Changes in cartilage proteoglycans associated with calcification

Summary The purposes of these experiments were to study the biosynthetic and postbiosynthetic relationships between proteoglycans in noncalcified growth cartilage and calcified cartilage in metaphysis from the costochondral junctions of immature rabbits. Based onin vivo experiments in which 35 S-sodium sulfate was injected into rabbits, it is shown that proteoglycans from the hypertrophic region becomes part of the calcified cartilage matrix which is to be incorporated into the metaphysis. The proteoglycan aggregates in the growth apparatus undergo partial disaggregation and degradation. There is approximately a 25% decrease in aggregation from regions of the rib distal to the metaphyseal-growth plate junction (69%) to the region proximal to it (50%). In contrast, in their final state in calcified cartilage, the proteoglycans are more completely disaggregated and the proteoglycans subunits are smaller, as adjudged from gel chromatography. Control experiments indicate that although some artifactual disaggregation is produced by the extraction process, it is not of the same magnitude as that seen in the actual isolation experiments nor are the subunits reduced in size.     

降钙素在体内体外实验中对兔关节炎关节软骨退变及软骨下骨骨代谢的影响

[目的]研究降钙素(calcitonin, CT)对骨性关节炎关节软骨退变和软骨下骨骨代谢的影响.[方法]30只3个月龄雌性日本大耳白兔随机分为三组,其中两组行右膝关节前交叉韧带切断术(anterior cruciate ligament transaction,ACLT),分为ACLT+CT组和ACLT+NS组,第3组为Sham组.ACLT+CT给予每日1次皮下注射降钙素5 IU/(kg·d),持续8周,ACLT+NS组给予同样剂量生理盐水.术后8周后处死所有动物.取股骨髁制成切片行MMP-13和Ⅱ型胶原免疫组化染色.取胫骨近端制成硬组织切片行骨形态计量学检测.体外实验中,取兔膝关节软骨,经消化、培养,将第3代软骨细胞分三组:向IL-1β组加入人重组IL-1β(10 ng/ml). IL-1β+CT组加入人重组IL-1β (10 ng/ml)2 d后,再向培养液中加入CT(50 ng/ml).正常组不加任何诱导剂和干扰剂培养.然后行MMP-13、Ⅱ型胶原免疫组化检测和Realtime RT-PCR法检测.[结果]Sham组和ACLT+CT组软骨下骨骨小梁相对体积和厚度等均显著高于ACLT+NS组.Sham组和ACLT+CT组的Ⅱ型胶原的光密度值均显著高于ACLT+NS组,而MMP-13的光密度值显著低于ACLT+NS组(P<0.05).正常组和IL-1β+CT组的Ⅱ型胶原光密度值均显著高于IL-1β组而MMP-13的光密度值都显著低于IL-1β组(P<0.05).在正常组和IL-1β+CT组中Ⅱ型胶原的mRNA含量均显著高于IL-1β组而MMP-13的mRNA含量均显著低于IL-1β组(P<0.05).[结论]降钙素5 IU/(kg·d)皮下注射能够增加ACLT兔膝关节软骨Ⅱ型胶原的分泌和抑制MMP-13的表达,并可能通过调节软骨下骨的骨代谢和微结构来保护关节软骨; CT(50 ng/ml)能增加体外培养的含有IL-1β(10 ng/ml)的软骨细胞中Ⅱ型胶原的含量和抑制MMP-13分泌.     

Transient expression of the diseased phenotype of osteoarthritic chondrocytes in engineered cartilage

Due to the degradation of osteoarthritic (OA) cartilage in post‐traumatic OA (PTOA), these tissues are challenging to study and manipulate in vitro. In this study, chondrocytes isolated from either PTOA (meniscal‐release (MR) model) or normal (contralateral limb) cartilage of canine knee joints were used to form micropellets to assess the maintenance of the OA chondrocyte phenotype in vitro. Media samples from the micropellet cultures were used to measure matrix metalloproteinase (MMP), chemokine, and cytokine concentrations. Significant differences in matrix synthesis were observed as a function of disease with OA chondrocytes generally synthesizing more extracellular matrix with increasing time in culture. No donor dependent differences were detected. Luminex multiplex analysis of pellet culture media showed disease and time‐dependent differences in interleukin (IL)‐8, keratinocyte chemoattractant (KC)‐like protein, MMP‐1, MMP‐2, and MMP‐3, which are differentially expressed in OA. This memory of their diseased phenotype persists for the first 2 weeks of culture. These results demonstrate the potential to use chondrocytes from an animal model of OA to study phenotype alterations during the progression and treatment of OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:829–836, 2017.

