Main and Minor Types of Collagens in the Articular Cartilage: The Role of Collagens in Repair Tissue Evaluation in Chondral Defects

Degenerative pathology of the musculoskeletal system is one of the main reasons for decreased mobility in patients of the older age group. Increasing the life expectancy leads to predominance non-epidemic pathology in all developed countries. Therefore, degenerative diseases of musculoskeletal system have not only medical significance but also social significance. Objective is studying the morphological features of synovial environment of the decompensated osteoarthritic (OA) knee joint. Structural features of subchondral bone, hyaline cartilage of the femur and tibia, the articular capsule, menisci and ligamentous apparatus of the knee joint were studied in 64 patients who underwent total knee arthroplasty at the Department of Traumatology and Orthopedics Bashkirian State Medical University in the period from 2015 to 2020. Material selection, preparation of histological samples, staining with hematoxylin-eosin, microscopy was performed. Adaptive signs of articular cartilage of the femoral condyles manifest in the form of cartilage tissue rearrangement, which are most pronounced in the central zone of the cartilage. At the same time, the phenomena of decompensation and significant areas of destruction are noted. Also, the subchondral bone was replaced with connective tissue with subsequent sclerosis. This sclerosis subsequently led to the decompensation of structures of the hyaline cartilage in the deep and middle zones. Destructive and dystrophic processes were noted in the knee joint menisci. Articular cartilage was replaced with granulation tissue with subsequent invasion of blood vessels. Cruciate ligaments in patients with OA show signs of adaptation due to expansion of endothenonium layers between bundles of collagen fibers and an increase in the diameter of blood vessels.

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Immunochemical and Mechanical Characterization of Cartilage Subtypes in Rabbit

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205000807 ◽

2002 ◽

Vol 50 (8) ◽

pp. 1049-1058 ◽

Cited By ~ 112

Author(s):

Andreas Naumann ◽

James E. Dennis ◽

Amad Awadallah ◽

David A. Carrino ◽

Joseph M. Mansour ◽

...

Keyword(s):

Articular Cartilage ◽

Cartilage Tissue ◽

Blue Staining ◽

Joint Analysis ◽

Type I ◽

Auricular Cartilage ◽

Collagen Types ◽

Type Vi Collagen ◽

Intense Staining ◽

Nasal Cartilage

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.

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Cryopreserved, Thin, Laser-Etched Osteochondral Allograft Maintains the Functional Components of Articular Cartilage After Two Years of Storage

10.21203/rs.3.rs-34944/v1 ◽

2020 ◽

Author(s):

Carolyn B Rorick ◽

Jordyn A Mitchell ◽

Ruth H Bledsoe ◽

Michael L Floren ◽

Ross M Wilkins

Keyword(s):

Flow Cytometry ◽

Articular Cartilage ◽

Growth Factor ◽

Treatment Options ◽

Osteochondral Allograft ◽

Protein Signaling ◽

Chondral Defects ◽

Healthy Cartilage ◽

Functional Components

Abstract Background : Despite improvements in treatment options and techniques, articular cartilage repair continues to be a challenge for orthopedic surgeons. This study provides data to support that the 2-year Cryopreserved, Thin, Laser-Etched Osteochondral Allograft (T-LE Allograft) embodies the necessary viable cells, protein signaling, and extracellular matrix (ECM) scaffold found in fresh cartilage in order to facilitate a positive clinical outcome for cartilage defect replacement and repair. Methods: Viability testing was performed by digestion of the graft, cells were counted using a trypan blue assay. Growth factor and ECM protein content was quantified using biochemical assays. A fixation model was introduced to assess tissue outgrowth capability and cellular metabolic activity in vitro . Histological and immunofluorescence staining were employed to confirm tissue architecture, cellular outgrowth, and presence of ECM. The effects of the T-LE Allograft to signal bone marrow-derived mesenchymal stem cell (BM-MSC) migration and chondrogenic differentiation were evaluated using in vitro co-culture assays. Immunogenicity testing was completed using flow cytometry analysis of cells obtained from digested T-LE Allografts and fresh articular cartilage. Results: Average viability of the T-LE Allograft post-thaw was found to be 94.97 ±3.38%, compared to 98.83 ±0.43% for fresh articular cartilage. Explant studies from the in vitro fixation model confirmed the long-term viability and proliferative capacity of these chondrocytes. Growth factor and ECM proteins were quantified for the T-LE Allograft revealing similar profiles to fresh articular cartilage. Cellular signaling of the T-LE Allograft and fresh articular cartilage both exhibited similar outcomes in co-culture for migration and differentiation of BM-MSCs. Flow cytometry testing confirmed the T-LE Allograft is immune-privileged as it is negative for immunogenic markers and positive for chondrogenic markers. Conclusions: Using our novel, proprietary cryopreservation method, the T-LE Allograft retains excellent cellular viability, with native-like growth factor and ECM composition of healthy cartilage after two years of storage at -80 o C. The successful cryopreservation of the T-LE Allograft alleviates the limited availably of conventionally used fresh osteochondral allograft (OCA), by providing a readily available and simple to use allograft solution. The results presented in this paper supports clinical data that the T-LE Allograft can be a successful option for repairing chondral defects.

