Narrow Results

This study was performed to assess the effect of intra-articularly-injected 3.0% povidone-iodine solution and 2% H₂O₂ on the joint cartilage. Ninety 12-week-old Spraque Dawley rats, weighing 250 g on average, were used; ten were used as controls without any treatment, and 80 were equally divided into four groups for the experiment. In group I, 0.05 ml of normal saline was injected twice into the right knee joints at intervals of one week each, and a running load over a treadmill was added until the animals were sacrificed. In group II, 0.05 ml of 2% H₂O₂ was injected twice into the right knees over the same interval. In group III, 0.05 ml of 2% H₂O₂ was injected twice into the right knees over the same weekly interval, and a running load was added until the knees were sacrificed. In group IV, 0.05 ml of 3.0% povidone-iodine was injected twice into the right knees at one week intervals and a running load was also added until their sacrifice. In each experimental group, five rats were sacrificed at two, four, six and eight weeks after the first intra-articular injection, respectively, and the medial femoral condyles of the joints were taken for histological study of the articular cartilage. After saline injection and running load were applied, stage-I cartilage degeneration was observed at eight weeks after the first saline injection. After H₂O₂ injection only, stage-I cartilage degeneration was observed at four and six weeks, and the changes progressed to Wagenhäuser stage-II degeneration at eight weeks. After H₂O₂ injection and running load were applied, there were no histologic changes by two weeks, but stage-II cartilage degeneration was induced as early as six weeks after the first H₂O₂ injection. After povidone-iodine injection and running load were applied, stage-I cartilage degeration was observed at two weeks, and was maintained up to six weeks. Stage-II degeneration was observed at eight weeks. The matriceal proteoglycan contents was relatively high in comparison with the other groups. Safranin-O stain reduction was observed in groups I, II, III and IV before the histological changes were being observed. From the results, it was found that 3.0% povidone-iodine and 2% H₂O₂ with running loads could induce cartilage damage, and that 3.0% povidone-iodine solution induced more damage to the cartilage than 2% H₂O₂.

Mineralization of the cartilage matrix in embryonic long bones and growth plates is preceded by hypertrophy of chondrocytes. We hypothesize that the swollen hypertrophic cells exert pressure on the matrix, and that this pressure plays a role in the cartilage mineralization process. For this study, we asked the following questions. First, does the ratio of cell volume to matrix volume (CV/MV) increase from the proliferation to the hypertrophic zone in embryonic long bones? Second, is there a correlation between cell-volume increase and the mineralization rate in embryonic and postnatal long bones? The CV/MV ratios in the proliferation and hypertrophic zones in embryonic mouse metatarsals at 17 days of gestational age were determined using morphometric analyses. Confocal laser scanning microscopy was used to determine chondrocyte volumes. Cell volumes in the proliferation and hypertrophic zones of embryonic mouse metatarsals at 17 days of gestational age were compared to the ones in the metatarsal growth plates of nine-day-old mice. The mineralization rate was determined using photographs at 24-hour intervals. The CV/MV increased significantly from the proliferation to the hypertrophic zone, from 1.30±0.15 (mean ± standard deviation) to 1.80±0.18. The relative increase in cell volume from the proliferation to the hypertrophic zone was 1.6 for embryonic cells, i.e. from 370±101 mm³ to 610±107 mm³, and 2.8 for postnatal cells, i.e. from 280±41 mm³ to 786±155 mm³(p<0.05). The mineralization rate was 295±47mm/24 hours and 382±149 mm/24 hours for embryonic and postnatal metatarsals, respectively (p<0.05). The finding that chondrocyte volume increase is accompanied by a higher mineralization rate supports the hypothesis that cell hypertrophy plays an important role during the mineralization process.

