Semiquantitative analysis of ECM molecules in the different cartilage layers in early and advanced osteoarthritis of the knee joint

The study was conducted to examine the expression of collagen type I and II in the different cartilage layers in relation to other ECM molecules during the progression of early osteoarthritic degeneration in human articular cartilage (AC). Quantitative real-time (RT)-PCR and colorimetrical techniques were used for calibration of Photoshop-based image analysis in detecting such lesions. Immunohistochemistry and histology were performed with 40 cartilage tissue samples showing mild (ICRS grade 1b) respectively moderate/advanced (ICRS grade 3a or 3b) (20 each) osteoarthritis compared with 15 healthy biopsies. Furthermore, we quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorimetrically. The digitized images of histology and immunohistochemistry stains were analyzed with Photoshop software. T-test and Spearman correlation analysis were used for statistical analysis. In the earliest stages of AC deterioration the loss of collagen type II was associated with the appearance of collagen type I, shown by increasing amounts of collagen type I mRNA. During subsequent stages, a progressive loss of structural integrity was associated with increasing deposition of collagen type I as part of a natural healing response. A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which then leads to an overall decrease. Analysis of proteoglycan showed losses of the overall content and a loss of the classical zonal formation. Correlation analysis of the proteoglycan Photoshop measurements with the RT-PCR revealed strong correlation for Safranin O and collagen type I, medium for collagen type II, alcian blue and glycoprotein but weak correlation with PCR aggrecan results. Photoshop based image analysis might become a valuable supplement for well known histopathological grading systems of lesioned articular cartilage. The evidence of collagen type I production early in the OA disease process coupled with the ability of chondrocytes to up-regulate collagen type II production suggests that therapeutic agents that suppress collagen type I production and increase collagen type II production may enable chondrocytes to generate a more effective repair response.     

Hepatitis C Related Chronic Liver Cirrhosis: Feasibility of Texture Analysis of MR Images for Classification of Fibrosis Stage and Necroinflammatory Activity Grade

Purpose

To assess the feasibility of texture analysis for classifying fibrosis stage and necroinflammatory activity grade in patients with chronic hepatitis C on T2-weighted (T2W), T1-weighted (T1W) and Gd-EOB-DTPA-enhanced hepatocyte-phase (EOB-HP) imaging.

Materials and methods

From April 2008 to June 2012, MR images from 123 patients with pathologically proven chronic hepatitis C were retrospectively analyzed. Texture parameters derived from histogram, gradient, run-length matrix, co-occurrence matrix, autoregressive model and wavelet transform methods were estimated with imaging software. Fisher, probability of classification error and average correlation, and mutual information coefficients were used to extract subsets of optimized texture features. Linear discriminant analysis in combination with 1-nearest neighbor classifier (LDA/1-NN) was used for lesion classification. In compliance with the software requirement, classification was performed based on datasets from all patients, the patient group with necroinflammatory activity grade 1, and that with fibrosis stage 4, respectively.

Results

Based on all patient dataset, LDA/1-NN produced misclassification rates of 28.46%, 35.77% and 20.33% for fibrosis staging and 34.15%, 25.20% and 28.46% for necroinflammatory activity grading in T2W, T1W and EOB-HP images. In the patient group with necroinflammatory activity grade 1, LDA/1-NN yielded misclassification rates of 5.00%, 0% and 12.50% for fibrosis staging in T2W, T1W and EOB-HP images respectively. In the patient group with fibrosis stage 4, LDA/1-NN yielded misclassification rates of 5.88%, 12.94% and 11.76% for necroinflammatory activity grading in T2W, T1W and EOB-HP images respectively.

Conclusion

Texture quantitative parameters of MR images facilitate classification of the fibrosis stage as well as necroinflammatory activity grade in chronic hepatitis C, especially after categorizing the input dataset according to the activity or fibrosis degree in order to remove the interference between the fibrosis stage and necroinflammatory activity grade on texture features.     

