Anik Chevrier

Chitosan-Glycerol Phosphate/Blood Implants Improve Hyaline Cartilage Repair in Ovine Microfracture Defects

BACKGROUND Microfracture is a surgical procedure that is used to treat focal articular cartilage defects. Although joint function improves following microfracture, the procedure elicits incomplete repair. As blood clot formation in the microfracture defect is an essential initiating event in microfracture therapy, we hypothesized that the repair would be improved if the microfracture defect were filled with a blood clot that was stabilized by the incorporation of a thrombogenic and adhesive polymer, specifically, chitosan. The objectives of the present study were to evaluate (1) blood clot adhesion in fresh microfracture defects and (2) the quality of the repair, at six months postoperatively, of microfracture defects that had been treated with or without chitosan-glycerol phosphate/blood clot implants, using a sheep model. METHODS In eighteen sheep, two 1-cm2 full-thickness chondral defects were created in the distal part of the femur and treated with microfracture; one defect was made in the medial femoral condyle, and the other defect was made in the trochlea. In four sheep, microfracture defects were created bilaterally; the microfracture defects in one knee received no further treatment, and the microfracture defects in the contralateral knee were filled with chitosan-glycerol phosphate/autologous whole blood and the implants were allowed to solidify. Fresh defects in these four sheep were collected at one hour postoperatively to compare the retention of the chitosan-glycerol phosphate/blood clot with that of the normal clot and to define the histologic characteristics of these fresh defects. In the other fourteen sheep, microfracture defects were made in only one knee and either were left untreated (control group; six sheep) or were treated with chitosan-glycerol phosphate/blood implant (treatment group; eight sheep), and the quality of repair was assessed histologically, histomorphometrically, and biochemically at six months postoperatively. RESULTS In the defects that were examined one hour postoperatively, chitosan-glycerol phosphate/blood clots showed increased adhesion to the walls of the defects as compared with the blood clots in the untreated microfracture defects. After histological processing, all blood clots in the control microfracture defects had been lost, whereas chitosanglycerol phosphate/blood clot adhered to and was partly retained on the surfaces of the defect. At six months, defects that had been treated with chitosan-glycerol phosphate/blood were filled with significantly more hyaline repair tissue (p < 0.05) compared with control defects. Repair tissue from medial femoral condyle defects that had been treated with chitosan-glycerol phosphate/blood contained more cells and more collagen compared with control defects and showed complete restoration of glycosaminoglycan levels. CONCLUSIONS Solidification of a chitosan-glycerol phosphate/blood implant in microfracture defects improved cartilage repair compared with microfracture alone by increasing the amount of tissue and improving its biochemical composition and cellular organization.

Meniscus structure in human, sheep, and rabbit for animal models of meniscus repair.

Meniscus injury is a frequently encountered clinical orthopedic issue and is epidemiologically correlated to osteoarthritis. The development of new treatments for meniscus injury is intimately related to the appropriateness of animal models for their investigation. The purpose of this study was to structurally compare human menisci to sheep and rabbit menisci to generate pertinent animal models for meniscus repair. Menisci were analyzed histologically, immunohistochemically, and by environmental scanning electron microscopy (ESEM). In all species, collagen I appeared throughout most menisci, but was absent from the inner portion of the tip in some samples. Collagen II was present throughout the inner main meniscal body, while collagen VI was found in pericellular and perivascular regions. The glycosaminoglycan‐rich inner portion of menisci was greater in area for rabbit and sheep compared to human. Cells were rounded in central regions and more fusiform at the surface, with rabbit being more cellular than sheep and human. Vascular penetration in rabbit was confined to the very outermost region (1% of meniscus length), while vessels penetrated deeper into sheep and human menisci (11–15%). ESEM revealed a lamellar collagenous structure at the articulating surfaces of sheep and human menisci that was absent in rabbit. Taken together, these data suggest that the main structural features that will influence meniscus repair—cellularity, vascularity, collagen structure—are similar in sheep and human but significantly different in rabbit, motivating the development of ovine meniscus repair models.

