Chitosan-glycerol phosphate/blood implants elicit hyaline cartilage repair integrated with porous subchondral bone in microdrilled rabbit defects

OBJECTIVE: We have previously shown that microfractured ovine defects are repaired with more hyaline cartilage when the defect is treated with in situ-solidified implants of chitosan-glycerol phosphate (chitosan-GP) mixed with autologous whole blood. The objectives of this study were (1) to characterize chitosan-GP/blood clots in vitro, and (2) to develop a rabbit marrow stimulation model in order to determine the effects of the chitosan-GP/blood implant and of debridement on the formation of incipient cartilage repair tissue. METHODS: Blood clots were characterized by histology and in vitro clot retraction tests. Bilateral 3.5 x 4 mm trochlear defects debrided into the calcified layer were pierced with four microdrill holes and filled with a chitosan-GP/blood implant or allowed to bleed freely as a control. At 1 day post-surgery, initial defects were characterized by histomorphometry (n=3). After 8 weeks of repair, osteochondral repair tissues between or through the drill holes were evaluated by histology, histomorphometry, collagen type II expression, and stereology (n=16). RESULTS: Chitosan-GP solutions structurally stabilized the blood clots by inhibiting clot retraction. Treatment of drilled defects with chitosan-GP/blood clots led to the formation of a more integrated and hyaline repair tissue above a more porous and vascularized subchondral bone plate compared to drilling alone. Correlation analysis of repair tissue between the drill holes revealed that the absence of calcified cartilage and the presence of a porous subchondral bone plate were predictors of greater repair tissue integration with subchondral bone (P<0.005), and of a higher total O'Driscoll score (P<0.005 and P<0.01, respectively). CONCLUSIONS: Chitosan-GP/blood implants applied in conjunction with drilling, compared to drilling alone, elicited a more hyaline and integrated repair tissue associated with a porous subchondral bone replete with blood vessels. Concomitant regeneration of a vascularized bone plate during cartilage repair could provide progenitors, anabolic factors and nutrients that aid in the formation of hyaline cartilage.     

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.     

Tibial subchondral bone size and knee cartilage defects: relevance to knee osteoarthritis

Unlike knee plain radiography which can only detect joint space narrowing and osteophytes, magnetic resonance imaging can directly visualize and analyse the whole knee structure, including bone size, cartilage defects and loss of cartilage volume. Tibial subchondral bone area expansion may be primary and is associated with risk factors such as age, body mass index (BMI), genetics and/or limb malalignment. It can lead to the development of knee defects, which may also be caused by demographic, anthropometric and environmental factors such as age, female sex, BMI and smoking as well as structural changes such as osteophytes, bone marrow lesions, meniscal tears, meniscal extrusion and ligament abnormalities. Once knee cartilage defects develop, they have a variable natural history but are associated with subsequent cartilage loss in a dose-response manner. Both tibial subchondral bone area and knee cartilage defects are quantitatively related to the severity of knee osteoarthritis (OA), and predictive of the need for knee joint replacement in subjects with knee OA independent of radiographic change. Taken as a whole, these studies suggest that tibial subchondral bone expansion and cartilage defect development represent important targets for the prevention of cartilage loss and joint replacement.     

Abnormal subchondral bone remodeling and its association with articular cartilage degradation in knees of type 2 diabetes patients

Type 2 diabetes(T2 D) is associated with systemic abnormal bone remodeling and bone loss. Meanwhile,abnormal subchondral bone remodeling induces cartilage degradation, resulting in osteoarthritis(OA).Accordingly, we investigated alterations in subchondral bone remodeling, microstructure and strength in knees from T2 D patients and their association with cartilage degradation. Tibial plateaus were collected from knee OA patients undergoing total knee arthroplasty and divided into non-diabetic(n = 70) and diabetes(n = 51) groups. Tibial plateaus were also collected from cadaver donors(n = 20) and used as controls.Subchondral bone microstructure was assessed using micro-computed tomography. Bone strength was evaluated by micro-finite-element analysis. Cartilage degradation was estimated using histology. The expression of tartrate-resistant acidic phosphatase(TRAP), osterix, and osteocalcin were calculated using immunohistochemistry. Osteoarthritis Research Society International(OARSI) scores of lateral tibial plateau did not differ between non-diabetic and diabetes groups, while higher OARSI scores on medial side were detected in diabetes group. Lower bone volume fraction and trabecular number and higher structure model index were found on both sides in diabetes group. These microstructural alterations translated into lower elastic modulus in diabetes group. Moreover, diabetes group had a larger number of TRAP~+ osteoclasts and lower number of Osterix~+ osteoprogenitors and Osteocalcin~+ osteoblasts. T2 D knees are characterized by abnormal subchondral bone remodeling and microstructural and mechanical impairments, which were associated with exacerbated cartilage degradation. In regions with intact cartilage the underlying bone still had abnormal remodeling in diabetes group, suggesting that abnormal bone remodeling may contribute to the early pathogenesis of T2 D-associated knee OA.     

