Catherine Marchand

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°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-1β 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.

Solidification mechanisms of chitosan-glycerol phosphate/blood implant for articular cartilage repair.

OBJECTIVE Chitosan-glycerol phosphate (chitosan-GP) is a unique polymer solution that is mixed with whole blood and solidified over microfractured or drilled articular cartilage defects in order to elicit a more hyaline repair cartilage. For clinical ease-of-use, a faster in situ solidification is preferred. Therefore, we investigated the mechanisms underlying chitosan-GP/blood implant solidification. METHODS In vitro solidification of chitosan-GP/blood mixtures, with or without added clotting factors, was evaluated by thromboelastography. Serum was analyzed for the onset of thrombin, platelet, and FXIII activation. In vivo solidification of chitosan-GP/blood mixtures, with and without clotting factors, was evaluated in microdrilled cartilage defects of adult rabbits (N=41 defects). RESULTS Chitosan-GP/blood clots solidified in an atypical biphasic manner, with higher initial viscosity and minor platelet activation followed by the development of clot tensile strength concomitant with thrombin generation, burst platelet and FXIII activation. Whole blood and chitosan-GP/blood clots developed a similar final clot tensile strength, while polymer-blood clots showed a unique, sustained platelet factor release and greater resistance to lysis by tissue plasminogen activator. Thrombin, tissue factor (TF), and recombinant human activated factor VII (rhFVIIa) accelerated chitosan-GP/blood solidification in vitro (P<0.05). Pre-application of thrombin or rhFVIIa+TF to the surface of drilled cartilage defects accelerated implant solidification in vivo (P<0.05). CONCLUSIONS Chitosan-GP/blood implants solidify through coagulation mechanisms involving thrombin generation, platelet activation and fibrin polymerization, leading to a dual fibrin-polysaccharide clot scaffold that resists lysis and is physically more stable than normal blood clots. Clotting factors have the potential to enhance the practical use, the residency, and therapeutic activity of polymer-blood implants.

Acute Osteoclast Activity following Subchondral Drilling Is Promoted by Chitosan and Associated with Improved Cartilage Repair Tissue Integration

Objective: Cartilage-bone integration is an important functional end point of cartilage repair therapy, but little is known about how to promote integration. We tested the hypothesis that chitosan-stabilized blood clot implant elicits osteoclasts to drilled cartilage defects and promotes repair and cartilage-bone integration. Design: Bilateral trochlear defects in 15 skeletally mature rabbit knees were microdrilled and then treated with chitosan–glycerol phosphate (GP)/blood implant with fluorescent chitosan tracer and thrombin to accelerate in situ solidification or with thrombin alone. Chitosan clearance, osteoclast density, and osteochondral repair were evaluated at 1, 2, and 8 weeks at the outside, edge, and through the proximal microdrill holes. Results: Chitosan was retained at the top of the drill holes at 1 week as extracellular particles became internalized by granulation tissue cells at 2 weeks and was completely cleared by 8 weeks. Osteoclasts burst-accumulated at microdrill hole edges at 1 week, in new woven bone at the base of the drill holes at 2 weeks, and below endochondral cartilage repair at 8 weeks. Implants elicited 2-fold more osteoclasts relative to controls (P < 0.001), a more complete drill hole bone repair, and improved cartilage-bone integration and histological tissue quality. Treated and control 8-week cartilage repair tissues contained 85% collagen type II. After 8 weeks of repair, subchondral osteoclast density correlated positively with bone-cartilage repair tissue integration (P < 0.0005). Conclusions: Chitosan-GP/blood implant amplified the acute influx of subchondral osteoclasts through indirect mechanisms, leading to significantly improved repair and cartilage-bone integration without inducing net bone resorption. Osteoclasts are cellular mediators of marrow-derived cartilage repair integration.

C3, C5, and factor B bind to chitosan without complement activation

Chitosan is a polycationic and biocompatible polysaccharide composed of glucosamine and N-acetyl glucosamine that is chemotactic for neutrophils and stimulates wound repair through mechanisms that remain unclear. It was previously shown that chitosan depletes complement proteins from plasma, suggesting that chitosan activates complement. Complement activation leads to cleavage of C5 to produce C5a, a neutrophil chemotactic factor. Here, we tested the hypothesis that chitosan generates C5a in human whole blood, citrated plasma, and serum. C5a fragment appeared in coagulating whole blood, and mixtures of chitosan-glycerol phosphate/whole blood, in parallel with platelet and thrombin activation. However, in plasma and serum, thrombin and chitosan-GP failed to generate C5a, although native C3, C5, and factor B adsorbed noncovalently to insoluble chitosan particles incubated in citrated plasma, serum, EDTA-serum and methylamine-treated plasma. By surface plasmon resonance, pure C3 adsorbed to chitosan. The profile of serum factors associating with chitosan was consistent with a model in which anionic blood proteins with a pI lower than the pK(0) 6.78 of chitosan (the upper limit of chitosan pK(a)) associate electrostatically with cationic chitosan particles. Zymosan, a yeast ghost particle, activated complement in serum and citrated plasma, but not in EDTA-serum or methylamine plasma, to generate fluid-phase C5a, while C3b formed covalent cross-links with zymosan-associated proteins and became rapidly cleaved to iC3b, with factor Bb stably associated. These data demonstrate that chitosan is a nonreactive biomaterial that does not directly activate complement, and provide a novel basis for predicting anionic serum protein-chitosan interactions.

Effect of chitosan and coagulation factors on the wound repair phenotype of bioengineered blood clots

Controlling the blood clot phenotype in a surgically prepared wound is an evolving concept in scaffold-guided tissue engineering. Here, we investigated the effect of added chitosan (80% or 95% Degree of Deacetylation, DDA) or coagulation factors (recombinant human Factor VIIa, Tissue Factor, thrombin) on inflammatory factors released by blood clots. We tested the hypothesis that 80% DDA chitosan specifically enhances leukotriene B4 (LTB4) production. Human or rabbit whole blood was combined with isotonic chitosan solutions, coagulation factors, or lipopolysaccharide, cultured in vitro at 37°C, and after 4hours the serum was assayed for LTB4 or inflammatory factors. Only 80% DDA chitosan clots produced around 15-fold more LTB4 over other clots including 95% DDA chitosan clots. All serum contained high levels of PDGF-BB and CXCL8. Normal clots produced very low type I cytokines compared to lipopolysaccharide clots, with even lower IL-6 and IL-12 and more CCL3/CCL4 produced by chitosan clots. Coagulation factors had no detectable effect on clot phenotype. Conclusion In blood clots from healthy individuals, 80% DDA chitosan has a unique influence of inducing more LTB4, a potent neutrophil chemoattractant, with similar production of PDGF-BB and CXCL8, and lower type I cytokines, compared to whole blood clots.