Postnatal development of collagen structure in ovine articular cartilage

Background  

Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Across species, adult AC shows an arcade-like structure with collagen predominantly perpendicular to the subchondral bone near the bone, and collagen predominantly parallel to the articular surface near the articular surface. Recent studies into collagen fibre orientation in stillborn and juvenile animals showed that this structure is absent at birth. Since the collagen structure is an important factor for AC mechanics, the absence of the adult Benninghoff structure has implications for perinatal AC mechanobiology. The current objective is to quantify the dynamics of collagen network development in a model animal from birth to maturity. We further aim to show the presence or absence of zonal differentiation at birth, and to assess differences in collagen network development between different anatomical sites of a single joint surface. We use quantitative polarised light microscopy to investigate properties of the collagen network and we use the sheep (Ovis aries) as our model animal.     

Regional differences of tibial and femoral cartilage in the chondrocyte gene expression,immunhistochemistry and composite in different stages of osteoarthritis

The function of articular cartilage as an avascular tissue is mainly served by collagen type II and proteoglycan molecules. Within this matrix homeostasis between production and breakdown of the matrix is exceptionally sensitive.The current study was conducted to identify regional differences in specific alterations in cartilage composition during the osteoarthritic process of the human knee joint. Therefor the changes in the expression of the key molecules of the extracellular matrix were measured in dependence of the anatomical side (femoral vs tibial) and associated with immunohistochemistry and quantitative measurement.60 serial osteochondral femoral condyle and the tibial plateau samples of patients undergoing implantation of total knee endoprosthesis of areas showing mild (Group A, macroscopically ICRS grade 1b) respectively advanced (Group B, macroscopically ICRS grade 3a/3b) (30 each) osteoarthritis according to the histological-histochemical grading system (HHGS) were compared with 20 healthy biopsies with immunohistochemistry and histology. We quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorometrically.In group A slightly increased colour intensity was found for collagen II in deeper layers, suggesting a persisting but initially still intact repair process. But especially on the medial tibia plateau the initial Col II increase in gene expression is followed by a decrease leading to the lowest over all Col II expression on the medial plateau, here especially in the central part. There in late stage diseases the collagen type I expression was also more pronounced. Markedly decreased safranin O staining intensity was observed in the radial zone and less reduced intensity in the transitional zone with loss of zonal anatomy in 40% of the specimens in group A and all specimens in group B. Correlation between colorometrically analysed proteoglycan GAG content and aggrecan Real Time PCR is mainly weak.Tibial and femoral cartilage in contrast to patellar cartilage both are preferential exposed to compressive stresses, but presence of menisci affects the load distribution at the tibial side, which creates varying conditions for the different cartilage surfaces in the knee.As directly measured Poissońs ratio in tibial cartilage is higher but Youn?s modulus is lower than in femoral cartilage, different resulting feedback amplification loops interact with proceeding cartilage damage. The initial loss of aggrecan may support Matrix metalloproteinases (Mmps) in the access to the collagen network and the considerably differing mechanical properties at both joint surfaces result in varying increased synthesis and release of matrix degrading enzymes.The present study has identified a selection of events which reflect the response of cartilage structure and composite, chondrocytes itself and their productivity to changes in mechanical stress depending on the anatomical site.     

Mechanical behavior of intact and low-grade degenerated cartilage.

Young's modulus, elastic and plastic deformation, mechanical hardness and load at failure were determined for low-grade degenerated hyaline cartilage in a porcine model. Osteochondral plugs from the medial condyle of 30 female pigs were used. Cartilage defects were classified using the International Cartilage Repair Society (ICRS) protocol. Mechanical hardness was measured using a Shore A testing device. Total stiffness and plastic deformation was evaluated in the range 50-200 N using a 5-mm indenter. The load at failure was then determined. ICRS grade I specimens showed significantly lower stiffness than grade 0 specimens. ICRS grade 0 specimen showed no significant plastic deformation within the load range 25-100 N. In degenerated cartilage, plastic deformation started at a significantly lower load (50 N). The Young's modulus at 25 N in ICRS grade 0 specimens (18.8 MPa) was significantly higher than in grade I (11.1 MPa) or grade II (10.5 MPa) specimens. Intact cartilage showed significantly higher tension at failure and mechanical Shore A hardness. Young's modulus and tension at failure showed strong correlation. Cartilage degeneration is associated with a significant loss of elasticity and mechanical stress resistance. Shore hardness measurement is an adequate method for rapid biomechanical evaluation of cartilage specimens.     