Elevated cross-talk between subchondral bone and cartilage in osteoarthritic joints

Osteoarthritis (OA) is a degenerative joint disease and one of the leading causes of disability in the United States and across the world. As a disease of the whole joint, OA exhibits a complicated etiology with risk factors including, but not limited to, ageing, altered joint loading, and injury. Subchondral bone is hypothesized to be involved in OA development. However, direct evidence supporting this is lacking. We previously detected measurable transport of solute across the mineralized calcified cartilage in normal joints, suggesting a potential cross-talk between subchondral bone and cartilage. Whether this cross-talk exists in OA has not been established yet. Using two models that induced OA by either ageing or surgery (destabilization of medial meniscus, DMM), we tested the hypothesis that increased cross-talk occurs in OA. We quantified the diffusivity of sodium fluorescein (mol. wt. 376Da), a marker of small-sized signaling molecules, within calcified joint matrix using our newly developed fluorescence loss induced by photobleaching (FLIP) method. Tracer diffusivity was found to be 0.30±0.17 and 0.33±0.20μm(2)/s within the calcified cartilage and 0.12±0.04 and 0.07±0.03μm(2)/s across the osteochondral interface in the aged (20-24-month-old, n=4) and DMM OA joints (5-month-old, n=5), respectively, which were comparable to the control values for the contralateral non-operated joints in the DMM mice (0.48±0.13 and 0.12±0.06μm(2)/s). Although we did not detect significant changes in tissue matrix permeability in OA joints, we found i) an increased number of vessels invading the calcified cartilage (and sometimes approaching the tidemark) in the aged (+100%) and DMM (+50%) joints relative to the normal age controls; and ii) a 60% thinning of the subchondral bone and calcified cartilage layers in the aged joints (with no significant changes detected in the DMM joints). These results suggested that the capacity for cross-talk between subchondral bone and articular cartilage could be elevated in OA. Further studies are needed to identify the direction of the cross-talk, the signaling molecules involved, and to test whether subchondral bone initiates OA development and could serve as a pharmaceutical target for OA treatment. This article is part of a Special Issue entitled "Osteoarthritis".     

Grafting of massive tibial subchondral bone defects in a caprine model using beta-tricalcium phosphate versus autograft

OBJECTIVE: This study evaluated the ability of beta-tricalcium phosphate particles (beta-TCP) and autograft (AUTO) to maintain joint surface morphology when used to supplement massive subchondral bone defects in a caprine model. DESIGN: This was a prospective, parallel arm study with 2 experimental arms and a control group. METHODS: Unilateral, 11 mm diameter, 25 mm deep cylindrical defects were created in tibial subchondral bone of anesthetized goats (n = 16) and filled with autograft or beta-tricalcium phosphate particles. The contralateral limbs served as internal controls. Goats were killed at 3 months and both tibiae harvested. Molds made of the tibial plateau surface were used to create positive casts from which medial and lateral tibial plateau surfaces of both experimental (beta-tricalcium phosphate particles, autograft) and control limbs were digitized in 3 dimensions. Mirror images of the medial condyle surface contours from the controls were superimposed onto the experimental surfaces and deviations were compared using a Student t test (alpha = 0.05). Tibiae were then cut sagittally into medial (biomechanics) and lateral (histology) halves. Compressive modulus within the defect area was assessed by indentation to 2.0 mm at 0.2 mm per second using a 6-mm diameter pin. Specimens from the lateral tibial plateau were processed for undecalcified histology and the area of bone within the defect region measured. The articular surface of 86% of the autograft and 0% of the beta-tricalcium phosphate particles group had degenerative changes, with 29% of autograft goats exhibiting large-scale plateau collapse. Mean surface deviation for autograft was significantly greater than for beta-tricalcium phosphate particles (2.19 +/- 1.49 mm versus 0.78 +/- 0.19 mm), as was maximum surface deviation (11.19 +/- 8.02 mm versus 4.39 +/- 1.33 mm) (P < 0.05). The compressive modulus within the defect area for control animals was significantly higher than the experimental groups (P < 0.05). Significantly more bone was regenerated within beta-tricalcium phosphate particle-grafted defects compared to autograft (P < 0.05). These results indicated that beta-tricalcium phosphate particles might be a useful graft material for local repair of load bearing skeletal sites such as depressed tibial plateau fractures.     

