Stereo Logical Analysis of Subchondral Angiogenesis Induced by Chitosan and Coagulation Factors in Microdrilled Articular Cartilage Defects

SummaryObjectiveCartilage repair elicited by bone marrow stimulation can be enhanced by a chitosan-glycerol phosphate (GP)/blood implant, through mechanisms involving therapeutic inflammatory angiogenesis. The implant is formed by in situ coagulation, which can be accelerated by adding coagulation factors. We hypothesized that coagulation factors enhance acute subchondral angiogenesis in repairing drilled defects.DesignFull-thickness cartilage defects were created bilaterally in 12 skeletally mature rabbit knee trochlea, microdrilled, then allowed to bleed as a control (N = 6) or treated with chitosan-GP/blood implant (N = 6), or implant solidified with thrombin (IIa), tissue factor (TF) with recombinant human factor VIIa (rhFVIIa), or rhFVIIa alone (N = 4 each condition). At 3 weeks post-operative, quantitative stereology was used to obtain blood vessel length (LV), surface (SV), and volume (VV) density at systematic depths in two microdrill holes per defect. Collagen type I, type II and glycosaminoglycan (GAG) percent stain in non-mineralized repair tissue were analysed by histomorphometry.ResultsAll drill holes were healing, and showed a depth-dependent increase in granulation tissue blood vessel density (Lv, Sv, and Vv, P < 0.005). Residual chitosan implant locally suppressed blood vessel ingrowth into the granulation tissue, whereas holes completely cleared of chitosan amplified angiogenesis vs microdrill-only (P = 0.049), an effect enhanced by IIa. Chitosan implant suppressed strong Col-I, Col-II, and GAG accumulation that occurred spontaneously in drill-only bone defects (P < 0.005) and coagulation factors did not alter this effect.ConclusionsSubchondral angiogenesis is promoted by chitosan implant clearance. Chitosan implant treatment suppresses fibrocartilage scar tissue formation, and promotes bone remodeling, which allows more blood vessel migration and woven bone repair towards the cartilage lesion area

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

Bone Marrow Stimulation Induces Greater Chondrogenesis in Trochlear Vs Condylar Cartilage Defects in Skeletally Mature Rabbits

SummaryObjectiveThe aim of this study was to compare the early repair response of cartilage defects in trochlea (TR) and medial femoral condyle (MFC) at 2–3 weeks after bone marrow stimulation.DesignBilateral full-thickness cartilage defects were generated in central trochlear groove and MFC of skeletally mature rabbits. Four subchondral perforations were made on each defect, either by microfracture to 2 mm deep, or by drilling to 2 mm or 6 mm deep. Rabbits were sacrificed either on Day 14 post-operatively or on Day 21. Defects were analyzed by histology, stereology, histomorphometry and micro-computed tomography (CT). Intact femurs (N = 4) served as controls.ResultsStromal cell density recruitment was similar in all defects, irrespective of defect location and surgical techniques used. There was a robust appearance of chondrocytes at Day 21 in TR defects with significantly higher volume fraction of chondrocytes in TR compared to MFC (P = 0.013). Chondrogenic foci were observed in marrow penetrating holes, with a significantly higher frequency and larger foci in TR vs MFC defects at Day 21 (P = 0.043 and P = 0.0014, respectively). Micro-CT analysis showed that deep drilling elicited significantly more mineralized bone fill compared to shallower perforations at 2 and 3 weeks repair (all at P ≤ 0.0008).ConclusionsBone marrow stimulation induced greater chondrogenesis in TR vs MFC defects in adult rabbits, with more chondrocytes and larger chondrogenic foci appearing in TR vs MFC on Day 21 post-operation