     

Increased expression and activation of cathepsin K in human osteoarthritic cartilage and synovial tissues

Few studies have analyzed Cathepsin K (CatK) expression in human osteoarthritic tissues. We investigated CatK expression and activation in human articular cartilage using clinical specimens. Human osteoarthritic cartilage was obtained during surgery of total hip arthroplasty (n = 10), and control cartilage was from that of femoral head replacement for femoral neck fracture (n = 10). CatB, CatK, CatL, CatS, and Cystatin C (CysC) expressions were evaluated immunohistochemically and by real‐time PCR. Intracellular CatK protein was quantified by ELISA. Intracellular CatK activity was also investigated. Osteoarthritis (OA) chondrocytes were strongly stained with CatK, particularly in the superficial layer and more damaged areas. CatB, CatL, CatS, and CysC were weakly stained. CatK mRNA expression was significantly higher in OA group compared to that in control group (p = 0.043), whereas those of CatB, CatL, CatS, and CysC did not differ significantly. Mean CatK concentration (4.83 pmol/g protein) in OA chondrocytes was higher than that (3.91 pmol/g protein) in control chondrocytes (p = 0.001). CatK was enzymatically more activated in OA chondrocytes as compared with control chondrocytes. This study, for the first time, revealed increased CatK expression and activation in human OA cartilage, suggesting possible crucial roles for it in the pathogenesis of osteoarthritic change in articular cartilage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:127–134, 2016.     

Alterations in structural macromolecules and chondrocyte deformations in lapine retropatellar cartilage 9 weeks after anterior cruciate ligament transection

The structural integrity and mechanical environment of the articular cartilage matrix directly affect chondrocyte deformations. Rabbit models of early osteoarthritis at 9 weeks following anterior cruciate ligament transection (ACLT) have been shown to alter the deformation behavior of superficial zone chondrocytes in mechanically loaded articular cartilage. However, it is not fully understood whether these changes in cell mechanics are caused by changes in structural macromolecules in the extracellular matrix. Therefore, the purpose of this study was to characterize the proteoglycan content, collagen content, and collagen orientation at 9 weeks post ACLT using microscopic techniques, and relate these changes to the altered cell mechanics observed upon mechanical loading of cartilage. At 9 weeks following ACLT, collagen orientation was significantly (p < 0.05) altered and proteoglycan content was significantly (p < 0.05) reduced in the superficial zone cartilage matrix. These structural changes either in the extracellular or pericellular matrix (ECM and PCM) were also correlated significantly (p < 0.05) with chondrocyte width and height changes, thereby suggesting that chondrocyte deformation response to mechanical compression in early OA changes primarily because of alterations in matrix structure. However, compared to the normal group, proteoglycan content in the PCM from the ACLT group decreased less than that in the surrounding ECM. Therefore, PCM could play a key role to protect excessive chondrocyte deformations in the ACLT group. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:342–350, 2018.     