Alterations in the collagen framework of osteoarthritic cartilage and subchondral bone

Summary Specimens obtained from human osteoarthritic knee joints and dog knees with experimentally induced osteoarthritis were used to study the collagenous framework of articular cartilage and subchondral bone in relation to osteoarthritic changes using scanning electron microscopy and light microscopy. Degenerative articular cartilage in osteoarthritic joints showed radial orientation of the collagen fibrils, which were usually discernible as fibrillar bundle formations. Cartilage with extensive lesions often showed cleavages or fissures down to the calcified layer. The cartilagenous collagen fibrils in osteoarthritic specimens merged into the subchondral bone plate making the tidemark and the osteochondral junction irregular or obscure. The trabecular orientation of subchondral bone changed with alteration in the articular cartilage and with reactive changes in the subchondral bone, showing the effect of cartilaginous degeneration on its ultrastructure.

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Résumé Des prélèvements obtenus à partir de genoux humains arthrosiques et de genoux de chien porteurs de lésions arthrosiques expérimentales ont permis l'étude de la charpente de collagène du cartilage articulaire et de l'os sous-chondral, par microscopie conventionnelle et par microscopie électronique.Dans les lésions dégénératives, les fibrilles de collagène du cartilage ont une orientation radiale, alors qu'elles sont habituellement groupées en faisceaux. En cas de lésions étendues, le cartilage présente souvent des clivages ou des fissures menant jusqu'aux couches calcifiées. Les fibrilles de collagène du cartilage arthrosique se fondent dans la plaque osseuse souschondrale, rendant la jonction ostéo-cartilagineuse irrégulière ou mal définie.L'orientation trabéculaire de l'os sous-chondral se modifie en fonction de l'altération du cartilage articulaire. Ces modifications réactionnelles montrent que, même au niveau ultrastructural, l'architecture de l'os sous-chondral reflète la dégénérescence cartilagineuse.

     

Role of subchondral bone in the initiation and progression of cartilage damage

Osteoarthrosis is a physiologic imbalance, a "joint failure" similar to "heart failure," in which mechanical factors play a role. The initiation and progression of cartilage damage are distinct phenomena. One of the mechanisms of initiation may be a steep stiffness gradient in the underlying subchondral bone. Progression of cartilage lesions probably requires stiffened subchondral bone. In such situations, transverse stresses at the base of the articular cartilage could cause deep horizontal splits in that tissue. The most likely cause of subchondral stiffening in an otherwise congruent joint is repeated failure of the musculoskeletal peak dynamic force attenuation mechanisms. The health and integrity of the overlying articular cartilage depends on the mechanical properties of its bony bed. In certain models of osteoarthrosis, alterations of the bony bed occur before the cartilage changes and suggest that this can occur in clinical conditions. Stiffening of the subchondral bone also can effect joint conformation, which involves deformation of articular cartilage and bone to create maximum contact areas under load.     

