Isolation Of Chondrocytes Research Articles

Inhibition of PA28γ expression can alleviate osteoarthritis by inhibiting endoplasmic reticulum stress and promoting STAT3 phosphorylation.

Osteoarthritis (OA) is a common degenerative disease. PA28γ is a member of the 11S proteasome activator and is involved in the regulation of several important cellular processes, including cell proliferation, apoptosis, and inflammation. This study aimed to explore the role of PA28γ in the occurrence and development of OA and its potential mechanism. A total of 120 newborn male mice were employed for the isolation and culture of primary chondrocytes. OA-related indicators such as anabolism, catabolism, inflammation, and apoptosis were detected. Effects and related mechanisms of PA28γ in chondrocyte endoplasmic reticulum (ER) stress were studied using western blotting, real-time polymerase chain reaction (PCR), and immunofluorescence. The OA mouse model was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity of 15 12-week-old male mice to reduce the expression of PA28γ. The degree of cartilage destruction was evaluated by haematoxylin and eosin (HE) staining, safranin O/fast green staining, toluidine blue staining, and immunohistochemistry. We found that PA28γ knockdown in chondrocytes can effectively improve anabolism and catabolism and inhibit inflammation, apoptosis, and ER stress. Moreover, PA28γ knockdown affected the phosphorylation of IRE1α and the expression of TRAF2, thereby affecting the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signalling pathways, and finally affecting the inflammatory response of chondrocytes. In addition, we found that PA28γ knockdown can promote the phosphorylation of signal transducer and activator of transcription 3 (STAT3), thereby inhibiting ER stress in chondrocytes. The use of Stattic (an inhibitor of STAT3 phosphorylation) enhanced ER stress. In vivo, we found that PA28γ knockdown effectively reduced cartilage destruction in a mouse model of OA induced by the DMM surgery. PA28γ knockdown in chondrocytes can inhibit anabolic and catabolic dysregulation, inflammatory response, and apoptosis in OA. Moreover, PA28γ knockdown in chondrocytes can inhibit ER stress by promoting STAT3 phosphorylation.

Dual Enzyme Sequential Digestion Protocol for Isolation of Human Primary Chondrocytes From the Articular Cartilage Derived From Knee Osteoarthritis Patients

Introduction: Majority of available protocols for isolation of chondrocytes from articular cartilage tissue rely on the enzymatic digestion of the tissue by collagenase type 2. The yield of chondrocytes in such protocols is low. Herein, we designed a novel indigenous sequential digestion by dual enzyme Pronase and Collagenase Type 1 for isolating human Chondrocytes from articular cartilage. Methods: Articular cartilage of Osteoarthritis (OA) patients undergoing total knee replacement were collected for the isolation of chondrocyte cells and subjected to sequential digestion by Pronase for three hours followed by Collagenase 1 overnight. Pellet of cells collected after digestion was plated on culture flask in 5% CO2 incubator. Results: From day three onwards, round to elongated cells adhered to the flask were visible which developed into elongated cell population of homogenous morphology, expressed Aggrecan (Agg), Collagen 2a (Col2a) and SRY-box transcription factor (Sox9) and had chondrogenic differentiation similar to a commercially available healthy chondrocyte. These cells were negative for Alizarin red stain, thus confirming the purity of chondrocytes. Conclusion: We have successfully established a sequential dual enzyme digestion-based culture technique for isolating the human chondrocytes from the articular cartilage biopsy derived from OA knee joints.