The objective of this study is to analyze the effects and benefits of subtotal synovectomy in the early stage of septic arthritis. seventy rabbits with septic arthritis of the left knee joint were treated at 24 or 72 hours after inoculation of Staphylococcus aureus, with different treatment modalities, including antibiotic therapy, arthrotomy, irrigation, and synovectomy. At the end of the 6th week, the knee joints were removed and examined both macroscopically and histologically. It was discovered that there was more significant degeneration at the articular surface of the femur than that of the tibia. antibiotic therapy alone was found to be insufficient to prevent the degeneration of articular cartilage. performing subtotal synovectomy had no statistically significant effect 24 hours after the inoculation of bacteria. However, adding subtotal synovectomy to the surgical drainage 72 hours after inoculation resulted in significantly lesser degeneration of the articular cartilage. Sufficient drainage and irrigation of the joint associated with antibiotic treatment seems to be an adequate choice of treatment at the very early stage of septic arthritis. However, in established septic arthritis, adding subtotal synovectomy to the surgical drainage resulted in significantly lesser degeneration of the articular cartilage.

Conventional histological assessment of tissue requires the removal of tissue from its physiological environment for pathological processing. In this study we report on the development and application of a laser scanning confocal arthroscope (LSCA) capable of in situ histological assessment by confocal microscopy.

The knee joints of three euthanasied adult Merino sheep were imaged using the LSCA and a combination of fluorescent contrasting agents. LSCA images of various tissues were examined for common histological characteristics.

The confocal histology images from the ovine knee acquired during the course of our study illustrated some of the major histological features of chondral and connective tissues.

Our study demonstrates the efficacy of the LSCA for the histological assessment of the chondral and connective tissues of the ovine knee. The LSCA provides an easy and rapid method for assessing the in situ structure of cartilage, muscle, tendon, ligament, meniscus and synovium in their native and unaltered physiological environment. By obviating the need for mechanical biopsy, the LSCA provides a method for the non-destructive assessment of the chondral and connective tissues of the knee, and may be of great benefit in the investigation of orthopedic diseases or in the assessment of newly emerging cartilage repair techniques.

The aim of this study is to investigate the structure and the collagen matrix of the superficial zone of articular cartilage using a 3D imaging technique. The split line thought to represent the orientation of the collagen fibres in the superficial zone was found using Hultkrantz's method. A semitransparent membrane was physically peeled off from the most superficial surface of bovine articular cartilage. Using fibre optic laser scanning confocal microscopy, the collagen matrix in normal cartilage, the membrane and the cartilage with the membrane peeled off were studied. The superficial zone was found to contain a more sophisticated 3D collagenous matrix than previously reported. The collagen matrix in the membrane consists of interwoven long collagen bundles, and the collagen fibres immediately subjacent to it align spatially in a predominantly oblique direction to the articular surface. The split line does not represent the orientation of the collagen in the membrane. This study presents a 3D visualization technique for a minimal-invasive examination of the 3D architecture of the collagen fibres in the superficial zone of articular cartilage, and offers a new insight into the 3D structure of the collagen matrix in the superficial zone of native cartilage.

An extractable bone-inducing agent has recently been identified in freeze-dried preparations of Saos-2 cultured human osteosarcoma cells. Although not all osteoinductive components of Saos-2 cell extracts have been identified, we have shown that Saos-2 cells express high levels of mRNA for bone morphogenetic proteins (BMPs)-1,2,3,4 and 6. Any or all of these BMPs (plus possible unknown factors) may be involved in ectopic bone induction, and may act as paracrine agents, conveying morphogenetic information to juxtaposed osteoprogenitor cells. Our objectives in this study were: 1) to determine whether Saos-2 cells secrete BIA into their culture medium; and 2) if secreted, to determine whether released bone-inducing agent is soluble and/or particulate and contains BMPs. Saos-2 cells were grown to confluence, and then overlaid with serum-free DMEM culture medium for 48 hours. The serum-free conditioned medium was then decanted and filtered through 0.45μ pore-size filters to retain any vesicles or other particulates released by the cells. Particulate protein retained on the 0.45μ filter (designated "retentate") was extracted into 6M urea and bioassayed for bone-inducing activity in Nu/Nu mice, along with soluble media protein that had passed through the 0.45μ filter (designated "filtrate") plus freeze-dried Saos-2 cells from which conditioned the culture medium was obtained. Results indicate that the bone-inducing agent of Saos-2 cells is not only retained by the cells, but is also secreted in both soluble and particulate forms into serum-free conditioned medium. Bone-inducing activity (per mg protein) is more concentrated in the particulate fraction, which is shown by electron microscopy to contain a mixture of vesicles (similar to matrix vesicles) plus electron dense granules (resembling ribosomes) and 10 mM microfilaments (of possible collagenous or cytoskeletal origin). BMP-1,2,3,4,6 and 7 were detected by western blots in both the soluble and particulate fractions of conditioned medium. Thus, it is indicated that Saos-2 cells secrete an osteoinductive factor which may function in vivo as a paracrine morphogenetic agent.