降钙素对大鼠骨关节炎关节软骨和软骨下骨的影响

目的 观察骨吸收抑制剂降钙素对大鼠前交叉韧带切断术后膝关节软骨和软骨下骨的影响,探讨软骨下骨在骨关节炎发病中的作用.方法 建立大鼠右膝前交叉韧带切断的骨关节炎模型,术后第二天隔日皮下注射降钙素,模型组和Sham 组注射等量的生理盐水,持续6 w,并于术后10 w取材.切片后作甲苯胺蓝染色和Ⅱ型胶原免疫组化染色,并测量平均灰度值,用于评估关节软骨中蛋白多糖和Ⅱ型胶原的含量,行HE染色用于软骨下骨组织形态计量分析.结果 甲苯胺蓝、Ⅱ型胶原免疫组化染色平均灰度值和骨组织形态计量学参数测量显示与Sham 组相比模型组关节软骨中蛋白多糖和Ⅱ型胶原丢失,软骨下骨骨形成增加,骨质硬化.早期皮下注射降钙素可抑制大鼠骨关节炎后期软骨下骨骨形成的增加,减少关节软骨中蛋白多糖和Ⅱ型胶原的丢失.结论 软骨下骨在骨关节炎发病中发挥重要作用,骨吸收抑制剂降钙素可作为治疗骨关节炎的有潜力的药物.     

Intraarticular location predicts cartilage filling and subchondral bone changes in a chondral defect

Background and purpose The natural history of, and predictive factors for outcome of cartilage restoration in chondral defects are poorly understood. We investigated the natural history of cartilage filling subchondral bone changes, comparing defects at two locations in the rabbit knee.

Animals and methods In New Zealand rabbits aged 22 weeks, a 4-mm pure chondral defect (ICRS grade 3b) was created in the patella of one knee and in the medial femoral condyle of the other. A stereo microscope was used to optimize the preparation of the defects. The animals were killed 12, 24, and 36 weeks after surgery. Defect filling and the density of subchondral mineralized tissue was estimated using Analysis Pro software on micrographed histological sections.

Results The mean filling of the patellar defects was more than twice that of the medial femoral condylar defects at 24 and 36 weeks of follow-up. There was a statistically significant increase in filling from 24 to 36 weeks after surgery at both locations.

The density of subchondral mineralized tissue beneath the defects subsided with time in the patellas, in contrast to the density in the medial femoral condyles, which remained unchanged.

Interpretation The intraarticular location is a predictive factor for spontaneous filling and subchondral bone changes of chondral defects corresponding to ICRS grade 3b. Disregarding location, the spontaneous filling increased with long-term follow-up. This should be considered when evaluating aspects of cartilage restoration.     

Intraarticular location predicts cartilage filling and subchondral bone changes in a chondral defect

Background and purpose

The natural history of, and predictive factors for outcome of cartilage restoration in chondral defects are poorly understood. We investigated the natural history of cartilage filling subchondral bone changes, comparing defects at two locations in the rabbit knee.

Animals and methods

In New Zealand rabbits aged 22 weeks, a 4-mm pure chondral defect (ICRS grade 3b) was created in the patella of one knee and in the medial femoral condyle of the other. A stereo microscope was used to optimize the preparation of the defects. The animals were killed 12, 24, and 36 weeks after surgery. Defect filling and the density of subchondral mineralized tissue was estimated using Analysis Pro software on micrographed histological sections.

Results

The mean filling of the patellar defects was more than twice that of the medial femoral condylar defects at 24 and 36 weeks of follow-up. There was a statistically significant increase in filling from 24 to 36 weeks after surgery at both locations.The density of subchondral mineralized tissue beneath the defects subsided with time in the patellas, in contrast to the density in the medial femoral condyles, which remained unchanged.

