The biomechanical role of the chondrocyte pericellular matrix in articular cartilage

The pericellular matrix (PCM) is a narrow tissue region that surrounds chondrocytes in articular cartilage. Previous parametric studies of cell-matrix interactions suggest that the mechanical properties of the PCM relative to those of the extracellular matrix (ECM) can significantly affect the micromechanical environment of the chondrocyte. The goal of this study was to use recently quantified mechanical properties of the PCM in a biphasic finite element model of the cell-PCM-ECM structure to determine the potential influence of the PCM on the mechanical environment of the chondrocyte under normal and osteoarthritic conditions. Our findings suggest that the mismatch between the Young's moduli of PCM and ECM amplifies chondrocyte compressive strains and exhibits a significant stress shielding effect in a zone-dependent manner. Furthermore, the lower permeability of PCM relative to the ECM inhibits fluid flux near the cell by a factor of 30, and thus may have a significant effect on convective transport to and from the chondrocyte. Osteoarthritic changes in the PCM and ECM properties significantly altered the mechanical environment of the chondrocyte, leading to approximately 66% higher compressive strains and higher fluid flux near the cell. These findings provide further support for a potential biomechanical role for the chondrocyte PCM, and suggest that changes in the properties of the PCM with osteoarthritis may alter the stress-strain and fluid flow environment of the chondrocytes.     

软骨细胞力学环境的跨尺度计算

目的通过跨尺度计算,比较表层和深层软骨细胞的周边力学环境。方法建立软骨细胞两相力学模型,将软骨两相力学模型里的结果映射到细胞模型对应边界上作为边界条件。计算细胞模型得到软骨细胞的周边力学环境并进行分析。结果深层软骨细胞及周边应力比表层细胞的小一半,但都远小于细胞外的应力。软骨细胞周围基质(pericellular matrix,PCM)承担了细胞外的高应力,显著降低了细胞内的应力。两处细胞周围的间隙流动方向完全相反。结论软骨承载能力使深层软骨细胞附近应力显著降低,保护了深层软骨细胞及软骨下骨。PCM承担了细胞外围高应力,保证了软骨细胞生存工作所需的低应力环境。两处细胞周围相反的间隙流动支持了由表层关节液渗透及软骨下骨营养泵入构成的软骨双向营养供给学说。     

Cartilage tissue engineering for laryngotracheal reconstruction: comparison of chondrocytes from three anatomic locations in the rabbit

Tissue engineering may provide a technique to generate cartilage grafts for laryngotracheal reconstruction in children. The present study used a rabbit model to characterize cartilage generated by a candidate tissue engineering approach to determine, under baseline conditions, which chondrocytes in the rabbit produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. We characterized tissue-engineered cartilage generated in perfused bioreactor chambers from three sources of rabbit chondrocytes: articular, auricular, and nasal cartilage. Biomechanical testing and histological, immunohistochemical, and biochemical assays were performed to determine equilibrium unconfined compression (Young's) modulus, and biochemical composition and structure. We found that cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness necessary for completion of the biomechanical testing, but five of six auricular samples completed the biomechanical testing (moduli of 210 +/- 93 kPa in two samples at 3 weeks and 100 +/- 65 kPa in three samples at 6 weeks). Auricular samples showed more consistent staining for proteoglycans and collagen II and had significantly higher glycosaminoglycan (GAG) content and concentration and higher collagen content than articular or nasal samples. In addition, the delayed gadolinium enhanced MRI of cartilage (dGEMRIC) method revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples. The results indicate that, for the candidate tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. The results also suggest that this and similar tissue engineering approaches must be optimized for each potential source of chondrocytes.     

关节软骨细胞和细胞周基质的压缩特性及其对软骨细胞代谢的影响

软骨细胞和细胞周基质(pericellular matrix, PCM)的力学特性对于关节软针的生理功能具有重要的意义。软骨细胞在压缩应力下表现出黏弹性固体材料特性，其压缩特性具有各向异性和多相性。PCM对软骨细胞具有明显的力学保护作用。压缩应力影响软骨细胞的代谢活动。软骨细胞和PCM的力学特性有许多仍未澄清，尚需进一步的研究。     

Biomechanical analysis of a chondrocyte-based repair model of articular cartilage.