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Functional roles of COMP and TSP-4 in articular cartilage and their relevance in osteoarthritis

10.21248/gups.61798 ◽

2021 ◽

Author(s):

◽

Kathrin Maly

Keyword(s):

Gene Expression ◽

Articular Cartilage ◽

Protein Level ◽

Functional Role ◽

Human Articular Cartilage ◽

Serum Samples ◽

Superficial Zone ◽

Ecm Proteins ◽

Healthy Cartilage

Osteoarthritis (OA) is a slowly progressing disease, resulting in the degradation of cartilage and the loss of joint functionality. The cartilage extracellular matrix (ECM) is degraded and undergoes remodelling in OA progression. Chondrocytes start to express degrading proteases but are also reactivated and synthesise ECM proteins. The spectrum of these newly synthesised proteins and their involvement in OA specific processes and cartilage repair is hardly investigated. Human articular cartilage obtained from OA patients undergoing knee replacement surgery was evaluated according to the OARSI histopathology grading system. Healthy, non-OA cartilage samples were used as controls. The expression and distribution of thrombospondin-4 (TSP-4) and the closely related COMP were analysed on the gene level by PCR and on the protein level by immunohistology and immunoblot assays. The potential of TSP-4 as a diagnostic marker was evaluated by immunoblot assays, using serum samples from OA patients and healthy individuals. The functional role of both proteins was further investigated in in vitro studies using chondrocytes isolated from femoral condyles of healthy pigs. The effect of COMP and TSP-4 on chondrocyte migration and attachment was investigated via transwell and attachment assays, respectively. Moreover, the potential of COMP and TSP-4 to modulate the chondrocyte phenotype by inducing gene expression, ECM protein synthesis and matrix formation was investigated by immunofluorescence staining and qPCR. The activation of cartilage relevant signalling pathways was investigated by immunoblot assays. These results showed for the first time the presence of TSP-4 in articular cartilage. Its amount dramatically increased in OA compared to healthy cartilage and correlated positively with OA severity. In healthy cartilage TSP-4 was primarily found in the superficial zone while it was wider distributed in the middle and deeper zones of OA cartilage. The amount of specific TSP-4 fragments was increased in sera of OA patients compared to healthy controls, indicating a potential to serve as an OA biomarker. COMP was ubiquitously expressed in healthy cartilage but degraded in early as well as re-expressed in late-stage OA. The overall protein levels between OA severity grades were comparable. Contrary to TSP-4, COMP was localised primarily in the upper zone of OA cartilage, in particular in areas with severe damage. COMP could attract chondrocytes and facilitated their attachment, while TSP-4 did not affect these processes. COMP and TSP 4 were generally weak inducers of gene expression, although both could induce COL2A1 and TSP-4 additionally COL12A1 and ACAN after 6 h. Correlating data were obtained on the protein level: COMP and TSP-4 promoted the synthesis and matrix formation of collagen II, collagen IX, collagen XII and proteoglycans. In parallel, both proteins suppressed chondrocyte hypertrophy and dedifferentiation by reducing collagen X and collagen I. By analysing the effect of COMP and TSP-4 on intracellular signalling, both proteins induced Erk1/2 phosphorylation and TSP-4 could further promote Smad2/3 signalling induced by TGF-β1. None of the two proteins had a direct or modulatory effect on Smad1/5/9 dependent signalling. In summary, COMP and TSP-4 contribute to ECM maintenance and repair by inducing the expression of essential ECM proteins and suppressing chondrocyte dedifferentiation. These effects might be mediated by Erk1/2 phosphorylation. The presented data demonstrate an important functional role of COMP and TSP-4 in both healthy and OA cartilage and provide a basis for further studies on their potential in clinical applications for OA diagnosis and treatment.