CELLULAR AND MOLECULAR ENDOTYPES FOR OSTEOARTHRITIS

The biological understanding for the disease progression osteoarthritis (OA) has uncovered specific biomarkers from either synovial fluid, articular chondrocytes or synoviocytes that can be used to diagnose the disease. Examples of these biomarkers include interleukin-1β (IL-1β) or collagen II fragments (1, 2). In parallel, isolation of chondrocytes or bone marrow derived mesenchymal stromal cells (MSCs) has yielded cell-based strategies that have shown long- term beneficial effects in a specific cohort of patients, specifically in traumatic cartilage lesions (2). This latter finding shows that patient stratification of OA is an important tool to both match patients for a specific treatment and to develop novel therapies, especially disease modifying drugs. In order to create disease stage specific therapies, the use of next generation analysis tools such as RNAseq and metabolomics, has the potential to decipher specific cellular and molecular endotypes. Alongside greater understanding of the clinical phenotype (e.g. imaging, pain, co- morbidities), therapies can be designed to alleviate the symptoms of OA at specific points of the disease in patients. This talk will outline the current biological understanding of OA and discuss how patient stratification could assist in the design of innovative therapies for the disease.Acknowledgements: This presentation was supported by the COST action, CA21110 – Building an open European Network on Osteoarthritis Research (NetwOArk)

Standardizing Chondrocyte Isolation and Articular Cartilage Decellularization: A Versatile Bioink for Tissue Engineering Applications.

The extracellular matrix (ECM) is a noncellular component of tissues that provides structural and biochemical support to cells. The purpose of decellularization is to provide a tissue-specific niche to preserve the architecture, composition, and signaling molecules of the ECM. The current protocol discusses the standardization of chondrocyte isolation and the preparation of acellular ECM as a bioink additive from human native articular cartilage. Isolated chondrocytes with bioink additives provide a tissue-specific microenvironment. Herein, we discuss a standardized protocol with multiple applications in the area of organ-on-a-chip model development, spheroid formation, microfluidics platform, bioprinting, and tissue engineering. Cartilage tissue engineering is complex owing to the heterogeneous complex proteins, which are a challenge to synthesize; hence, this protocol in many ways offers cues to exploit the acellular ECM for multiple ongoing research studies.

Poster 114: Viability and Expression of Chondrocytes Harvested During Arthroscopic Treatment of Femoroacetabular Impingement

Poster 114: Viability and Expression of Chondrocytes Harvested During Arthroscopic Treatment of Femoroacetabular Impingement

Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

Perspective in Achieving Stratified Articular Cartilage Repair Using Zonal Chondrocytes.

Articular cartilage is composed of superficial, medial, and deep zones, which endow the tissue with biphasic mechanical properties to withstand shearing force and compressional loading. The tissue has very limited self-healing capacity once it is damaged due to its avascular nature. To prevent the early onset of osteoarthritis, surgical intervention is often needed to repair the injured cartilage. Current noncell-based and cell-based treatments focus on the regeneration of homogeneous cartilage to achieve bulk compressional properties without recapitulating the zonal matrix and mechanical properties, and often oversight in aiding cartilage integration between host and repair cartilage. It is hypothesized that achieving zonal architecture in articular cartilage tissue repair could improve the structural and mechanical integrity and thus the life span of the regenerated tissue. Engineering stratified cartilage constructs using zonal chondrocytes have been hypothesized to improve the functionality and life span of the regenerated tissues. However, stratified articular cartilage repair has yet to be realized to date due to the lack of an efficient zonal chondrocyte isolation method and an expansion platform that would allow both cell propagation and phenotype maintenance. Various attempts and challenges in achieving stratified articular cartilage repair in a clinical setting are evaluated. In this review, different perspectives on achieving stratified articular cartilage repair using zonal chondrocytes are described. The effectiveness of different zonal chondrocyte isolation and zonal chondrocyte phenotype maintenance methodologies during expansion are compared, with the focus on recent advancements in zonal chondrocyte isolation and expansion that could present a possible strategy to overcome the limitation of applying zonal chondrocytes to facilitate zonal architecture development in articular cartilage regeneration. Impact Statement The zonal properties of articular cartilage contribute to the biphasic mechanical properties of the tissues. Recapitulation of the zonal architecture in regenerated articular cartilage has been hypothesized to improve the mechanical integrity and life span of the regenerated tissue. This review provides a comprehensive discussion on the current state of research relevant to achieving stratified articular cartilage repair using zonal chondrocytes from different perspectives. This review further elaborates on a zonal chondrocyte production pipeline that can potentially overcome the current clinical challenges and future work needed to realize stratified zonal chondrocyte implantation in a clinical setting.