Articular cartilage is a vital component of human knee joints by providing a low-friction and wear-resistant surface in knee joints and distributing stresses to tibia. The degeneration or damage of articular cartilage will incur acute pain on the human knee joints. Hence, to understand the mechanism of normal and pathological functions of articular cartilage, it is very important to investigate the contact mechanics of the human knee joints. Experimental research has difficulties in reproducing the physiological conditions of daily activities and measuring the key factors such as contact-stress distributions inside knee joint without violating the physiological environment. On the other hand, numerical approaches such as finite element (FE) analysis provide a powerful tool in the biomechanics study of the human knee joint. This article presents a two-dimensional (2D) FE model of the human knee joints that includes the femur, tibia, patella, quadriceps, patellar tendon, and cartilages. The model is analyzed with dynamic loadings to study stress distribution in the tibia and contact area during contact with or without articular cartilage. The results obtained in this article are very helpful to find the pathological mechanism of knee joint degeneration or damage, and thus guide the therapy of knee illness and artificial joint replacement.

Background. As articular cartilage is unable to repair itself, there is a tremendous clinical need for a tissue engineered replacement tissue. Current tissue engineering efforts using the self-assembly process have demonstrated promising results, but the biomechanical properties remain at roughly 50% of native tissue. Methodology/Principal Findings. The objective of this study was to determine the feasibility of using exogenous crosslinking agents to enhance the biomechanical properties of a scaffoldless cartilage tissue engineering approach. Four crosslinking agents (glutaraldehyde, ribose, genipin, and methylglyoxal) were applied each at a single concentration and single application time. It was determined that ribose application resulted in a significant 69% increase in Young's modulus, a significant 47% increase in ultimate tensile strength, as well as a trend toward a significant increase in aggregate modulus. Additionally, methylglyoxal application resulted in a significant 58% increase in Young's modulus. No treatments altered the biochemical content of the tissue. Conclusions/Significance. To our knowledge, this is the first study to examine the use of exogenous crosslinking agents on any tissue formed using a scaffoldless tissue engineering approach. In particular, this study demonstrates that a one-time treatment with crosslinking agents can be employed effectively to enhance the biomechanical properties of tissue engineered articular cartilage. The results are exciting, as they demonstrate the feasibility of using exogenous crosslinking agents to enhance the biomechanical properties without the need for increased glycosaminoglycan (GAG) and collagen content.

The purpose of this study was to investigate the attachment and proliferation of cells on selective laser-sintered (SLS) polycaprolactone (PCL) scaffolds coated with gelatin for cartilage tissue engineering using chondrocytes isolated from the articular cartilage of swine. Scaffolds without modification were used as control groups. Cell proliferation was measured by cell count 1, 3 and 5 days after cell seeding into the scaffolds. The biocompatibility of the scaffold was examined by scanning electron microscopy (SEM). The PCL scaffolds coated with gelatin had higher hydrophilicity. The results provided a useful strategy for modifying the microenvironments to increase cell attachment, growth and the formation of extracellular matrix on scaffolds for cartilage tissue engineering.