Depth of Subchondral Perforation Influences the Outcome of Bone Marrow Stimulation Cartilage Repair

Subchondral drilling and microfracture are bone marrow stimulation techniques commonly used for the treatment of cartilage defects. Few studies to date have examined the technical variants which may influence the success of the cartilage repair procedures. This study compared the effect of hole depth (6 mm vs. 2 mm) and hole type (drill vs. microfracture) on chondral defect repair using a mature rabbit model. Results from quantitative histomorphometry and histological scoring showed that deeper versus shallower drilling elicited a greater fill of the cartilage defect with a more hyaline character in the repair matrix indicated by significant improvement (p = 0.021) in the aggregate measure of increased cartilage defect fill, increased glycosaminoglycan and type II collagen content and reduced type I collagen content of total soft repair tissue. Compared to microfracture at the same 2 mm depth, drilling to 2 mm produced a similar quantity and quality of cartilage repair (p = 0.120) according to the aggregate indicator described above. We conclude that the depth of bone marrow stimulation can exert important influences on cartilage repair outcomes.

Characterization of Subchondral Bone Repair for Marrow-Stimulated Chondral Defects and Its Relationship to Articular Cartilage Resurfacing

Background Microfracture and drilling are bone marrow–stimulation techniques that initiate cartilage repair by providing access to cell populations in subchondral bone marrow. This study examined the effect of hole depth and of microfracture versus drilling on subchondral bone repair and cartilage repair in full-thickness chondral defects. Hypotheses Repaired subchondral bone does not reconstitute its native structure and exhibits atypical morphologic features. Drilling deeper induces greater bone remodeling and is related to improved cartilage repair. Study Design Controlled laboratory study. Methods Trochlear cartilage defects debrided of the calcified layer were prepared bilaterally in 16 skeletally mature rabbits. Drill holes were made to a depth of 2 mm or 6 mm and microfracture holes to 2 mm. Animals were sacrificed 3 months postoperatively, and joints were scanned by micro–computed tomography before histoprocessing. Bone repair was assessed with a novel scoring system and by 3-dimentional micro–computed tomography and compared with intact controls. Correlation of subchon-dral bone features to cartilage repair outcome was performed. Results Although surgical holes were partly repaired with mineralized tissue, atypical features such as residual holes, cysts, and bony overgrowth were frequently observed. For all treatment groups, repair led to an average bone volume density similar to that of the controls but the repair bone was more porous and branched as shown by significantly higher bone surface area density and connectivity density. Deeper versus shallower drilling induced a larger region of repairing and remodeling subchondral bone that positively correlated with improved cartilage repair. Conclusion Incomplete reconstitution of normal bone structure and continued remodeling occurred in chondral defects 3 months after bone marrow stimulation. Deep drilling induced a larger volume of repairing and remodeling bone, which appeared beneficial for chondral repair. Clinical Relevance Bone marrow stimulation does not reconstitute normal bone structure. Strategies that increase subchondral bone involvement in marrow stimulation could further benefit cartilage repair.

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.

High Frequency Acoustic Parameters of Human and Bovine Articular Cartilage following Experimentally-Induced Matrix Degradation:

Matrix degradation and proteoglycan loss in articular cartilag eare features of early osteoarthritis. To determine the effect of matrix degradation and proteoglycan loss on ultrasound propagation in cartilage, we used papain and interleukin-1α to degrade the matrix proteoglycans of human and bovine cartilage samples, respectively. There is also minor collagen alteration associated with these chemical degradation methods. We compared the speed of sound and frequency dependent attenuation (20–40 MHz) of control and experimental paired samples. We found that a loss of matrix proteoglycans and collagen disruption resulted in a 20–30% increase in the frequency dependent attenuation and a 2% decrease in the speed of sound in both human and bovine cartilage. We conclude that the frequency dependent attenuation and speed of sound in articular cartilage are sensitive to experimental modification of the matrix proteoglycans and collagen. These findings suggest that ultrasound can potentially be used to detect morphologic changes in articular cartilage associated with the progression of osteoarthritis.