Chondrocyte Isolation from Loose Bodies-An Option for Reducing Donor Site Morbidity for Autologous Chondrocyte Implantation.

Loose bodies (LBs) from patients with osteochondritis dissecans (OCD) are usually removed and discarded during surgical treatment of the defect. In this study, we address the question of whether these LBs contain sufficient viable and functional chondrocytes that could serve as a source for autologous chondrocyte implantation (ACI) and how the required prolonged in vitro expansion affects their phenotype. Chondrocytes were isolated from LBs of 18 patients and compared with control chondrocyte from non-weight-bearing joint regions (n = 7) and bone marrow mesenchymal stromal cells (BMSCs, n = 6) obtained during primary arthroplasty. No significant differences in the initial cell yield per isolation and the expression of the chondrocyte progenitor cell markers CD44 + /CD146+ were found between chondrocyte populations from LBs (LB-CH) and control patients (Ctrl-CH). During long-term expansion, LB-CH exhibited comparable viability and proliferation rates to control cells and no ultimate cell cycle arrest was observed within 12 passages respectively 15.3 ± 1.1 mean cumulative populations doublings (CPD). The chondrogenic differentiation potential was comparable between LB-CH and Ctrl-CH, but both groups showed a significantly higher ability to form a hyaline cartilage matrix in vitro than BMSC. Our data suggest that LBs are a promising cell source for obtaining qualitatively and quantitatively suitable chondrocytes for therapeutic applications, thereby circumventing donor site morbidity as a consequence of the biopsies required for the current ACI procedure.

Optimization of 3D autologous chondrocyte-seeded polyglycolic acid scaffolds to mimic human ear cartilage.

Auricular reconstruction in children with microtia is one of the more complex procedures in plastic surgery. Obtaining sufficient native material to build an ear requires harvesting large fragments of rib cartilage in children. Herein, we investigated how to optimize autologous chondrocyte isolation, expansion and re-implantation using polyglycolic acid (PGA) scaffolds for generating enough cartilage to recapitulate a whole ear starting from a small ear biopsy. Ear chondrocytes isolated from human microtia subjects grew slower than microtia rib or healthy ear chondrocytes and displayed a phenotypic shift due to the passage number. Rabbit ear chondrocytes co-cultured with mesenchymal stem cells (MSC) at a 50 : 50 ratio recapitulated the cartilage biological properties in vitro. However, PGA scaffolds with different proportions of rabbit chondrocytes and MSC did not grow substantially in two months when subcutaneously implanted in immunosuppressed mice. In contrast, rabbit chondrocyte-seeded PGA scaffolds implanted in immunocompetent rabbits formed a cartilage 10 times larger than the original PGA scaffold. This cartilage mimicked the biofunctional and mechanical properties of an ear cartilage. These results indicate that autologous chondrocyte-seeded PGA scaffolds fabricated following our optimized procedure have immense potential as a solution for obtaining enough cartilage for auricular reconstruction and opens new avenues to redefine autologous cartilage replacement.

Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome.

The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome. Cartilage tissues were transferred into a hypoxia chamber (4% O2) immediately after being removed from OA patients and minced finely. Collagenase II at concentrations of 0.02%, 0.1%, 0.25%, 0.5%, 1%, and 2% was applied for 0.5, 1, 2, 4, and 18h to digest the minced tissue. Actinomycin D (ActD) was added to test its capacity in stabilizing the transcriptome. Cell yield, viability, cell size, and transcriptome were determined using counter chamber, flow cytometry, and RNA sequencing (RNA-seq). Collagenase II at 2% concentration released small chondrocytes from cartilage matrix during the first digestion hour and started to release large cells thereafter, reaching a complete release at 4h. During 4-h digestions, collagenase II at 2% and 1% but not at lower concentrations yielded maximal release also of the large chondrocyte population. RNA-seq analysis revealed that a 4-h digestion period with 1% or 2% collagenase II plus Actinomycin D optimally preserved the transcriptome. Thus, this study provides an isolation protocol for single chondrocytes from human articular cartilage optimized for transcriptome preservation and RNA-seq analysis.