Bone marrow stimulation techniques, such as abrasion arthroplasty or microfracture, have been widely used for repairing cartilage; however, the mechanical stress analysis of these surgical techniques has not been fully investigated. In this study, finite element analysis was used to investigate stresses produced in complex structures (e.g., cartilage, subchondral bone and trabecular bone) using 2D knee structural models. Abrasion arthroplasty creates global damages only in subchondral bone, but, microfracture technique creates local damages in both trabecular and subchondral regions. Although stresses do not significantly change in trabecular bones as 50% recovery occurs in both abrasion and microfacture samples, significant changes are observed in both subchondral bone and cartilage layer depending on the procedure. The maximum stress levels in the microfractured bone represent approximately a 10.48% increase in cartilage and a 38.25% increase in subchondral bones compared to normal conditions. After 150% recovery, however, all three layers increase their stress levels in microfractured samples. Therefore, the 2D computational analysis results suggest that the microfracture technique should be cautiously used.

The articular cartilage in the human knee plays an important role to ensure a lifetime knee function for an individual. Due to damage of the cartilage in the knee, the coefficient of friction (COF) increases even after treatments for the cartilage, due to the poor self-healing ability of the cartilage, resulting in decreased knee life. As the mechanism of the function of the knee joint is similar to a bearing, a model based on the regression model of cylindrical bearing life has been developed. The model is used to evaluate the effect of the COF on the lifetime of the knee. The results show the correlation between the life of the knee and the COF of the knee cartilage. The knee lifetime depends on the ratio of the COF of the healthy cartilage to the damaged cartilage. The results demonstrate the effect of the COF on the knee lifetime, which is an exponential decrease in the life of the knee for both males and females.

Native subject-specific knee geometries are usually based on CT and MRI images reconstruction. Unfortunately, while the definition of bone geometries using CTs is quite consistent, MRIs are often hardly readable, due to the usual lower resolution, and the final shape of cartilage and menisci is not consequently detailed enough. Moreover, further smoothing techniques, necessary to efficiently use these structures for numerical modeling, could result in bad interfaces and/or geometry inaccuracies. In this study a CAD-based approach to generate 3D cartilages and menisci geometries, avoiding the use of MRIs, was proposed and tested versus the traditional methods that use MRIs segmentation. The femoral, tibial and patellar cartilage layers were generated as offset from the bone geometries, the menisci were obtained by an extrusion based on tibia borders. Such geometries were compared to the reconstructions obtained from MRIs of healthy knee specimens. Overlapping the resulting geometries with the ones traditionally reconstructed, volumes differ from 2% to 14%. By using the new methodology, the geometries are obtained in 75% less time. The CAD-based methods shown in this pilot study is able to generate faster and accurate subject-specific knee cartilage layers and menisci geometries and can be suitable to be applied for numerical modeling.

Extraskeletal chondroma comprises synovial chondromatosis, intracapsular chondroma and soft tissue chondroma, its presentation in the hand, however, is very rare. A 42-year-old woman presented with a mass around right fourth metacarpophalangeal (MCP) joint. She had no pain or discomfort in activities. The radiographs showed soft tissue swelling, but no calcification or ossifying lesions. The magnetic resonance imaging (MRI) showed a lobulated juxta-cortical encircling mass existing around the fourth MCP joint. We did not suspect any cartilage-forming tumour in MRI. The mass was easily removed because there was no adhesion with surrounding tissues and the specimen had the appearance of a cartilage. The histological diagnosis was chondroma. Based on the tumour location and histological results, we diagnosed it as intracapsular chondroma. Although intracapsular chondroma is very rare-ly seen in the hands, it is important to consider an intracapsular chondroma when differentiating a tumour in the hand because it is difficult to diagnose one in an imaging examination.

Level of Evidence: Level V (Therapeutic)

The most exciting discoveries in the last two decades have been the mapping, sequencing, and understanding of genes in our body. In this chapter, we will discuss how to specifically map expression profiles of gene products — mRNA, protein, and reporter gene (X-gal) — using in situ hybridization, immunohistochemistry, and X-gal staining, respectively.