膝关节原发性骨关节炎软骨和软骨下骨病理改变的定量研究

目的观察膝关节原发性骨关节炎(osteoarthritis,OA)胫骨平台软骨和软骨下骨病理改变特点,对比内、外侧平台软骨和软骨下骨结构参数,探讨钙化层和软骨下骨在OA发病机制中的作用。方法取2009年10月-2011年5月行人工全膝关节置换术治疗的30例30膝原发性OA患者自愿捐赠的新鲜胫骨平台标本进行实验。其中男11例,女19例;年龄55～78岁,平均65.1岁。病程10～25年,平均16.6年;患膝内翻畸形1～23°,平均9.3°。大体观察胫骨平台后在内、外侧中央负重区取材,常规制备脱钙石蜡切片,行HE和番红O/固绿染色,观察关节软骨退变特点,参照Mankin评分标准评分并分期;观察钙化层及软骨下骨病理改变。应用Image Pro Plus 6.0图像分析软件测量软骨和软骨下骨结构参数,包括软骨全层(total articular cartilage,TAC)厚度、钙化层(articular calcified cartilage,ACC)厚度、ACC/TAC比值、软骨下骨板(subchondral bone plate,SCP)厚度以及骨小梁体积分数(trabecular bone volume,BV/TV)。结果大体观察内侧平台软骨退变较外侧严重,内侧平台软骨Mankin评分为(12.4±1.1)分,显著高于外侧平台的(8.3±1.6)分(t=12.173,P=0.000)。根据Mankin评分结果在60个标本中,14个为OA早期,可见软骨浅表层裂隙、潮线复制和软骨下骨增厚;19个为OA中期,可见软骨深层裂隙、多发软骨下骨吸收陷窝和明显增厚的软骨下骨;27个为OA晚期,可见软骨全层缺失、软骨内化骨和"象牙化"软骨下骨。软骨和软骨下骨结构参数测定示:内侧平台TAC厚度显著低于外侧平台,ACC/TAC比值、BV/TV及SCP厚度显著高于外侧平台,差异均有统计学意义(P<0.05)。内、外侧平台ACC厚度比较,差异无统计学意义(P>0.05)。结论钙化层和软骨下骨可能在OA发生与进展中发挥了重要作用。     

In situ measurement of transport between subchondral bone and articular cartilage

Subchondral bone and articular cartilage play complementary roles in load bearing of the joints. Although the biomechanical coupling between subchondral bone and articular cartilage is well established, it remains unclear whether direct biochemical communication exists between them. Previously, the calcified cartilage between these two compartments was generally believed to be impermeable to transport of solutes and gases. However, recent studies found that small molecules could penetrate into the calcified cartilage from the subchondral bone. To quantify the real‐time solute transport across the calcified cartilage, we developed a novel imaging method based on fluorescence loss induced by photobleaching (FLIP). Diffusivity of sodium fluorescein (376 Da) was quantified to be 0.07 ± 0.03 and 0.26 ± 0.22 µm²/s between subchondral bone and calcified cartilage and within the calcified cartilage in the murine distal femur, respectively. Electron microscopy revealed that calcified cartilage matrix contained nonmineralized regions (～22% volume fraction) that are either large patches (53 ± 18 nm) among the mineral deposits or numerous small regions (4.5 ± 0.8 nm) within the mineral deposits, which may serve as transport pathways. These results suggest that there exists a possible direct signaling between subchondral bone and articular cartilage, and they form a functional unit with both mechanical and biochemical interactions, which may play a role in the maintenance and degeneration of the joint. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1347–1352, 2009     

Detectable reporter gene expression following transduction of adenovirus and adeno‐associated virus serotype 2 vectors within full‐thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg‐Gly‐Asp (RGD)‐modified Ad, adeno‐associated viral serotype 2 (AAV2), and self‐complementary AAV2 (scAAV2) vectors within full‐thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real‐time RT‐PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p ≤ 0.026). Ad and Ad‐RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full‐thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:149–155, 2010     

Formation of cartilage repair tissue in articular cartilage defects pretreated with microfracture and covered with cell‐free polymer‐based implants

The aim of our study was to evaluate the mid‐term outcome of a cell‐free polymer‐based cartilage repair approach in a sheep cartilage defect model in comparison to microfracture treatment. Cell‐free, freeze‐dried implants (chondrotissue®) made of a poly‐glycolic acid (PGA) scaffold and hyaluronan were immersed in autologous serum and used for covering microfractured full‐thickness articular cartilage defects of the sheep (n = 4). Defects treated with microfracture only served as controls (n = 4). Six months after implantation, cartilage implants and controls were analyzed by immunohistochemical staining of type II collagen, histological staining of proteoglycans, and histological scoring. Histological analysis showed the formation of a cartilaginous repair tissue rich in proteoglycans. Histological scoring documented significant improvement of repair tissue formation when the defects were covered with the cell‐free implant, compared to controls treated with microfracture. Immunohistochemistry showed that the cell‐free implant induced cartilaginous repair tissue and type II collagen. Controls treated with microfracture showed marginal formation of a mixed‐type repair tissue consisting of cartilaginous tissue and fibro‐cartilage. Covering of microfractured defects with the cell‐free polymer‐based cartilage implant is suggested to be a promising treatment option for cartilage defects and improves the regeneration of articular cartilage. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1353–1360, 2009     