Temporal and spatial modulation of chondrogenic foci in subchondral microdrill holes by chitosan-glycerol phosphate/blood implants

OBJECTIVE Subchondral drilling initiates a cartilage repair response involving formation of chondrogenic foci in the subchondral compartment. The purpose of this study was to structurally characterize these sites of chondrogenesis and to investigate the effects of chitosan-glycerol phosphate (GP)/blood implants on their formation. METHOD Thirty-two New Zealand White rabbits received bilateral cartilage defects bearing four subchondral drill holes. One knee per rabbit was treated by solidifying a chitosan-GP/blood implant over the defect. After 1-56 days of repair, chondrogenic foci were characterized by histostaining and immunostaining. Collagen fiber orientation was characterized by polarized light microscopy. RESULTS Glycosaminoglycan and collagen type II were present throughout the foci while the upper zone expressed collagen type I and the lower zone collagen type X. Large chondrogenic foci had a stratified structure with flatter cells closer to the articular surface, and round or hypertrophic chondrocytes deeper in the drill holes that showed signs of calcification after 3 weeks of repair in control defects. Markers for pre-hypertrophic chondrocytes (Patched) and for proliferation (Ki-67) were detected within foci. Some cells displayed a columnar arrangement where collagen was vertically oriented. For treated defects, chondrogenic foci appeared 1-3 weeks later, foci were nascent and mature rather than resorbing, and foci developed closer to the articular surface. CONCLUSIONS Chondrogenic foci bear some similarities to growth cartilage and can give rise to a repair tissue that has similar zonal stratification as articular cartilage. The temporal and spatial formation of chondrogenic foci can be modulated by cartilage repair therapies.

Optimization of Histoprocessing Methods to Detect Glycosaminoglycan, Collagen Type II, and Collagen Type I in Decalcified Rabbit Osteochondral Sections

Abstract Rabbit models frequently are used as a small animal cartilage repair model, where hyaline articular cartilage repair can be identified by the presence of type II collagen, the absence of type I collagen, and an abundant glycosaminoglycan component (GAG). Staining methods for these three extracellular matrix components should therefore function in the same sample after histologic processing. The goal of the current study was to optimize histoprocessing techniques to maximally retain GAG, while still enabling the immunodetection of both collagen type II and collagen type I in decalcified rabbit osteochondral sections. We compared four different fixation methods and two different decalcification procedures in both paraffin and cryostat sections using a histological grading scale for five tissue characteristics, including chondrocyte morphology, bone marrow structure, cartilage GAG staining with Safranin O, cartilage immunostaining for type II collagen, and bone matrix immunostaining for type I collagen. We found that addition of ruthenium hexaammine trichloride to the fixation solution provided the best retention of chondrocyte morphology. However, ruthenium hexaammine trichloride interfered with immunodetection of type II collagen, an effect not observed for any of the other fixation conditions. Additionally, paraffin embedding was found to suppress immunodetection of type I collagen in bone matrix compared with cryosectioning. Nonetheless, bone marrow structure was better preserved in paraffin sections. Taken together, our results show that fixation in formalin or paraformaldehyde, followed by decalcification in acid with trace fixative, and then cryosectioning permitted 1) adequate preservation of chondrocyte morphology, 2) Safranin O staining of GAG in cartilage matrix, 3) immunostaining of type II collagen in cartilage matrix, and 4) immunostaining of type I collagen in bone matrix. (The J Histotechnol 28:165, 2005) Submitted March 28, 2005; accepted July 7, 2005

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.

Stereological analysis of subchondral angiogenesis induced by chitosan and coagulation factors in microdrilled articular cartilage defects

OBJECTIVE Cartilage 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. DESIGN Full-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 (L(V)), surface (S(V)), and volume (V(V)) 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. RESULTS All 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. CONCLUSIONS Subchondral 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.