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Cryopreserved, Thin, Laser-Etched Osteochondral Allograft Maintains the Functional Components of Articular Cartilage After Two Years of Storage

10.21203/rs.3.rs-34944/v2 ◽

2020 ◽

Author(s):

Carolyn B Rorick ◽

Jordyn A Mitchell ◽

Ruth H Bledsoe ◽

Michael L Floren ◽

Ross M Wilkins

Keyword(s):

Flow Cytometry ◽

Articular Cartilage ◽

Growth Factor ◽

Treatment Options ◽

Osteochondral Allograft ◽

Protein Signaling ◽

Chondral Defects ◽

Healthy Cartilage ◽

Functional Components

Abstract Background : Despite improvements in treatment options and techniques, articular cartilage repair continues to be a challenge for orthopedic surgeons. This study provides data to support that the 2-year Cryopreserved, Thin, Laser-Etched Osteochondral Allograft (T-LE Allograft) embodies the necessary viable cells, protein signaling, and extracellular matrix (ECM) scaffold found in fresh cartilage in order to facilitate a positive clinical outcome for cartilage defect replacement and repair. Methods: Viability testing was performed by digestion of the graft, cells were counted using a trypan blue assay. Growth factor and ECM protein content was quantified using biochemical assays. A fixation model was introduced to assess tissue outgrowth capability and cellular metabolic activity in vitro . Histological and immunofluorescence staining were employed to confirm tissue architecture, cellular outgrowth, and presence of ECM. The effects of the T-LE Allograft to signal bone marrow-derived mesenchymal stem cell (BM-MSC) migration and chondrogenic differentiation were evaluated using in vitro co-culture assays. Immunogenicity testing was completed using flow cytometry analysis of cells obtained from digested T-LE Allografts and fresh articular cartilage. Results: Average viability of the T-LE Allograft post-thaw was found to be 94.97 ±3.38%, compared to 98.83 ±0.43% for fresh articular cartilage. Explant studies from the in vitro fixation model confirmed the long-term viability and proliferative capacity of these chondrocytes. Growth factor and ECM proteins were quantified for the T-LE Allograft revealing similar profiles to fresh articular cartilage. Cellular signaling of the T-LE Allograft and fresh articular cartilage both exhibited similar outcomes in co-culture for migration and differentiation of BM-MSCs. Flow cytometry testing confirmed the T-LE Allograft is immune-privileged as it is negative for immunogenic markers and positive for chondrogenic markers. Conclusions: Using our novel, proprietary cryopreservation method, the T-LE Allograft retains excellent cellular viability, with native-like growth factor and ECM composition of healthy cartilage after two years of storage at -80 o C. The successful cryopreservation of the T-LE Allograft alleviates the limited availably of conventionally used fresh osteochondral allograft (OCA), by providing a readily available and simple to use allograft solution. The results presented in this paper supports clinical data that the T-LE Allograft can be a successful option for repairing chondral defects.