Interpretation

The intraarticular location is a predictive factor for spontaneous filling and subchondral bone changes of chondral defects corresponding to ICRS grade 3b. Disregarding location, the spontaneous filling increased with long-term follow-up. This should be considered when evaluating aspects of cartilage restoration.Focal articular cartilage injuries of the knee are common (Hjelle et al. 2002, Aroen et al. 2004) and they can impair patients'' quality of life as much as severe osteoarthritis (Heir et al. 2010). The literature concerning the natural history of focal cartilage defects in patients, and the intrinsic factors affecting it, is limited (Linden 1977, Messner and Gillquist 1996, Drogset and Grontvedt 2002, Shelbourne et al. 2003, Loken et al. 2010). In experimental studies evaluating cartilage restoration in general, the importance of intrinsic factors such as the depth and size of the lesion and the time from when the lesion was made to evaluation have been emphasized (Shapiro et al. 1993, Hunziker 1999, Lietman et al. 2002). Which part of the joint is affected and whether or not the defect is weight-bearing are also of interest (Hurtig 1988, Frisbie et al. 1999). Most of these studies have, however, concerned defects penetrating the subchondral mineralized tissues corresponding to ICRS grade 4 (Brittberg and Winalski 2003). Access to bone marrow elements in these defects might be one of the strongest predictive factors for filling of the defect, making the importance of other factors difficult to evaluate (Hunziker 1999).In experimental studies on pure chondral defects that do not penetrate the subchondral mineralized tissues, corresponding to ICRS grade 3b (Brittberg and Winalski 2003), the type of animal studied, the size of the lesion, and the location of the defects vary, and there is limited data on the influence of these parameters on outcome (Breinan et al. 2000). The information on spontaneous filling comes mainly from observations of untreated defects serving as controls (Grande et al. 1989, Brittberg et al. 1996, Breinan et al. 1997, 2000, Frisbie et al. 1999, 2003, Dorotka et al. 2005) and the information on subchondral bone changes is even more limited (Breinan et al. 1997, Frisbie et al. 1999). Although most human focal cartilage lesions are located on the medial femur condyle (Aroen et al. 2004), there have been few experimental studies involving untreated ICRS grade 3b defects on the medial femur condyle (Dorotka et al. 2005). According to a PubMed search, the rabbit knee is the most widely used experimental animal model for cartilage restoration (Årøen 2005). The locations of ICRS grade 3 chondral defects in the rabbit knee evaluated for spontaneous changes have included the patella (Grande et al. 1989, Brittberg et al. 1996) and, in one study, defects at the distal surface of the femur (Mitchell and Shepard 1976). The latter report did not, however, include quantitative data.To our knowledge, the influence of the intraarticular location on the outcome of cartilage restoration and subchondral bone changes has not been thoroughly studied. Thus, the main purpose of our study was to test the hypothesis that the intraarticular location influences the spontaneous filling of a chondral defect that does not penetrate the subchondral bone. Secondly, we wanted to evaluate whether the intraarticular location would influence changes in the subchondral bone and degenerative changes as evaluated from macroscopic appearance and proteoglycan content of synovial fluid (Messner et al. 1993a).     

Changes in articular cartilage and subchondral bone histomorphometry in osteoarthritic knee joints in humans

In this study, we have examined the correlation between the histological and histochemical changes of articular cartilage and bone parameters of the underlying subchondral bone. The aim was to elucidate patterns of bone parameter changes within different depths of subchondral bone in the joints with macroscopically normal cartilage and in joints with osteoarthritis (OA). Ten tibial plateaus were taken from patients during total knee replacement surgery due to severe OA. They were compared with 10 sets of tibial condyles obtained from autopsy subjects with no history of bone or joint disease. The cylindrical cartilage-bone samples were taken out from the anterior, posterior, external, and internal areas of the condyles for cartilage assessment (Mankin score) and subchondral bone histomorphometry. Four histomorphometric parameters were measured: bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.S). Our study showed that subchondral bone from the OA group had significantly higher bone volume (54.1 +/- 10.6%) than control group (37.8 +/- 8.1%) (P < 0.01). In addition, trabecular parameters from the OA subchondral bone showed a smaller number of sparsely distributed and thicker trabecules than in control group (P < 0.05). Medial and lateral condyle from the control group did not differ significantly, while medial condyle from OA group showed a high increase of bone volume (62.8 +/- 13.3) and consecutively different trabecular parameters when compared with the lateral condyle from the same group. Also, it was shown that there are regional differences (anterior, posterior, internal, and external) in bone parameters between both condyles within both, control and OA groups. Comparison of bone parameters from three different stage of articular cartilage degeneration (Mankin score) showed that higher degree of cartilage degeneration is followed by significant changes in subchondral bone architecture. Furthermore, we have found that progression of cartilage degeneration leads to changes in bone parameters which affected deeper levels of subchondral bone. According to these results, it can be suggested that changes in histomorphometric parameters of subchondral bone are secondary to cartilage damage and proceed deeper into subchondral bone with increasing cartilage degeneration.