The objective of this study was to evaluate the biomechanical properties of newly formed cartilaginous tissue synthesized from isolated chondrocytes. Cartilage from articular joints of lambs was either digested in collagenase to isolated chondrocytes or cut into discs that were devitalized by multiple freeze-thaw cycles. Isolated cells were incubated in suspension culture in the presence of devitalized cartilage matrix for 3 weeks. Multiple chondrocyte/matrix constructs were assembled with fibrin glue and implanted subcutaneously in nude mice for up to 6 weeks. Testing methods were devised to quantify integration of cartilage pieces and mechanical properties of constructs. These studies showed monotonic increase with time in tensile strength, fracture strain, fracture energy, and tensile modulus to values 5-10% of normal articular cartilage by 6 weeks in vivo. Histological analysis indicated that chondrocytes grown on dead cartilage matrix produced new matrix that integrated individual cartilage pieces with mechanically functional tissue.     

Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilage

Numerous studies have reported generation of cartilage-like tissue from chondrocytes and stem cells, using pellet cultures, bioreactors and various biomaterials, especially hydrogels. However, one of the primary unsolved challenges in the field has been the inability to produce tissue that mimics the highly organized zonal architecture of articular cartilage; specifically its spatially varying mechanical properties and extra-cellular matrix (ECM) composition. Here we show that different combinations of synthetic and natural biopolymers create unique niches that can "direct" a single marrow stem cell (MSC) population to differentiate into the superficial, transitional, or deep zones of articular cartilage. Specifically, incorporating chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) into PEG hydrogels (PEG:CS:MMP-pep) induced high levels of collagen II and low levels of proteoglycan expression resulting in a low compressive modulus, similar to the superficial zone. PEG:CS hydrogels produced intermediate-levels of both collagen II and proteoglycans, like the transitional zone, while PEG:hyaluronic acid (HA) hydrogels induced high proteoglycan and low collagen II levels leading to high compressive modulus, similar to the deep zone. Additionally, the compressive moduli of these zone-specific matrices following cartilage generation showed similar trend as the corresponding zones of articular cartilage, with PEG:CS:MMP-pep having the lowest compressive modulus, followed by PEG:CS while PEG:HA had the highest modulus. These results underscore the potential for composite scaffold structures incorporating these biomaterial compositions such that a single stem-progenitor cell population can give rise to zonally-organized, functional articular cartilage-like tissue.     

Glycosaminoglycans in the pericellular matrix of chondrons and chondrocytes

This is the first study to quantitate and profile the glycosaminoglycan (GAG) composition of the pericellular matrix (PCM) of chondrons and chondrocytes using the highly sensitive technique; fluorophore-assisted carbohydrate electrophoresis (FACE). Bovine articular chondrocytes and chondrons were isolated enzymatically. High cell yield and viability were obtained for both preparations. Chondrons had strong immunofluorescent labeling for keratan sulphate and chondroitin-6 sulphate but no labeling for hyaluronan. We compared the immunofluorescent data with FACE. The quantities of total keratan sulphate were determined to be 0.013 +/- 0.002 pg cell(-1) and 0.032 +/- 0.003 pg cell(-1) in the chondrocyte and chondron preparations, respectively. Four internal keratan sulphate sugars were detected (gal beta 1,4glcNAc6S, gal6S beta 1,4glcNAc6S, glcNAc beta 1,3gal and glcNAc6S beta 1,3gal) for both preparations but they were present at significantly higher concentrations in chondron preparations (P < 0.01). Total chondroitin sulphate (CS) was determined to be 0.054 +/- 0.004 pg cell(-1) and 0.077 +/- 0.005 pg cell(-1) for chondrocyte and chondron preparations, respectively. Unsulphated CS disaccharide levels were similar but chondrons had significantly more chondroitin-4 sulphated disaccharides and chondroitin-6 sulphated disaccharides (P < 0.05). Hyaluronan acid was present at low concentrations (0.010 +/- 0.001 pg cell(-1)) in both chondrocytes and chondrons. In this study, enzyme digestion coupled with FACE separation revealed new information about the differences in GAGs from isolated chondrocyte and chondron preparations. Further investigation of the differences in GAGs from chondrocytes and chondrons from different zones of articular cartilage may be useful for tissue engineering approaches.     

Engineering articular cartilage with spatially-varying matrix composition and mechanical properties from a single stem cell population using a multi-layered hydrogel