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Strains in Stratified Agarose Constructs as Determined by Displacement-Encoded MRI

ASME 2012 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2012-80143 ◽

2012 ◽

Author(s):

Adam Griebel ◽

C. C. van Donkelaar ◽

Corey P. Neu

Keyword(s):

Tissue Engineering ◽

Cartilage Tissue Engineering ◽

Cartilage Tissue ◽

Mechanical Stimuli ◽

Healthy Cartilage ◽

Mechanical Quality ◽

Different Strains ◽

Engineered Cartilage ◽

External Stimuli

Osteoarthritis (OA) is a debilitating disease for which no satisfactory treatment exists. Tissue engineering-based strategies have shown considerable potential for repair. Agarose is frequently used as a scaffold material, as chondrocytes maintain their phenotype and cells remain responsive to mechanical stimuli. To improve the mechanical quality of tissue engineered cartilage, recent studies aimed to reproduce the depth-dependent structure of healthy cartilage. One approach to achieve this is by applying depth-dependent mechanical stimuli via cyclically sliding a glass cylinder over the cell-seeded agarose construct [1,2]. The different strains applied to the surface and the deeper regions are expected to induce stratified matrix synthesis and therefore stratified tissue stiffness. Consequently, with the same external stimuli, the internal strain distribution may alter with ongoing tissue development. Such effect is important to understand in order to optimize mechanical loading regimes for cartilage tissue engineering.

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Detection of MMP-13 Activity on Intentionally Strain-Released Type-II Collagen Network in Bovine Articular Cartilage

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53913 ◽

2011 ◽

Author(s):

Nazli Caner ◽

Jeffrey W. Ruberti

Keyword(s):

Articular Cartilage ◽

Degradation Kinetics ◽

Type Ii Collagen ◽

Healthy Tissue ◽

Cartilage Explants ◽

Cartilage Tissue ◽

Mechanical Stimuli ◽

Type Ii ◽

Collagen Network ◽

Mechanical Tension

Articular cartilage is a specialized avascular connective tissue found at the contact regions of diarthrodial joints. Cartilage has few cells (< 5% of the volume), though these cells can maintain the balance of turnover in healthy tissue, when the tissue is damaged, they are not able to repair the defects [1–3]. Extra cellular matrix (ECM) in cartilage comprises water, collagen (principally type II), proteoglycans and noncollagenous proteins. The type II collagen network, which is the dominant structural protein in cartilage ECM, constrains the expansion of the resident PGs and is generally held in mechanical tension. In osteoarthritis (OA), the balance of cartilage tissue production/degradation is thought to be affected by abnormal mechanical stimuli leading to net matrix resorption through production of excess degradative enzymes (e.g. matrix metalloproteinases (MMP) and aggrecanases) [4–8]. In OA tissue the amount of MMP-13 is thought to be increased relative to healthy tissue. OA typically occurs in older adults where, as cartilage ages, there is a marked decrease in the fixed charge density (FCD), the hydration and, consequently, mechanical tension on the collagen type II network [9–11]. We have hypothesized that loss of tension on the collagen network accelerates degradation by MMP. Detection of the effect of MMP on loaded, native cartilage could lead to insight about cartilage degradation kinetics in OA. However, it is quite difficult to controllably deliver MMP to cartilage, to activate the MMP during detensioning of the collagen network and to detect the effect on the cartilage mechanics (because cost limits the amount of MMP used). We have developed a transpirational enzyme loading method which is capable of precisely dosing bovine cartilage explants with a small, known quantity of MMP-13. Following enzyme insertion, we are able to detect the activity of the MMP on osmotically compressed cartilage (i.e. cartilage with a detensioned collagen network) via a simple hydration measurement.

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Chondrogenic Differentiation of Human Adipose-Derived Stem Cells: A New Path in Articular Cartilage Defect Management?

BioMed Research International ◽

10.1155/2014/740926 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 17

Author(s):

Jan-Philipp Stromps ◽

Nora Emilie Paul ◽

Björn Rath ◽

Mahtab Nourbakhsh ◽

Jürgen Bernhagen ◽

...

Keyword(s):

Stem Cells ◽

Articular Cartilage ◽

Cartilage Tissue Engineering ◽

Cartilage Tissue ◽

Cartilage Defects ◽

Adipose Derived Stem Cells ◽

Age Related ◽

Healthy Cartilage ◽

Control And Prevention ◽

Articular Cartilage Defects

According to data published by the Centers for Disease Control and Prevention, over 6 million people undergo a variety of medical procedures for the repair of articular cartilage defects in the U.S. each year. Trauma, tumor, and age-related degeneration can cause major defects in articular cartilage, which has a poor intrinsic capacity for healing. Therefore, there is substantial interest in the development of novel cartilage tissue engineering strategies to restore articular cartilage defects to a normal or prediseased state. Special attention has been paid to the expansion of chondrocytes, which produce and maintain the cartilaginous matrix in healthy cartilage. This review summarizes the current efforts to generate chondrocytes from adipose-derived stem cells (ASCs) and provides an outlook on promising future strategies.