The Potential Value of Preoperative MRI Texture and Shape Analysis in Grading Meningiomas: A Preliminary Investigation

OBJECT: Preoperative knowledge of meningioma grade is essential for planning treatment and surgery. The purpose of this study was to investigate the diagnostic value of MRI texture and shape analysis in grading meningiomas. METHODS: A surgical database was reviewed to identify meningioma patients who had undergone tumor resection between January 2015 and December 2016. Preoperative MR images were retrieved and analyzed. Texture and shape analysis was conducted to quantitatively evaluate tumor heterogeneity and morphology. Three machine learning classifiers were trained with these features to build classification models. The performance of the features and classification models was assessed. RESULTS: A total of 131 patients were included in this study: 21 with high-grade meningiomas and 110 with low-grade meningiomas. Three texture features were selected: Horzl_RLNonUni, S(2,2)SumOfSqs, and WavEnHL_s-3; three shape features were selected: GeoFv, GeoW4, and GeoW5b. The Mann–Whitney test indicated that all six features were significantly different between high-grade and low-grade meningiomas. AUC values were generally greater than 0.50 (range, 0.73 to 0.88). Sensitivities and specificities ranged from 47.62% to 90.48% and 69.09% to 96.36%, respectively. Among the nine classification models obtained, the one built by training the SVM classifier with all six features achieved the best performance, with a sensitivity, specificity, diagnostic accuracy, and AUC of 0.86, 0.87, 0.87, and 0.87, respectively. CONCLUSIONS: Texture and shape analysis, especially when combined with a SVM classifier, can provide satisfactory performance in the preoperative determination of meningioma grade and is thus potentially useful for clinical application.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

Articular cartilage tensile integrity: modulation by matrix depletion is maturation-dependent

Articular cartilage function depends on the molecular composition and structure of its extracellular matrix (ECM). The collagen network (CN) provides cartilage with tensile integrity, but must also remodel during growth. Such remodeling may depend on matrix molecules interacting with the CN to modulate the tensile behavior of cartilage. The objective of this study was to determine the effects of increasingly selective matrix depletion on tensile properties of immature and mature articular cartilage, and thereby establish a framework for identifying molecules involved in CN remodeling. Depletion of immature cartilage with guanidine, chondroitinase ABC, chondroitinase AC, and Streptomyces hyaluronidase markedly increased tensile integrity, while the integrity of mature cartilage remained unaltered after depletion with guanidine. The enhanced tensile integrity after matrix depletion suggests that certain ECM components of immature matrix serve to inhibit CN interactions and may act as modulators of physiological alterations of cartilage geometry and tensile properties during growth/maturation.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

Collagenous network in cartilage of human femoral condyles. A light microscopic and scanning electron microscopic study

To determine the spatial arrangement of collagen fibrils in articular cartilage of the human femoral head, three healthy femoral heads, obtained at necropsy, were examined by light microscopy and scanning electron microscopy. Light microscopic observations revealed no collagen fibril organization. Scanning electron microscopic observations showed a fine fibrillar texture throughout the articular cartilage. At the articular surface, smooth and fibrillated areas were detectable. Underneath the articular surface, the collagen network in the superficial zone showed a tighter appearance when compared with the homogeneous collagen network of the matrix in the deeper zones. The calcified cartilage zone was well demarcated from the uncalcified cartilage. The arcade model of Benninghoff [Z. Zellforsch. Mikrosk. Anat. 2: 783-862 (1925)] could not be confirmed. It was concluded that the organization of collagen fibrils in hyaline cartilage shows a three-dimensional network of randomly oriented fibrils.     