Despite significant advances in stem cell differentiation and tissue engineering, directing progenitor cells into three-dimensionally (3D) organized, native-like complex structures with spatially-varying mechanical properties and extra-cellular matrix (ECM) composition has not yet been achieved. The key innovations needed to achieve this would involve methods for directing a single stem cell population into multiple, spatially distinct phenotypes or lineages within a 3D scaffold structure. We have previously shown that specific combinations of natural and synthetic biomaterials can direct marrow-derived stem cells (MSC) into varying phenotypes of chondrocytes that resemble cells from the superficial, transitional, and deep zones of articular cartilage. In this current study, we demonstrate that layer-by-layer organization of these specific biomaterial compositions creates 3D niches that allow a single MSC population to differentiate into zone-specific chondrocytes and organize into a complex tissue structure. Our results indicate that a three-layer polyethylene glycol (PEG)-based hydrogel with chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) incorporated into the top layer (superficial zone, PEG:CS:MMP-pep), CS incorporated into the middle layer (transitional zone, PEG:CS) and hyaluronic acid incorporated in the bottom layer (deep zone, PEG:HA), creates native-like articular cartilage with spatially-varying mechanical and biochemical properties. Specifically, collagen II levels decreased gradually from the superficial to the deep zone, while collagen X and proteoglycan levels increased, leading to an increasing gradient of compressive modulus from the superficial to the deep zone. We conclude that spatially-varying biomaterial compositions within single 3D scaffolds can stimulate efficient regeneration of multi-layered complex tissues from a single stem cell population.     

Relationship among biomechanical, biochemical, and cellular changes associated with osteoarthritis

Articular cartilage that lines the surface of long bones is a multilayered material. The superficial layer consists of collagen fibrils and chondrocytes that run parallel to the joint surface. In the deeper layers, the collagen fibrils are more randomly arranged and support vertical units termed chondrons containing rows of chondrocytes. In the deepest layers, the collagen fibrils run almost vertically and ultimately insert into the underlying subchondral bone. Osteoarthritis (OA) is a disease that affects articular cartilage and is characterized by enzymatic and mechanical breakdown of the extracellular matrix, leading to cartilage degeneration, exposure of subchondral bone, pain, and limited joint motion. Changes in mechanical properties of articular cartilage associated with OA include decreases in modulus and ultimate tensile strength. These changes parallel the changes observed after enzymatic degradation of either collagen or proteoglycans in cartilage. Results of recent viscoelastic studies on articular cartilage suggest that the elastic modulus of collagen and fibril lengths decrease in OA and are associated with a loss of the superficial zone and a decreased ability of articular cartilage to store elastic energy during locomotion. It is suggested that osteoarthritic changes to cartilage involve enzymatic degradation of matrix components and fibril fragmentation that is promoted by subsequent mechanical loading.     

不同病理学等级关节炎软骨的三相力学特性

目的基于超声膨胀观测技术与三相理论提取关节软骨的轴向弹性模量,探索不同等级关节炎软骨的三相力学特性。方法对不同阶段的兔子关节炎软骨样本进行病理学分级,基于高频瞬态超声测量技术获取软骨因自由膨胀引起的组织应变量,结合组织的固定电荷密度和水体积分数,运用三相模型估计软骨的轴向弹性模量,并进行相关性分析。结果正常软骨与关节炎软骨的轴向弹性模量存在着明显差异(P<0.05),不同等级软骨样本的轴向弹性模量存在明显差异(P<0.05)。正常软骨组织的平均弹性模量为(15.87±6.30)MPa,随着关节炎的发生与关节炎程度的加重,软骨的弹性模量逐渐减小,1、2、3级软骨样本的轴向弹性模量分别为(11.33±5.21)、(9.15±5.68)、(6.05±4.99)MPa。结论不同病理学关节炎等级软骨样本的三相力学特性有明显差异,该研究为应用软骨组织的力学属性定量评判关节炎的严重程度提供了新的思路。     

Composition of the pericellular matrix modulates the deformation behaviour of chondrocytes in articular cartilage under static loading

The aim was to assess the role of the composition changes in the pericellular matrix (PCM) for the chondrocyte deformation. For that, a three-dimensional finite element model with depth-dependent collagen density, fluid fraction, fixed charge density and collagen architecture, including parallel planes representing the split-lines, was created to model the extracellular matrix (ECM). The PCM was constructed similarly as the ECM, but the collagen fibrils were oriented parallel to the chondrocyte surfaces. The chondrocytes were modelled as poroelastic with swelling properties. Deformation behaviour of the cells was studied under 15% static compression. Due to the depth-dependent structure and composition of cartilage, axial cell strains were highly depth-dependent. An increase in the collagen content and fluid fraction in the PCMs increased the lateral cell strains, while an increase in the fixed charge density induced an inverse behaviour. Axial cell strains were only slightly affected by the changes in PCM composition. We conclude that the PCM composition plays a significant role in the deformation behaviour of chondrocytes, possibly modulating cartilage development, adaptation and degeneration. The development of cartilage repair materials could benefit from this information.     

Mechanical shear properties of cell-polymer cartilage constructs.