Electromechanical probe and automated indentation maps are sensitive techniques in assessing early degenerated human articular cartilage

Recent advances in the development of new drugs to halt or even reverse the progression of Osteoarthritis at an early‐stage requires new tools to detect early degeneration of articular cartilage. We investigated the ability of an electromechanical probe and an automated indentation technique to characterize entire human articular surfaces for rapid non‐destructive discrimination between early degenerated and healthy articular cartilage. Human cadaveric asymptomatic articular surfaces (four pairs of distal femurs and four pairs of tibial plateaus) were used. They were assessed ex vivo: macroscopically, electromechanically, (maps of the electromechanical quantitative parameter, QP, reflecting streaming potentials), mechanically (maps of the instantaneous modulus, IM), and through cartilage thickness. Osteochondral cores were also harvested from healthy and degenerated regions for histological assessment, biochemical analyses, and unconfined compression tests. The macroscopic visual assessment delimited three distinct regions on each articular surface: Region I was macroscopically degenerated, region II was macroscopically normal but adjacent to regions I and III was the remaining normal articular surface. Thus, each extracted core was assigned to one of the three regions. A mixed effect model revealed that only the QP (p < 0.0001) and IM (p < 0.0001) were able to statistically discriminate the three regions. Effect size was higher for QP and IM than other assessments, indicating greater sensitivity to distinguish early degeneration of cartilage. When considering the mapping feature of the QP and IM techniques, it also revealed bilateral symmetry in a moderately similar distribution pattern between bilateral joints. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:858–867, 2017.

     

Local tissue properties of human osteoarthritic cartilage correlate with magnetic resonance T1rho relaxation times

The objective of this study is to examine the local relationship between T_1ρ relaxation times and the mechanical behavior of human osteoarthritic articular cartilage using high‐resolution magnetic resonance imaging (MRI) and local in situ microindentation. Seven human tibial plateaus were obtained from patients who underwent total knee arthroplasty due to severe osteoarthritis (OA). Three to six sites were selected from each sample for visual classification using the ICRS Outerbridge scale (a total of 36 sites). Samples were imaged by MR, and the local distribution of T_1ρ relaxation times were obtained at these selected sites. The elastic and viscoelastic characteristics of the tissue were quantified nondestructively using dynamic microindentation to measure peak dynamic modulus, energy dissipation, and phase angle. Measured Outerbridge scores, MR T_1ρ relaxation times, and mechanical properties were highly heterogeneous across each cartilage surface. Site‐specific measures of T_1ρ relaxation times correlated significantly with the phase angle (p < 0.001; R = 0.908), a viscoelastic mechanical behavior of the cartilage. The novel combination of high‐resolution MR imaging and microindentation allows the investigation of the local relationship between quantitative MRI and biomechanical properties in highly heterogeneous OA cartilage. These findings suggest that MRI T_1ρ can provide a functional assessment of articular cartilage. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1312–1319, 2011     

Effects of ACL graft placement on in vivo knee function and cartilage thickness distributions

Injuries to the anterior cruciate ligament (ACL) frequently lead to early‐onset osteoarthritis. Despite advancement in surgical techniques, ACL reconstruction has a limited ability to prevent these degenerative changes. While previous studies have investigated knee function after ACL reconstruction, in vivo investigations of the effects of graft placement on in vivo joint function and cartilage health are limited. This review presents a series of studies that used novel imaging and 3D modeling techniques to determine the in vivo placement of the ACL graft on the femur using two different ACL reconstruction techniques. These techniques resulted in two distinct graft placement groups: one where the ACL was placed anatomically near the center of the native ACL footprint and another where the graft was placed anteroproximally on the femur, centered outside the ACL footprint. We quantified the effects of graft placement on graft deformation during in vivo loading and how these variables affected knee motion. Finally, we quantified whether femoral placement of the graft affected cartilage thickness. Our results demonstrate that achieving anatomic graft placement on the femur is critical to restoring native ACL function and normal knee kinematics. Knees with grafts that more closely restored normal ACL function, and thus knee motion, experienced less focal cartilage thinning than did those that experienced abnormal knee motion. These results suggest that achieving anatomic graft placement is a critical factor in restoring normal knee motion and potentially slowing the development of degenerative changes after ACL reconstruction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1160–1170, 2017.