Chondrocyte Isolation and Expansion.

Chondrocyte isolation requires a combination of enzymatic and mechanical digestion of cartilaginous tissues in order to release the chondrocytes. Extracted primary chondrocytes will then adhere to standard tissue culture plastics, typically in small clusters, over a period of a few days in monolayer culture. Chondrocyte populations are expanded in a basal medium containing serum, supplemented with ascorbic acid, antibiotics, and sometimes antifungal agents and growth factors. Here we describe the standard research grade and good manufacturing practice (GMP) protocols used for the isolation and expansion of chondrocytes by the Oswestry/Keele University Orthopaedic Research (OsKOR) group and John Charnley GMP and MHRA licensed laboratory, both based at the RJAH Orthopaedic Hospital, Oswestry, UK.

Isolation of Murine Articular Chondrocytes for Single-Cell RNA or Bulk RNA Sequencing Analysis.

Single-cell RNA sequencing (scRNA-seq) is highly dependent on cellular composition of a tissue of interest. For soft tissues, isolation of individual cells from the extracellular matrix (ECM) while retaining viability and minimizing degradation within subpopulations is well established. In contrast, articular cartilage is comprised of sparsely positioned chondrocytes embedded within a dense ECM high in glycosaminoglycans, proteoglycans, and many fibrous proteins such as collagens, elastin, fibronectin, and laminins. This densely packed ECM makes it difficult to isolate viable chondrocytes for further single-cell analysis. This protocol highlights a successful technique optimized for isolating chondrocytes from the articulated joints of rodent animal models using a series of enzymatic digestions and chondrocyte enrichment using a double negative selection process through florescence-activated cell sorting (FACS).

Primary growth plate chondrocyte isolation, culture, and characterization from the modern broiler

Lameness is a leading cause of animal welfare and production concerns for the poultry industry as fast-growing, high-yielding broilers seem more susceptible to bone disease and infections. A major limitation to the study of these disorders is the lack of a chicken immortalized chondrocyte cell. Primary cell isolation is a valid and complex method for establishing a relevant in vitro model for diseases. In this study, isolation and high-density culturing of primary chondrocytes form 1-d old chicks was followed by confirmation of cell type, identification of optimal phenotypic expression, and evaluation of cells functionality. mRNA expression, as well as protein production and secretion, of COLI, COLII, Sox9, ACAN, and COLXA1 on day 3 (d3), d7, d11, d14, d18, and d21 in culture showed that avian growth plate chondrocytes under these conditions exhibit optimal phenotypes from d3 to d7. This is evident by a shift from COLII dominant expression in early-culture to COLI dominant expression by late-culture in conjunction with a loss of other chondrocyte markers Sox9, ACAN, and COLXA1. Additionally, morphological changes seen through live cell imaging coincide with the shift of phenotype in mid- to late-culture periods indicating a dedifferentiated phenotype. The functionality of the cultured cells was confirmed using Brefeldin-A treatment which significantly reduced secretion of COLII by d7 chondrocytes. These results provide a foundation for future research utilizing avian primary chondrocytes with optimal phenotypes for disease modeling or passaging.