Contribution of postnatal collagen reorientation to depth-dependent mechanical properties of articular cartilage

The collagen fibril network is an important factor for the depth-dependent mechanical behaviour of adult articular cartilage (AC). Recent studies show that collagen orientation is parallel to the articular surface throughout the tissue depth in perinatal animals, and that the collagen orientations transform to a depth-dependent arcade-like structure in adult animals. Current understanding on the mechanobiology of postnatal AC development is incomplete. In the current paper, we investigate the contribution of collagen fibril orientation changes to the depth-dependent mechanical properties of AC. We use a composition-based finite element model to simulate in a 1-D confined compression geometry the effects of ten different collagen orientation patterns that were measured in developing sheep. In initial postnatal life, AC is mostly subject to growth and we observe only small changes in depth-dependent mechanical behaviour. Functional adaptation of depth-dependent mechanical behaviour of AC takes place in the second half of life before puberty. Changes in fibril orientation alone increase cartilage stiffness during development through the modulation of swelling strains and osmotic pressures. Changes in stiffness are most pronounced for small stresses and for cartilage adjacent to the bone. We hypothesize that postnatal changes in collagen fibril orientation induce mechanical effects that in turn promote these changes. We further hypothesize that a part of the depth-dependent postnatal increase in collagen content in literature is initiated by the depth-dependent postnatal increase in fibril strain due to collagen fibril reorientation.     

Trabecular meshwork's collagen network formation is inhibited by non-pigmented ciliary epithelium-derived extracellular vesicles

The present research aims to determine whether the application of non-pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non-pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non-pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non-pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net-like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open-angle glaucoma abnormal matrix deposition of the extracellular matrix.     

Postnatal development of depth-dependent collagen density in ovine articular cartilage

Background  

Articular cartilage (AC) is the layer of tissue that covers the articulating ends of the bones in diarthrodial joints. Adult AC is characterised by a depth-dependent composition and structure of the extracellular matrix that results in depth-dependent mechanical properties, important for the functions of adult AC. Collagen is the most abundant solid component and it affects the mechanical behaviour of AC. The current objective is to quantify the postnatal development of depth-dependent collagen density in sheep (Ovis aries) AC between birth and maturity. We use Fourier transform infra-red micro-spectroscopy to investigate collagen density in 48 sheep divided over ten sample points between birth (stillborn) and maturity (72 weeks). In each animal, we investigate six anatomical sites (caudal, distal and rostral locations at the medial and lateral side of the joint) in the distal metacarpus of a fore leg and a hind leg.     

Microstructural Modeling of Collagen Network Mechanics and Interactions with the Proteoglycan Gel in Articular Cartilage

Cartilage matrix mechanical function is largely determined by interactions between the collagen fibrillar network and the proteoglycan gel. Although the molecular physics of these matrix constituents have been characterized and modern imaging methods are capable of localized measurement of molecular densities and orientation distributions, theoretical tools for using this information for prediction of cartilage mechanical behavior are lacking. We introduce a means to model collagen network contributions to cartilage mechanics based upon accessible microstructural information (fibril density and orientation distributions) and which self-consistently follows changes in microstructural geometry with matrix deformations. The interplay between the molecular physics of the collagen network and the proteoglycan gel is scaled up to determine matrix material properties, with features such as collagen fibril pre-stress in free-swelling cartilage emerging naturally and without introduction of ad hoc parameters. Methods are developed for theoretical treatment of the collagen network as a continuum-like distribution of fibrils, such that mechanical analysis of the network may be simplified by consideration of the spherical harmonic components of functions of the fibril orientation, strain, and stress distributions. Expressions for the collagen network contributions to matrix stress and stiffness tensors are derived, illustrating that only spherical harmonic components of orders 0 and 2 contribute to the stress, while orders 0, 2, and 4 contribute to the stiffness. Depth- and compression-dependent equilibrium mechanical properties of cartilage matrix are modeled, and advantages of the approach are illustrated by exploration of orientation and strain distributions of collagen fibrils in compressed cartilage. Results highlight collagen-proteoglycan interactions, especially for very small physiological strains where experimental data are relatively sparse. These methods for determining matrix mechanical properties from measurable quantities at the microscale (composition, structure, and molecular physics) may be useful for investigating cartilage structure-function relationships relevant to load-bearing, injury, and repair.     

