Comparison of the efficiency of laminin versus fibronectin as a differential adhesion assay for isolation of human articular cartilage derived chondroprogenitors.

Purpose: Cartilage repair following trauma or degeneration is poor, making cell-based therapy an important avenue of treatment. Chondrocytes and mesenchymal stem cells have been extensively studied as potential candidates, although tendency toward hypertrophy and formation of mixed hyaline-fibrocartilage necessitates further optimization. Chondroprogenitors, isolated using fibronectin adhesion assay are reported to show reduced hypertrophy and enhanced chondrogenesis. Laminin, an essential component of extracellular matrix, has been shown to positively modulate chondrocyte proliferation, migration, and survival. The aim of our study was to evaluate the effect of laminin as a differential adhesion assay and obtain an enriched population of chondroprogenitors and assess its efficiency when compared to progenitors obtained via fibronectin.Materials and methods: Chondrocytes were isolated from three osteoarthritic knee joints and subjected to fibronectin and laminin adhesion to obtain chondroprogenitors. After expansion in culture, they were assessed for differences in their biological characteristics based on growth kinetics, surface marker expression, gene expression for assessing markers of chondrogenesis and hypertrophy, and potential for tri-lineage differentiation.Results: Our results showed that cells isolated by laminin and fibronectin both displayed comparable characteristics except in terms of proliferative potential (higher in laminin), gene expression of COL2A1 (lower in laminin) and trilineage potential where the laminin group showed higher osteogenic and adipogenic differentiation.Conclusion: This was the first attempt to successfully isolate human articular cartilage derived chondroprogenitor clones using laminin, which retained stem cell like characteristics. Further evaluation to optimize this method will help enhance chondroprogenitor characteristics, for use in cartilage repair.

Optimization of immunohistochemical detection of collagen type II in osteochondral sections by comparing decalcification and antigen retrieval agent combinations.

Bone containing tissues such as osteochondral joint are resistant to routine tissue processing, therefore require decalcification. This technique causes removal of mineral salts, but in the process may macerate the organic tissue, hence the need for tissue fixation. Such severe processing demands careful antigen retrieval to necessitate optimal staining. The aim of our study was to compare five different antigen retrieval protocols (heat retrieval and protein digestion) following decalcification of rabbit knee joints using two different techniques (20% formic acid and 10% ethylenediamine-tetra acetic acid: EDTA). Osteochondral sections were compared based on time required for decalcification, ease of sectioning, morphological integrity using HE staining and antigen preservation (Collagen type II) using immunohistochemistry. The two decalcification solutions did not impair the tissue morphology and ease of sectioning. Joints processed with formic acid decalcified four times faster than EDTA. Among the five antigen retrieval approaches, maximal collagen II uptake with minimal nonspecific staining was found with protein digestion (pronase and hyaluronidase) in both formic acid and EDTA sections. For osteo-chondral sections, we recommend using 10% EDTA for decalcification and pronase plus hyaluronidase for antigen retrieval if maintaining tissue morphology is crucial, whereas if time is of the essence, 20% FA with pronase plus hyaluronidase is the faster option while still preserving structural integrity. Clin. Anat. 33:343-349, 2020. © 2019 Wiley Periodicals, Inc.

Red cell indexes made easy using an interactive animation: do students and their scores concur?

A good understanding of red cell indexes can aid medical students in a considerable manner, serving as a basis to unravel both concepts in red cell physiology and abnormalities associated with the same. In this study, we tried to assess whether an interactive animation was helpful in improving student comprehension and understanding of red cell indexes compared with conventional classroom teaching. Eighty-eight first-year MBBS students participated, of which 44 were assigned to group A and 44 were assigned to group B after randomization. After further creation of smaller groups, students were provided with 45 min to revise red cell indexes, after which they were required to complete a multimodal questionnaire. Group A subgroups used written material for revision, whereas group B subgroups had access to an interactive animation. After completion of the questionnaire, group A students also used the animation after which feedback was collected from all students. Efficacy of the animation to improve learning and retention was demonstrated, as group B students scored significantly higher than group A students on the questionnaire ( P = 0.0003). A clear majority of the students agreed/strongly agreed that the animation was easy to operate, conveyed important concepts efficiently, and improved their knowledge of related clinical aspects as well. From the results and feedback, we found that the animation was a simple, well-received model, which, by significantly improving student performance, corroborated our hypothesis that inclusion of interactive animation into student curriculum can advance their academic attainment, compared with didactic teaching alone.