     

Hepatocyte growth factor in human osteoarthritic cartilage

OBJECTIVE: Hepatocyte growth factor/scatter factor is a potent mitogen, morphogen and motogen for a variety of mainly epithelial cells. Hepatocyte growth factor is synthesized by mesenchymal cells and can be found in various tissues. The objective of this study was to investigate the expression and distribution patterns of this pleiotropic growth factor and its receptor, the product of the proto-oncogene c-met in normal and osteoarthritic human knee cartilage. METHODS: Five normal and 14 osteoarthritic human cartilage samples graded histomorphologically by Mankin Score, were studied by radioactive in-situ hybridization and immunohistochemistry for the expression of Hepatocyte growth factor and the c-met receptor. RESULTS: Hepatocyte growth factor could be found by immunohistochemistry in the territorial matrix surrounding the chondrocytes of calcified cartilage and within the deep zone of normal cartilage. Chondrocytes of these cartilage zones showed also positive c-met receptor-staining. Moreover, a small number of chondrocytes in the superficial and intermediate zone showed c-met staining. In accordance with the increased hepatocyte growth factor staining of osteoarthritic cartilage, an enhanced expression of hepatocyte growth factor-RNA by chondrocytes of the deep zone as well as the deeper mid zone was observed. Contrary to normal cartilage, c-met was identified immunohistochemically in osteoarthritic chondrocytes of all cartilage zones. CONCLUSION: These results indicate that hepatocyte growth factor seems to be acting in an autocrine/paracrine manner in normal and osteoarthritic cartilage. The ubiquitous presence of the HGF/HGF-receptor complex in osteoarthritic chondrocytes suggests that hepatocyte growth factor may contribute to the altered metabolism in osteoarthritic cartilage.     

Correlation of histopathology and sulfated proteoglycans in human osteoarthritic hip cartilage

The histopathologic characteristics, in vitro proteoglycan and glycosaminoglycan biosynthesis, and proteoglycan content of osteoarthritic (OA) cartilage tissue types from human femoral heads obtained at the time of total joint replacement were compared. Articular cartilage from fibrillated or discolored cartilage surfaces demonstrated overlapping histopathologic patterns, while cartilage from osteophytic areas was distinct. ³⁵SO₄ from each of these three tissue types was found in two peaks of radioactivity on a Sepharose CL-2B column. The average partition coefficient (K_av) of the first peak (peak I) was 0.07, while that of the second (peak II) was 0.63. Proteoglycan monomer predominated in discolored, fibrillated, and osteophytic OA cartilage in peak I. The hydrodynamic size on Sepharose CL-2B of the synthetic proteoglycan monomer was the same for discolored, fibrillated, and osteophytic samples (K_av, 0.25–0.28). Discolored and fibrillated tissues showed a similar percentage of proteoglycan monomer in peak II, whereas osteophyte was reduced in proteoglycan monomer content in peak II. In addition, the endogenous proteoglycans extracted from each cartilage area were generally of a smaller hydrodynamic size than the newly synthesized peak I or proteoglycan monomer. Glycosaminoglycans were predominantly chondroitin 6-sulfate. These results indicated that OA discolored and fibrillated cartilage tissue types from defined topographical areas of human femoral heads possessed neither unique histopathologic nor synthetic or endogenous proteoglycan characteristics. Osteophytic cartilage appeared more histopathologically distinct than either discolored or fibrillated OA cartilage, but synthesized proteoglycan monomer with similar hydrodynamic size to the other cartilage tissue types.