Voxel-Based Texture Analysis of the Brain

This paper presents a novel voxel-based method for texture analysis of brain images. Texture analysis is a powerful quantitative approach for analyzing voxel intensities and their interrelationships, but has been thus far limited to analyzing regions of interest. The proposed method provides a 3D statistical map comparing texture features on a voxel-by-voxel basis. The validity of the method was examined on artificially generated effects as well as on real MRI data in Alzheimer''s Disease (AD). The artificially generated effects included hyperintense and hypointense signals added to T1-weighted brain MRIs from 30 healthy subjects. The AD dataset included 30 patients with AD and 30 age/sex matched healthy control subjects. The proposed method detected artificial effects with high accuracy and revealed statistically significant differences between the AD and control groups. This paper extends the usage of texture analysis beyond the current region of interest analysis to voxel-by-voxel 3D statistical mapping and provides a hypothesis-free analysis tool to study cerebral pathology in neurological diseases.     

Accumulation of advanced glycation end products decreases collagen turnover by bovine chondrocytes

The integrity of the collagen network is essential for articular cartilage to fulfill its function in load support and distribution. Damage to the collagen network is one of the first characteristics of osteoarthritis. Since extensive collagen damage is considered irreversible, it is crucial that chondrocytes maintain a functional collagen network. We investigated the effects of advanced glycation end products (AGEs) on the turnover of collagen by articular cartilage chondrocytes. Increased AGE levels (by culturing in the presence of ribose) resulted in decreased collagen synthesis (P < 0.05) and decreased MMP-mediated collagen degradation (P < 0.02). The latter could be attributed to increased resistance of the collagen network to MMPs (P < 0.05) as well as the decreased production of MMPs by chondrocytes (P < 0.02). Turnover of a protein is determined by its synthesis and degradation rates and therefore these data indicate that collagen turnover is decreased at enhanced AGE levels. Since AGE levels in human cartilage increase approximately 50 fold between age 20 and 80, cartilage collagen turnover likely decreases with increasing age. Impaired collagen turnover adversely affects the capacity of chondrocytes to remodel and/or repair its extracellular matrix. Consequently, age-related accumulation of AGE (via decreased collagen turnover) may contribute to the development of cartilage damage in osteoarthritis.     

3D non-affine finite strains measured in isolated bovine annulus fibrosus tissue samples

Understanding of the mechanics of disc tissue calls for measurement of strains in physiological conditions. Because the intervertebral disc is gripped between two vertebrae, the swelling is constrained in vivo, resulting in a intradiscal pressure of 0.1–0.2?MPa in supine position. The excision of isolated disc tissue samples results often in non-physiological swelling. The purpose of the present study is to measure 3D finite strains in isolated bovine disc tissue specimens under physiological osmolarity and pressure, particularly around discontinuities of the collagen network. The collagen is stained by means of CNA35 probe, and the (dead) cells are stained by means of propidium iodide. The tissue is observed under confocal microscopy, under an externally applied pressure generated by a PEG solution. The 3D finite strains are obtained through correlation of the texture of the 3D images. The correlation technique yields principal strains in all areas except within collagen-free areas. The deformation is strongly non-affine. Especially around discontinuities, the strain field is non-homogeneous. Macroscopic strains as computed from finite element analysis of whole discs are insufficient to predict microstrains around clefts or cells. Because of the small number of specimens, the present results should be considered preliminary.     

