Subchondral pre-solidified chitosan/blood implants elicit reproducible early osteochondral wound-repair responses including neutrophil and stromal cell chemotaxis, bone resorption and repair, enhanced repair tissue integration and delayed matrix deposition

Abstract

BackgroundIn this study we evaluated a novel approach to guide the bone marrow-driven articular cartilage repair response in skeletally aged rabbits. We hypothesized that dispersed chitosan particles implanted close to the bone marrow degrade in situ in a molecular mass-dependent manner, and attract more stromal cells to the site in aged rabbits compared to the blood clot in untreated controls.MethodsThree microdrill hole defects, 1.4 mm diameter and 2 mm deep, were created in both knee trochlea of 30 month-old New Zealand White rabbits. Each of 3 isotonic chitosan solutions (150, 40, 10 kDa, 80% degree of deaceylation, with fluorescent chitosan tracer) was mixed with autologous rabbit whole blood, clotted with Tissue Factor to form cylindrical implants, and press-fit in drill holes in the left knee while contralateral holes received Tissue Factor or no treatment. At day 1 or day 21 post-operative, defects were analyzed by micro-computed tomography, histomorphometry and stereology for bone and soft tissue repair.ResultsAll 3 implants filled the top of defects at day 1 and were partly degraded in situ at 21 days post-operative. All implants attracted neutrophils, osteoclasts and abundant bone marrow-derived stromal cells, stimulated bone resorption followed by new woven bone repair (bone remodeling) and promoted repair tissue-bone integration. 150 kDa chitosan implant was less degraded, and elicited more apoptotic neutrophils and bone resorption than 10 kDa chitosan implant. Drilled controls elicited a poorly integrated fibrous or fibrocartilaginous tissue.ConclusionsPre-solidified implants elicit stromal cells and vigorous bone plate remodeling through a phase involving neutrophil chemotaxis. Pre-solidified chitosan implants are tunable by molecular mass, and could be beneficial for augmented marrow stimulation therapy if the recruited stromal cells can progress to bone and cartilage repair.

Chondrogenic Regeneration Using Bone Marrow Clots and a Porous Polycaprolactone-Hydroxyapatite Scaffold by Three-Dimensional Printing

“…6,19 The sulfated glycosaminoglycan (GAG) content of engineered tissues was quantified by ELISA using dimethylmethylene blue (DMMB; Sigma Aldrich) colorimetric assay with chondroitin sulfate from shark cartilage to generate a standard curve. Total DNA content was measured using Quit-iT dsDNA kit (Invitrogen) with salmon test DNA to generate a standard curve.…”

“…6,20,21 In brief, bone marrow was aspirated from the posterior superior iliac crest, and mononuclear cells (MNCs) were isolated by density gradient centrifugation. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% HEPES buffer, and 1% antibiotics-antimycotics in a 37°C, 5% CO 2 incubator.…”

“…4 Previous evidence proved that bone marrow clots (MC) on the surface of the microfracture lesions provide an optimal environment for cartilaginous tissue repair. 5,6 However, this technique results in a repaired tissue with lower mechanical strength. This tissue is easily worn by daily activities, causing symptoms to recur and necessitating a second repair.…”

Enhanced Anchorage of Tissue-Engineered Cartilage Using an Osteoinductive Approach

“…However, other laboratories have attempted to augment the interfacial mechanical properties of engineered cartilage and subchondral bone using biomaterials such as chitosan particles [48], porous calcium polyphosphate [49], chitosan-chondroitin sulfate [50]. One group even used HA in conjunction with non-photopolymerizable alginate hydrogels and reported on its shear mechanical properties [18,51].…”

“…Elsewhere, Hoemann et al [17] indicated that Chitosan glycerol phosphate when mixed with blood in the cartilage defect space not only formed greatly improved integration with sub-chondral bone, but also resulted in enhanced hyaline cartilage tissue formation. Indeed, in vivo (primarily in the rabbit model) cartilage tissue engineering studies have shown convincing bone marrow stem cell migratory responses towards the ostechondral interface and subsequent tissue remodeling when chitosan was utilized [47,48].…”