A Pre-Clinical Animal Study for Zonal Articular Cartilage Regeneration Using Stratified Implantation of Microcarrier Expanded Zonal Chondrocytes

ObjectiveThe zonal properties of articular cartilage critically contribute to the mechanical support and lubrication of the tissue. Current treatments for articular cartilage have yet to regenerate this zonal architecture, thus compromising the functional efficacy of the repaired tissue and leading to tissue degeneration in the long term. In this study, the efficacy of zonal cartilage regeneration through bilayered implantation of expanded autologous zonal chondrocytes was investigated in a porcine chondral defect model.DesignAutologous chondrocytes extracted from articular cartilage in the non-weight bearing trochlea region of the knee were subjected to an expansion-sorting strategy, integrating dynamic microcarrier (dMC) culture, and spiral microchannel size-based zonal chondrocyte separation. Zonal chondrocytes were then implanted as bilayered fibrin hydrogel construct in a porcine knee chondral defect model. Repair efficacy was compared with implantation with cell-free fibrin hydrogel and full thickness (FT) cartilage-derived heterogenous chondrocytes. Cartilage repair was evaluated 6 months after implantation.ResultsSufficient numbers of zonal chondrocytes for implantation were generated from the non-weight bearing cartilage. Six-month repair outcomes showed that bilayered implantation of dMC-expanded zonal chondrocytes resulted in substantial recapitulation of zonal architecture, including chondrocyte arrangement, specific Proteoglycan 4 distribution, and collagen alignment, that was accompanied by healthier underlying subchondral bone.ConclusionThese results demonstrate that with appropriate expansion and isolation of zonal chondrocytes, the strategy of stratified zonal chondrocyte implantation represents a significant advancement to Autologous Chondrocyte Implantation-based cartilage regeneration, with the potential to improve the long-term integrity of the regenerated tissues.

Tiao-bu-fei-shen formula promotes downregulation of the caveolin 1-p38 mapk signaling pathway in COPD - Associated tracheobronchomalacia cell model

Ethnopharmacological relevanceThe Tiao-bu-fei-shen (TBFS) formula, extensively used in Traditional Chinese Medicine (TCM), can enhance therapeutic efficacy and reduce the frequency of acute exacerbations of lung–kidney Qi deficiency in patients with chronic obstructive pulmonary disease (COPD). According to both TCM theory and long-term observation of practice, TBFS has become an effective treatment for COPD-associated tracheobronchomalacia (TBM). Aim of the studyTo investigate the mechanism of the TBFS formula in treating COPD-associated TBM based on caveolin 1–p38 MAPK signaling and apoptosis. Materials and methodsA rat COPD model was prepared by exposure to smoking combined with tracheal lipopolysaccharide injection. The trachea or bronchus chondrocytes from COPD rats were isolated, cultured, and treated with 10 ng/mL IL-1β for 24 h to develop a model of COPD-associated TBM. Normal rats were administered TBFS to prepare drug-containing serum, and CCK8 assays were used to screen the optimal drug-containing serum concentration and SB203580 dose. TBFS drug-containing serum and SB203580 were processed separately for the control, model, drug-containing serum, blocker, and drug-containing serum combined with blocker groups. Flow cytometry and CCK8 assays were used to detect apoptosis and proliferative activity. Toluidine blue staining and immunohistochemistry were used to analyze the chondrocyte proteoglycan and type II collagen content. Western blotting was used to detect the expression of caveolin 1, p-p38 MAPK, TNF-α, IL-1β, MMP-13, Bax, and Bcl-2 proteins. Quantitative PCR was used to detect the expression of caveolin 1, p38 MAPK, IL-1β, MMP-13, Bax, Bcl-2, and miR-140–5p. ResultsThe isolation and identification of bronchial chondrocytes from COPD rats revealed that 10 ng/mL IL-1β can produce a stable COPD-associated TBM model. Screened via the CCK8 method, fourth-generation bronchial chondrocytes were determined as the optimal cells, and 5 μM SB203580 and 5% low-dose drug-containing serum were the optimal intervention doses. The experimental chondrocytes of each group were treated separately for 48 h. Toluidine blue staining and immunohistochemical analysis revealed that TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 can effectively increase the proteoglycan and type II collagen content after chondrocyte degradation. Flow cytometry of cells treated with SB203580 and TBFS drug-containing serum combined with SB203580 revealed significantly reduced cell apoptosis and enhanced cell proliferation activity. Western blot and qPCR analyses revealed that the TBFS drug-containing serum, SB203580, and TBFS drug-containing serum combined with SB203580 effectively inhibit the expression of caveolin 1, p-p38 MAPK, MMP-13, IL-1β, TNF-α, and Bax proteins while promoting Bcl −2 protein expression. Treatment with TBFS drug-containing serum and SB203580 effectively inhibited the expression of MMP-13, p38 MAPK, caveolin 1, and Bax genes, and promoted the expression of Bcl-2 and miR-140–5p genes. ConclusionsA concentration of 10 ng/mL of IL-1β can generate a stable COPD-associated TBM cell model. TBFS can improve the proteoglycan and type II collagen content, increase cell activity, and reduce the amount of chondrocyte apoptosis. The role of TBFS may be related to mechanisms of inhibiting the expression of the key signaling molecules caveolin 1 and p-p38 MAPK in the caveolin 1–p38 MAPK signaling pathway, thereby reducing the expression of the downstream effector products MMP-13, IL-1β, and TNF-α, while inhibiting the expression of the apoptotic gene Bax and improving the expression of Bcl-2 and miR-140–5p genes.