Three dimensional patient-specific collagen architecture modulates cartilage responses in the knee joint during gait

Site-specific variation of collagen fibril orientations can affect cartilage stresses in knee joints. However, this has not been confirmed by 3-D analyses. Therefore, we present a novel method for evaluation of the effect of patient-specific collagen architecture on time-dependent mechanical responses of knee joint cartilage during gait. 3-D finite element (FE) models of a human knee joint were created with the collagen architectures obtained from T₂ mapped MRI (patient-specific model) and from literature (literature model). The effect of accuracy of the implementation of collagen fibril architecture into the model was examined by using a submodel with denser FE mesh. Compared to the literature model, fibril strains and maximum principal stresses were reduced especially in the superficial/middle regions of medial tibial cartilage in the patient-specific model after the loading response of gait (up to ?413 and ?26%, respectively). Compared to the more coarsely meshed joint model, the patient-specific submodel demonstrated similar strain and stress distributions but increased values particularly in the superficial cartilage regions (especially stresses increased >60%). The results demonstrate that implementation of subject-specific collagen architecture of cartilage in 3-D modulates location- and time-dependent mechanical responses of human knee joint cartilage. Submodeling with more accurate implementation of collagen fibril architecture alters cartilage stresses particularly in the superficial/middle tissue.     

Full-thickness cartilage defects are repaired via a microfracture technique and intraarticular injection of the small-molecule compound kartogenin

IntroductionMicrofracture does not properly repair full-thickness cartilage defects. The purpose of this study was to evaluate the effect of intraarticular injection of the small-molecule compound kartogenin (KGN) on the restoration of a full-thickness cartilage defect treated with microfracture in a rabbit model.MethodsFull-thickness cartilage defects (3.5 mm in diameter and 3 mm in depth) were created in the patellar groove of the right femurs of 24 female New Zealand White rabbits. The rabbits were divided into two groups (12 in each group) based on postsurgery treatment differences, as follows: microfracture plus weekly intraarticular injection of KGN (group 1) and microfracture plus dimethyl sulfoxide (group 2). Six rabbits from each group were illed at 4 and 12 weeks after surgery, and their knees were harvested. The outcome was assessed both macroscopically, by using the International Cartilage Repair Society (ICRS) macroscopic evaluation system, and histologically, by using the modified O’Driscoll histologic scoring system. Immunohistochemistry for type II and I collagen was also conducted.ResultsAt 4 weeks, group 1 showed better defect filling and a greater number of chondrocyte-like cells compared with group 2. At 12 weeks, group 1 showed statistically significantly higher ICRS scores and modified O’Driscoll scores compared with group 2. More hyaline cartilage-like tissue was found in the defects of group 1 at 12 weeks.ConclusionsIntraarticular injection of KGN enhances the quality of full-thickness cartilage defects repair after microfracture, with better defect filling and increased hyaline-like cartilage formation.     

Icariin accelerates cartilage defect repair by promoting chondrogenic differentiation of BMSCs under conditions of oxygen‐glucose deprivation

This study explored the role played by combined ICA and bone mesenchymal stem cells (BMSCs) in repairing rabbit knee cartilage defects. Firstly, rabbit BMSCs were isolated and used to construct an in vitro cellular model of oxygen‐glucose deprivation/reoxygenation (OGD/R). Subsequently, ICA processing, Alcian blue staining, immunofluorescence and Western blot studies were performed to evaluate the ability of BMSCs to display signs of chondrogenic differentiation. Furthermore, a rabbit knee cartilage injury model was established in vivo. International Cartilage Repair Society (ICRS) macroscopic evaluations, H&E, Alcian blue and EdU staining, as well as immunohistochemistry, were analysed cartilage repair and pathological condition of the knee cartilage tissue. Our in vitro results showed that ICA promoted the chondrogenic differentiation of BMSCs, as well as aggrecan (AGR), bone morphogenetic protein 2 (BMP2) and COL2A1 protein expression in BMSCs. In vivo experiments showed that rabbits in the BMSCs or ICA treatment group had higher ICRS scores and displayed a better restoration of cartilage‐like tissue and chondrocyte expression on the surface of their cartilage defects. In conclusion, ICA or BMSCs alone could repair rabbit knee cartilage damage, and combined treatment with ICA and BMSCs showed a better ability to repair rabbit knee cartilage damage.