Cardiac Autonomic Activity, Personality Traits, and Academic Performance in First-Year Medical Students: A Gender-Specific Relation.

Background It is not always the sincere or hardworking or intelligent student that gets the highest grades. Exploring unknown dimensions that may distinguish academic performance in adolescents/youth migrating from a high school study environment to that of a professional school and in a learning environment without parental supervision for the first time remains important. We hypothesized that cardiac autonomic activity influenced by cognitive domain factors and emotions would predict academic success in them. Further, we investigated which of their personality traits related to academic performance. Exploratory gender-based analysis was included. Methods A prospective cohort study measured first-year medical students' resting heart rate, heart rate variability (HRV), and personality traits (from the self-reported NEO Five-Factor Inventory-3). Spearman's correlation coefficient tested the correlation between the year-end final aggregate marks and assessed parameters, including subgroup analysis based on gender. Regression analyses of variables with academic marks were performed in the entire cohort. Results The aggregate marks of 81 volunteering students (Mage = 18.7, SD = 0.8 years; 42 females, 39 males) as a cohort did not correlate with their resting heart rate or HRV indices. Subgroup analysis revealed a positive correlation between marks and high-frequency power (r = 0.33, p= 0.03) and total power (r = 0.37, p= 0.02) of HRV in females. The marks positively correlated with the personality conscientiousness score (r = 0.32, p= 0.04) and extraversion score (r = 0.34, p= 0.03) in females. Multivariable regression analysis in the entire cohort revealed no significant interactions. Conclusion Academic performance was significantly related to cardiac autonomic modulation and personality traits of conscientiousness and extraversion in female but not male first-year medical students. These results indicate a gender-specific difference in the relation between scholastic performance and HRV in adolescents/youth transiting from high school to professional schools and entering a study environment without parental supervision for the first time. Further our data expands the knowledge base of educational psychology among them.

Pondering the Potential of Hyaline Cartilage-Derived Chondroprogenitors for Tissue Regeneration: A Systematic Review.

OBJECTIVE: Chondroprogenitors have recently gained prominence due to promising results seen in in vitro and animal studies as a potential contender in cell-based therapy for cartilage repair. Lack of consensus regarding nomenclature, isolation techniques, and expansion protocols create substantial limitations for translational research, especially given the absence of distinct markers of identification. The objective of this systematic review was to identify and collate information pertaining to hyaline cartilage-derived chondroprogenitors, with regard to their isolation, culture, and outcome measures. DESIGN: As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a web-based search of Scopus and PubMed databases was performed from January 2000 to May 2020, which yielded 509 studies. A total of 65 studies were identified that met the standardized inclusion criteria which comprised of, but was not limited to, progenitors derived from fibronectin adhesion, migrated subpopulation from explant cultures, and single-cell sorting. RESULT: Literature search revealed that progenitors demonstrated inherent chondrogenesis and minimal tendency for hypertrophy. Multiple sources also demonstrated significantly better outcomes that bone marrow-derived mesenchymal stem cells and comparable results to chondrocytes. With regard to progenitor subgroups, collated evidence points to better and consistent outcomes with the use of migratory progenitors when compared to fibronectin adhesion assay-derived progenitors, although a direct comparison between the two cell populations is warranted. CONCLUSION: Since chondroprogenitors exhibit favorable properties for cartilage repair, efficient characterization of progenitors is imperative, to complete their phenotypic profile, so as to optimize their use in translational research for neocartilage formation.

An assessment of bone marrow mesenchymal stem cell and human articular cartilage derived chondroprogenitor cocultures vs. monocultures.