Cartilage damage is irreversible due to its properties of avascularity and lack of undifferentiated cells. Studying the biology of chondrocytes and cartilage is therefore critical to understand the underlying mechanism and to explore the potential repair approaches. It has been reported that three-dimensional (3D) chondrocyte culture behaves very differently from two-dimensional (2D) monolayer culture. Therefore, a native 3D culture of chondrocytes is valuable for such a research purpose, and may also be an option for repairing cartilage defects. This chapter describes a detailed protocol for high-yield chondrocyte isolation and the modified pellet culture technique (using a 3D chondrocyte culture), which can synthesize a bioengineered tissue up to 8 mm in diameter. The authors aim at providing a platform for researchers to study chondrocyte behavior in a 3D environment and to explore the application of scaffold-free tissue for transplantation into a large cartilage defect model.

Perhaps the most difficult task in bone histology work is the preparation of a good specimen. It is impossible to obtain a good image of bone or cartilage without careful preparation and processing of specimens. This chapter will describe very basic skills in the preparation of specimens. These techniques include the paraffin method, plastic method, and frozen method.

Bone or cartilage cells are essentially colorless, and it is difficult to distinguish their morphologies under a light microscope. To better visualize these structures, various staining techniques have been developed; due to page constraints, we will only describe the most common methods for readers. These staining assays include hematoxylin and eosin (H&E) stain, used for general histology of the cell; Safranin O stain, used for the cartilage; and von Kossa and van Gieson stains as well as modified Goldner's trichrome stain, used for nondecalcified tissues.

Attrition and eventual loss of articular cartilage are crucial elements in the pathophysiology of osteoarthritis. Preventing the breakdown of cartilage is believed to be critical in order to preserve the functional integrity of a joint. Magnetic resonance imaging (MRI) and advanced digital postprocessing techniques have opened novel possibilities for in vivo quantitative analysis of cartilage morphology, structure, and function in health and disease. Techniques of semiquantitative scoring of human knee cartilage pathology and quantitative assessment of human cartilage have recently been developed. Though cartilage represents a thin layer of material relative to the size of voxels typically used for MRI, cartilage thickness and volume have been quantified in human and in small animals. MRI-detected cartilage loss has been shown to be more sensitive than radiography-detected joint space narrowing. Progress made in MRI technology in the last few years allows longitudinal studies of knee cartilage with an accuracy good enough to follow disease-caused changes and to evaluate the therapeutic effects of chondroprotective drugs.

The etiology of most of the degenerative changes in the spine continues to remain obscure. However, several lines of evidence suggest that genetic factors may play an important role in the onset of degenerative changes, in addition to various environmental factors. We have generated transgenic mice expressing mutant αl(IX) collagen in the cartilage matrix. They developed progressive intervertebral disc degeneration with age as well as joint degeneration. Both radiologic and histologic studies indicated that cervical and lumbar disc degeneration was more advanced in the transgenic mice than in control littermates. The initial degenerative changes included shrinkage and replacement of the nucleus pulposus with consolidated fibrous tissue, that resulted in a loss of nuclear-anular demarcation. Partial disruption in the lamellar structure of the anulus fibrosus also occurred at this stage. With age, the disc degeneration progressively advanced and sometimes caused herniation of disc material and mild osteophyte formation. These findings imply that genetic abnormalities of cartilage matrix components, such as type IX collagen, may be responsible for certain degenerative diseases in the spine.

Hydroxyapatite is the well known bioactive ceramic used in medical and dental fields as blocks and particles, while chondroitin-sulfate is a dominant polysaccharide component of cartilage. Novel composite materials consisting of hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂] and chondroitin-sulfate were synthesized by a coprecipitation method with a H₃PO₄ solution and a Ca(OH)₂ suspension, one of which contain chondroitin-sulfate, and were consolidated under a cold isostatic pressure. The composites were evaluated by X-ray diffractometry, Fourier transformed infrared spectroscopy and transmission electron microscopy. In the composites, hydroxyapatite nanocrystals contained calcium-deficiency and a small amount of carbonate ions like human bone minerals and their c-axes were aligned along chondroitin-sulfate molecules by a self-assembly mechanism.