Cartilaginous constructs were created by using bovine chondrocytes on synthetic, biodegradable scaffolds made of fibrous polyglycolic acid (PGA). The constructs have previously been shown to resemble natural articular cartilage biochemically and histologically. The mechanical properties of articular cartilage mainly depend on the swollen extracellular matrix (ECM), which is a gel consisting of collagen fibers and proteoglycans in a fluid phase of water and electrolytes. The biomechanical properties of the constructs and the build-up of the ECM were studied using dynamic, nondestructive measurements in shear. A small, harmonic strain, lambda < or = 5 x 10(-4), was applied to the sample, and the resulting stress was recorded and used for calculating the complex shear modulus G*. The applied strain was much smaller than that used in confined compression. The shear modulus G* correlated well with both the collagen and glycosaminoglycan content of the constructs but did not reach the same level as in natural cartilage. Collagen is the dominant component contributing to the shear strength of cartilage, and G* was shown to depend approximately quadratically on the collagen content of the constructs. The difference in G* between the constructs and natural cartilage was shown to depend on both the biochemical composition and the microstructure of the constructs. () ()     

Lack of oxygen in articular cartilage: consequences for chondrocyte biology

Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment. While huge progress has been performed on deciphering intracellular hypoxia signalling the last few years, nothing was known about the particular case of the chondrocyte biology in response to hypoxia. Recent findings in this growing field showed crucial influence of the hypoxia signalling on chondrocytes physiology and raised new potential targets to repair cartilage and maintain tissue integrity. This review will thus focus on describing hypoxia‐mediated chondrocyte function in the native articular cartilage.     

大骨节病关节软骨胶原表型表达和软骨细胞异常分化的研究

目的探讨大骨节病关节软骨胶原表型表达的变化特点和软骨细胞异常分化在发病中的意义。方法用单克隆免疫组化法测定５例大骨节病关节软骨Ⅰ、Ⅱ、Ⅲ、Ⅵ、Ⅹ型胶原表型的表达。结果（１）关节软骨表层的Ⅱ型胶原表型表达减少；（２）Ⅰ、Ⅲ和Ⅵ型胶原表型表达见于关节软骨全层，而Ⅹ型胶原仅限于关节软骨钙化层和深层软骨细胞团周围；（３）软骨细胞团有Ⅰ、Ⅱ、Ⅲ、Ⅵ型胶原表型表达，而软骨细胞坏死区内无任何胶原表型表达。结论大骨节病关节软骨胶原表型表达类似于原发性骨关节病的变化，但在关节软骨表层Ⅰ型胶原表型表达增强以及软骨坏死区内无任何胶原表型表达不同于原发性骨关节病。     

Measurement of the layered compressive properties of trypsintreated articular cartilage: An ultrasound investigation

An ultrasound-compression system has been developed for the study of the layered biomechanical properties of articular cartilage. Cartilage specimens harvested from the bovine patella groove, with and without trypsin digestion, were tested using this system. It was noted that a large ultrasound reflection can be detected in the interface of the trypsin digestion front. This ultrasound reflection signal was used to differentiate the deformations of different portions of the cartilage throughout its depth when a load was applied. The equilibrium compression moduli of the digested, undigested and entire portions of articular cartilage were measured. The modulus of the cartilage without any digestion was 660±230kPa. After 1h digestion with 1 mg ml⁻¹ trypsin solution, the thickness of the digested portion was 0.50±0.06 mm, and the modulus of the entire cartilage layer changed to 125±42 kPa. The moduli of the digested and undigested portions were 58±24 kPa and 470±31 kPa, respectively. Similar results were obtained for the cartilage with trypsin digestion for 2 h.     

Biomimetic Pericellular Microniche Inspired by Chondron enhances the Cartilage Matrix Accumulation

Cartilage repair is currently an inevitable and significant issue due to the prevalence of cartilage defects or osteoarthritis. However, it remains a big challenge worldwide because of the avascular and aneural nature of cartilage. In this paper, a novel biomimetic pericellular microniche (BPM), inspired by chondron composed of chondrocyte and its surrounding pericellular matrix (PCM), is proposed to offer a promising solution to achieve cartilage repair. BPM, with a diameter ≈64 µm, not only mimicked the structure of the chondron but also mimicked the key components of the PCM by artificially synthesizing brush-like molecules hyaluronic acid-grafted-(poly-2-acrylamide-2-methylpropanesulfonic acid sodium salt) (HA/PA) and hyaluronic acid-grafted-(poly-2-methacryloyloxyethyl phosphorylcholine) (HA/PM). Chondrocyte-laden BPM is constructed successfully through microfluidic and showed excellent injectability. The encapsulated chondrocyte showed high viability in BPM. F-Actin and H&E reflected that chondrocytes would grow out of BPM and adhere to the BPM surface through the interaction between CD44 and hyaluronic acid. RT-qPCR and immunofluorescence staining indicated that the gene expression of type II collagen and aggrecan are upregulated significantly in the BPM. Therefore, the construction of BPM provides a promising strategy for cartilage tissue engineering.     