The Endplate Role in Degenerative Disc Disease Research: The Isolation of Human Chondrocytes from Vertebral Endplate-An Optimised Protocol.

Background: Degenerative disc disease is a progressive and chronic disorder with many open questions regarding its pathomorphological mechanisms. In related studies, in vitro organ culture systems are becoming increasingly essential as a replacement option for laboratory animals. Live disc cells are highly appealing to study the possible mechanisms of intervertebral disc (IVD) degeneration. To study the degenerative processes of the endplate chondrocytes in vitro, we established a relatively quick and easy protocol for isolating human chondrocytes from the vertebral endplates. Methods: The fragments of human lumbar endplates following lumbar fusion were collected, cut, ground and partially digested with collagenase I in Advanced DMEM/F12 with 5% foetal bovine serum. The sediment was harvested, and cells were seeded in suspension, supplemented with special media containing high nutrient levels. Morphology was determined with phalloidin staining and the characterisation for collagen I, collagen II and aggrecan with immunostaining. Results: The isolated cells retained viability in appropriate laboratory conditions and proliferated quickly. The confluent culture was obtained after 14 days. Six to 8 h after seeding, attachments were observed, and proliferation of the isolated cells followed after 12 h. The cartilaginous endplate chondrocytes were stable with a viability of up to 95%. Pheno- and geno-typic analysis showed chondrocyte-specific expression, which decreased with passages. Conclusions: The reported cell isolation process is simple, economical and quick, allowing establishment of a viable long-term cell culture. The availability of a vertebral endplate cell model will permit the study of cell properties, biochemical aspects, the potential of therapeutic candidates for the treatment of disc degeneration, and toxicology studies in a well-controlled environment.

Costal cartilage ensures low degradation of DNA needed for genetic identification of human remains retrieved at different decomposition stages and different postmortem intervals

