae compared with treated defects. Low osteoclast numbers after 6.5 months repair suggested that bone was no longer remodeling. The subchondral bone plate surrounding the defects exhibited a significant thickening compared with age-matched intact trochlea. These data suggest that debridement and drilling can lead to long-term subchondral bone changes outside the cartilage defect. Compared with drilled controls, chitosan implants solidified with thrombin elicited a more hyaline and structurally integrated osteochondral unit, features needed for long-term durability.

Mesenchymal stem cell transplantation to promote bone healing.

An overall decline in the availability of osteogenic precursor cells and growth factors in the bone marrow microenvironment have been associated with impaired bone formation and osteopenia in humans. The objective of the current study was to determine if transplantation of mesenchymal stromal cells (MSC) from a healthy, young donor mouse into an osteopenic recipient mouse could enhance osseointegration of a femoral implant. MSC harvested from normal young adult mice differentiated into bone forming osteoblasts when cultured on implant grade titanium surfaces ex vivo and promoted bone formation around titanium-coated rods implanted in the femoral canal of osteopenic recipient mice. Micro computed tomographic imaging and histological analyses showed more, better quality, bone in the femur that received the MSC transplant compared with the contra-lateral control femur that received carrier alone. These results provide pre-clinical evidence that MSC transplantation promotes peri-implant bone regeneration and suggest the approach could be used in a clinical setting to enhance bone regeneration and healing in patients with poor quality bone.

Excess polycation mediates efficient chitosan-based gene transfer by promoting lysosomal release of the polyplexes.

The optimal ratio of the polycation's amine to DNA phosphate group (N:P) for efficient polymer-based transfection always employs excess polycation versus DNA. Most of the excess polycation remains free in solution, unassociated with the polyplexes, but is essential for efficient transfection. The mechanism by which excess polycation increases transfection efficiency is not identified. We hypothesised that excess chitosan facilitates intracellular lysosomal escape of the polyplexes. We highlight here the essential role of excess chitosan by rescuing poorly transfecting low N:P ratio polyplexes, by adding free chitosan before or after polyplex addition to cells. We examined polyplex uptake, the kinetics of rescue, intracellular trafficking, and the effects of lysosomotropic agents. We found the facilitating role of excess chitosan to be downstream of cellular uptake. Live-cell confocal quantification of intracellular trafficking revealed prolonged colocalisation of low N:P polyplexes within lysosomes, compared to shorter residence times for both rescued or N:P 5 samples, followed by observation of free pDNA in the cytosol. These data demonstrate that excess polycation mediates enhanced transfection efficiency by promoting the release of polyplexes from the endo-lysosomal vesicles, revealing a critical intracellular barrier overcome by excess polycation and suggesting possible avenues for further optimisation of polymer-based gene delivery.

Scaffold-guided subchondral bone repair: implication of neutrophils and alternatively activated arginase-1+ macrophages.

BACKGROUND: Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1, release angiogenic factors, and could be potential mediators of trabecular bone repair. HYPOTHESIS: Chitosan-glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (+/-blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37 degrees C were analyzed by proteomics and neutrophil chemotaxis assays. RESULTS: Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB, IL-8/CXCL8, MCP-1/CCL2, and no IL-1beta or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (+/-blood) implants attracted more neutrophils (P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls (P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue. CONCLUSION AND CLINICAL RELEVANCE: AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone.

Effect of chitosan particles and dexamethasone on human bone marrow stromal cell osteogenesis and angiogenic factor secretion.

Chitosan is a polysaccharide scaffold used to enhance cartilage repair during treatments involving bone marrow stimulation, and it is reported to increase angiogenesis and osteogenesis in vivo. Here, we tested the hypotheses that addition of chitosan particles to the media of human bone marrow stromal cell (BMSC) cultures stimulates osteogenesis by promoting osteoblastic differentiation and by favoring the release of angiogenic factors in vitro. Confluent BMSCs were cultured for 3 weeks with 16% fetal bovine serum, ascorbate-2-phosphate and disodium beta-glycerol phosphate, in the absence or presence of dexamethasone, an anti-inflammatory glucocorticoid commonly used as an inducer of BMSC osteoblast differentiation in vitro. As expected, dexamethasone slowed cell division, stimulated alkaline phosphatase activity and enhanced matrix mineralization. Added chitosan particles accumulated intra- and extracellularly and, while not affecting most osteogenic features, they inhibited osteocalcin release to the media at day 14 and interfered with mineralized matrix deposition. Interestingly, dexamethasone promoted cell attachment and suppressed the release and activation of matrix metalloprotease-2 (MMP-2). While chitosan particles had no effect on the release of angiogenic factors, dexamethasone significantly inhibited (p<0.05 to p<0.0001) the release of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukins 1beta, 4, 6, and 10 (IL-1beta, IL-4, IL-6, IL-10), and a host of other inflammatory factors that were constitutively secreted by BMSCs. These results demonstrate that chitosan particles alone are not sufficient to promote osteoblast differentiation of BMSCs in vitro, and suggest that chitosan promotes osteogenesis in vivo through indirect mechanisms. Our data further show that continuous addition of dexamethasone promotes osteoblastic differentiation in vitro partly by inhibiting gelatinase activity and by suppressing inflammatory cytokines which result in increased cell attachment and cell cycle exit.