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The Effect of Varying Concentrations and Application Periods of Chondroitinase ABC on Tissue-Engineered Cartilage

Columbia Undergraduate Research Journal ◽

10.52214/curj.v1i1.4108 ◽

2014 ◽

Vol 1 (1) ◽

Author(s):

Eric Tong ◽

Grace D. O'Connell ◽

Terri-Ann N. Kelly ◽

Clark T. Hung

Keyword(s):

Mechanical Properties ◽

Articular Cartilage ◽

Treatment Option ◽

Type Ii Collagen ◽

Treated Group ◽

Cartilage Tissue ◽

Type Ii ◽

Chondroitinase Abc ◽

Engineered Cartilage

Osteoarthritis, a chronic malady characterized by joint pain and swelling, is caused by damage to articular cartilage and is perpetuated by low-grade inflammation. Treatments for osteoarthritis do exist, but many treatments focus on coping with the disease rather than curing it. Surgical options that replace damaged cartilage tissue with that of donor cartilage tissue or cartilage tissue from other parts of articular joints face complications especially when the tissue is not of the correct size or does not have native-like properties. A more suitable treatment option for osteoarthritis is to develop an in vitro tissue-engineered cartilage construct that can be grown using the patient’s own cells and to surgically remove the patient’s damaged cartilage and replace it with the tissue-engineered cartilage. A challenge in developing such a treatment option is producing tissue-engineered cartilage with mechanical properties akin to those of native human articular cartilage. This challenge may be overcome by maximizing the production of type II collagen by the chondrocytes in vitro. One way to maximize collagen production is through the application of chondroitinase ABC, an enzyme which temporarily suppresses proteoglycans in the cartilage matrix to create more space for type II collagen to develop. In this study, two two levels of cABC treatment were applied (“high” and “low”) to cartilage tissue constructs. The “low” cABC treated group received daily feeding of 0.075 U/mL from day 14 to 21 followed by a replacement of chondrogenic media without cABC. The “high” cABC treated group received a single addition of 0.15 U/mL from day 14 to 16 followed by a replacement of chondrogenic media without cABC. At the end of 42 days, the constructs were subjected to mechanical testing and biochemical analyses. These analyses showed that the high cABC treatment yielded more native-like mechanical properties when compared to the low cABC treatment and the control results. Biochemical and histological analyses confirmed that the proteoglycan and collagen II content were higher in the low and high cABC treated groups when compared to the control. All analyses show that the most efficient application of chondroitinase ABC is through a two day duration treatment of a higher concentration (0.15 U/mL).

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THE NEW PLASTIC METHOD OF ARTICULAR HYALINE CARTILAGE DEFECTS WITH COMBINED CELLULAR-TISSUE GRAFT

Traumatology and Orthopedics of Russia ◽

10.21823/2311-2905-2010-0-1-150-155 ◽

2010 ◽

Vol 16 (1) ◽

pp. 150-155

Author(s):

G. P. Kotelnikov ◽

L. T. Volova ◽

Yu. V. Lartsev ◽

D. A. Dolgushkin ◽

M. A. Terteryan

Keyword(s):

Cell Culture ◽

Articular Cartilage ◽

Subchondral Bone ◽

Hyaline Cartilage ◽

Costal Cartilage ◽

Cellular Tissue ◽

New Method ◽

Cartilage Tissue ◽

Cartilage Defects ◽

Porous Carrier

The new method of restoration of injured articular cartilage using plasty of intraoperative bone-cartilage defects with combined allograft based on biologic porous carrier and cell culture from stroma of costal cartilage. The transplantation of allogenic culture of chondroblast cells on demineralized spongy substance «Lioplast» ensure еру the formation of hyaline cartilage tissue on the site of defect and amplified development of microcirculatory network in subchondral bone. The heterotopic principle obtaining cellular material allows to perform low-traumatic operations.

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