Abstract Introduction The study aimed to evaluate if costal cartilage is a good source of DNA for genetic individual identification tests performed in forensic autopsies. Materials and Methods The study included samples of costal cartilage collected from 80 cadavers retrieved from different environments: indoors (flat/hospital), outdoors (primarily in the forest), a coal mine, a fire site, uninhabited buildings, a basement, bodies of fresh water, exhumation sites, and unknown locations. After isolation of DNA chondrocytes, T. Large autosomal chromosome (214 bp), T. Small autosomal chromosome (80 bp), and the Y chromosome (75 bp; for male cadavers), sequences were amplified using real-time PCR. Additionally, 23 autosomal short tandem repeat (STR) loci and 16 Y chromosome STR loci were amplified using multiplex PCR. Forensic DNA typing was done using capillary electrophoresis and all results were analyzed. Results There was no statistically significant difference in DNA concentration after T. Large, T. Small autosomal chromosome and the Y chromosome amplification between samples collected from cadavers retrieved from different environments. The DNA degradation index was the same regardless of the postmortem interval. The results show that it is possible to generate a full genetic profile from costal cartilage samples collected from cadavers retrieved from different environments and at different times elapsed after death. Conclusions The results suggest that costal cartilage can be routinely collected during forensic autopsies, especially from cadavers at the advanced decomposition stage.

Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering.

The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity. Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

Lower androgen levels promote abnormal cartilage development in female patients with adolescent idiopathic scoliosis

BackgroundAdolescent idiopathic scoliosis (AIS) is a disease characterized by changes in the three-dimensional structure of the spine. Studies have shown that the development of AIS might be associated with genetic, biomechanics, endocrine factors and abnormal bone or cartilage development.MethodsBlood samples collected from 301 female patients (161 females with AIS and 140 females without AIS) were used for genotyping. Forty-eight serum samples from 161 females with AIS and 40 serum samples from 140 females without AIS were subjected to enzyme-linked immunosorbent assays (ELISAs). We also evaluated 32 facet joints (18 females with AIS and 14 females without AIS from the 301 female patients) using immunohistochemistry, Western blotting, and isolation of human primary chondrocytes, among other methods. We treated the AIS primary chondrocytes with dihydrotestosterone (DHT) to verify the relationship among androgen, the androgen receptor (AR), and its downstream pathway proteins.ResultsThe serum androgen level in the AIS group was significantly decreased (1.94±0.09 vs. 2.284±0.103) compared with that in the non-AIS (control) group. The single nucleotide polymorphism genotyping results showed that the mutation rates of rs6259 between the AIS and control groups were significantly different (G/G genotype: 48.4% vs. 42.1%, G/A genotype: 40.4% vs. 35.7%, P<0.05). The levels of interleukin (IL)-6 and metalloproteinase (MMP)-13 were increased in the cartilage of AIS patients, and these patients also exhibited decreased AR levels. The cell experiment results showed that androgen reduced the degree of abnormal cartilage development in female AIS patients through the AR/IL-6/signal transducer and activator of transcription 3 (STAT3) signaling pathway.ConclusionsOur study provides a new perspective on the pathogenesis of AIS and indicates that decreased androgen levels in female AIS patients play a potential role in the development of AIS via the AR/IL-6/STAT3 signaling pathway.

Comparison of articular and auricular cartilages: decellularization, cell proliferation rate, and infiltration in scaffolds

Various treatment approaches are applied to repair damaged cartilage. However, a developing field of tissue engineering holds a realistic promise to replace injured cartilage tissue using the patient’s cells. Using one type of chondrocytes to repair functionally different and far localized cartilage can be feasible from a clinical perspective. Toward this ultimate goal, we aimed to implement key protocols utilized in tissue engineering of articular (AR) and auricular (AU) cartilages. The experiments were performed according to established protocols that include chondrocyte isolation, assessment of the cell proliferation rates, the degree of cell infiltration in three-dimensional scaffolds, and cartilage decellularization efficacy. The data pointed to significant discrepancies in the size and in vitro chondrocytes proliferation rate isolated from distinct types of cartilage, with AR chondrocytes being 55% larger (p < 0.01) while having a slower rate of proliferation. Both collagen- and alginate-based scaffolds showed relevant properties for cell infiltration. Lastly, we have shown that the AR and AU cartilages are decellularized to a different degree (17 ± 5.5% vs. 42 ± 8.5%, p < 0.01) while using the same SDS-based decellularization protocol. This study will contribute to the global efforts to rebuild damaged cartilage with the help of tissue engineering.