基于纳米压痕法的关节软骨保湿测量技术

目的 在保持关节软骨表面湿润的情况下,进行纳米压痕实验,研究具有一定生物活性的关节软骨微结构的力学性能。方法 通过实验的方法,评估冷镶嵌法和保湿法对保持软骨在体力学性能的优劣,并应用保湿法得到不同保护液下软骨微结构的力学性能。结果 冷镶嵌法测得的软骨表层的弹性模量远大于保湿法测得的数值,蒸馏水保护下的软骨表层和深层的弹性模量明显高于壳多糖溶液和生理盐水保护下的软骨的弹性模量。结论 保湿法更有利于保持生物材料在体时的力学性能和保持生物材料的生物活性,通过保湿法也可以证明壳多糖溶液及生理盐水均可有效保持软骨的力学性能。     

In vitro chondrocyte behavior on porous biodegradable poly (e-caprolactone)/polyglycolic acid scaffolds for articular chondrocyte adhesion and proliferation

In this study, poly(e-caprolactone)/polyglycolic acid (PCL/PGA) scaffolds for repairing articular cartilage were fabricated via solid-state cryomilling along with compression molding and porogen leaching. Four distinct scaffolds were fabricated using this approach by four independent cryomilling times. These scaffolds were assessed for their suitability to promote articular cartilage regeneration with in vitro chondrocyte cell culture studies. The scaffolds were characterized for pore size, porosity, swelling ratio, compressive, and thermal properties. Cryomilling time proved to significantly affect the physical, mechanical, and morphological properties of the scaffolds. In vitro bovine chondrocyte culture was performed dynamically for 1, 7, 14, 28, and 35 days. Chondrocyte viability and adhesion were tested using MTT assay and scanning electron microscopy micrographs. Glycosaminoglycan (GAG) and DNA assays were performed to investigate the extracellular matrix (ECM) formation and cell proliferation, respectively. PCL/PGA scaffolds demonstrated high porosity for all scaffold types. Morphological analysis and poly(ethylene oxide) continuity demonstrated the existence of a co-continuous network of interconnected pores with pore sizes appropriate for tissue engineering and chondrocyte ingrowth. While mean pore size decreased, water uptake and compressive properties increased with increasing cryomilling times. Compressive modulus of 12, 30, and 60 min scaffolds matched the compressive modulus of human articular cartilage. Viable cells increased besides increase in cell proliferation and ECM formation with progress in culture period. Chondrocytes exhibited spherical morphology on all scaffold types. The pore size of the scaffold affected chondrocyte adhesion, proliferation, and GAG secretion. The results indicated that the 12 min scaffolds delivered promising results for applications in articular cartilage repair.     

Cell-based bonding of articular cartilage: An extended study

This study evaluated the biomechanical characteristics of newly formed cartilaginous tissue synthesized from isolated chondrocytes and seeded onto devitalized cartilage in an extended study in vivo. Cartilage from porcine articular joints was cut into regular discs and devitalized by multiple freeze-thaw cycles. Articular chondrocytes were enzymatically isolated and incubated in suspension culture in the presence of devitalized cartilage discs for 21 days. This procedure allowed the isolated chondrocytes to adhere to the devitalized matrix surfaces. Chondrocyte-matrix constructs were assembled with fibrin glue and implanted in dorsal subcutaneous pockets in nude mice for up to 8 months. Histological evaluation and biomechanical testing were performed to quantify the integration of cartilage pieces and the mechanical properties of the constructs over time. Histological analysis indicated that chondrocytes grown on devitalized cartilage discs produced new matrix that bonded and integrated individual cartilage elements with mechanically functional tissue. Biomechanical testing demonstrated a time dependent increase in tensile strength, failure strain, failure energy, and tensile modulus to values 5-30% of normal articular cartilage by 8 months in vivo. The values recorded at 4 months were not statistically different from those collected at the latest time point, indicating that the limits of the biomechanical property values were reached after four months from implantation.     

Review. Articular cartilage chondrons: form, function and failure

The chondrocyte and its pericellular microenvironment together represent the chondron, historically considered the primary structural, functional and metabolic unit of articular and other hyaline cartilages. This review summarises research over the last 10 years to establish the molecular anatomy, functional properties and metabolic contribution of the chondron in articular cartilage homeostasis, and its failure during the initiation and progression of degenerative osteoarthritis.