BACKGROUND: Cell based therapy in cartilage repair predominantly involves the use of chondrocytes and mesenchymal stromal cells (MSC). Co-culture systems, due to their probable synergistic effect on enhancement of functional chondrogenesis and reduction in terminal differentiation have also been attempted. Chondroprogenitors, derived from articular cartilage and regarded as MSCs, have recently garnered interest for consideration in cartilage regeneration to overcome limitations associated with use of conventional cell types. The aim of this study was to assess whetherco-culturing bone marrow (BM)-MSCs and chondroprogenitors at different ratios would yield superior results in terms of surface marker expression, gene expression and chondrogenic potential. METHODS: Human BM-MSCs and chondroprogenitors obtained from three osteoarthritic knee joints and subjected to monolayer expansion and pellet cultures (10,000 cells/cm2) as five test groups containing either monocultures or co-cultures (MSC: chondroprogenitors) at three different ratios (75:25, 50:50 and 25:75) were utilized. RESULTS: Data analysis revealed that all groups exhibited a high expression of CD166, CD29 and CD49e. With regard to gene expression, high expression of SOX9, Aggrecan and Collagen type I; a moderate expression of Collagen type X and RUNX2; with a low expression of Collagen type II was seen. Analysis of pellet culture revealed that chondroprogenitor monoculture and chondroprogenitor dominant coculture, exhibited a subjectively larger pellet size with higher deposition of Collagen type II and glycosaminoglycan. CONCLUSION: In conclusion, this study is suggestive of chondroprogenitor monoculture superiority over MSCs, either in isolation or in a coculture system and proposes further analysis of chondroprogenitors for cartilage repair.

Comparative analysis of human bone marrow mesenchymal stem cells, articular cartilage derived chondroprogenitors and chondrocytes to determine cell superiority for cartilage regeneration.

INTRODUCTION: Chondroprogenitors, a promising therapeutic modality in cell-based therapy, are routinely isolated from articular cartilage by fibronectin differential adhesion assay. However, there is paucity of information regarding their biological profile and the lack of a marker that can reliably distinguish them from cultured chondrocytes due to possible dedifferentiation. Since chondroprogenitors have been classified as mesenchymal stem cells(MSCs), the aim of our study was to compare bone marrow-MSCs, chondroprogenitors and chondrocytes, and assess superiority for cartilage repair. An additional objective was to also compare CD49b as a differentiating marker for isolating chondroprogenitors as a recent report demonstrated significantly high expression in the surfaceome of migratory articular chondroprogenitors. METHODS: Bone marrow aspirate and articular cartilage was obtained from three osteoarthritic knee joints. Study arms included a) bone marrow-MSCs, b) chondroprogenitors, c) cultured chondrocytes, d) chondrocytes cultured with additional growth factors and e) CD49b + sorted chondroprogenitors. Assessment parameters included population doubling, surface expression for positive, negative MSC markers and potential markers of chondrogenesis (CD29, CD49e, CD49b, CD166 and CD146), RT-PCR for markers of chondrogenesis and hypertrophy and trilineage differentiation. RESULTS AND CONCLUSION: Chondroprogenitors exhibited efficient chondrogenesis (SOX-9 and COL2A1) and significantly lower tendency for hypertrophy (RUNX2), which was also reflected in trilineage differentiation where progenitors displayed minimal calcified matrix, efficient glycosaminoglycan deposition and high collagen type II uptake. CD49b did not serve as a marker for isolation as sorted chondroprogenitors performed significantly poorer when compared to fibronectin assay derived cells. Emphasis on preclinical studies utilizing progenitors of higher purity is the future direction.

In vitro chondrogenic differentiation of human articular cartilage derived chondroprogenitors using pulsed electromagnetic field.

BACKGROUND: The ability to grow new cartilage remains the standard goal of any treatment strategy directed at cartilage repair. Chondroprogenitors have garnered interest due to their applicability in cell therapy. Pulsed electromagnetic field (PEMF) favors chondrogenesis by possible upregulation of genes belonging to TGFß superfamily. Since TGFß is implicated in chondrogenic signalling, the aim of the study was to evaluate the ability of PEMF to induce chondrogenesis via endogenous TGFß production in chondroprogenitors vs differentiation using chondrogenic medium inclusive of TGFß. METHODS: Chondroprogenitors were harvested from three non-diseased human knee joints via fibronectin assay. Passage 3 pellets were subjected to four different culture conditions: a) negative control contained chondrogenic medium without TGFß2, b) positive control contained medium with TGFß2, c) PEMF 1 contained medium of negative control plus single exposure to PEMF and d) PEMF 2 contained medium of negative control plus multiple exposures to PEMF. Following differentiation (day 21), pellets were assessed for gene expression of ACAN, SOX9, COL2A1, TGFß1, TGFß2, and TGFß3. Alcian blue staining to detect glycosaminoglycan deposition was also performed. Medium supernatant was used to detect endogenous latent TGF-ß1 levels using ELISA. RESULTS: All study arms exhibited comparable gene expression without any significant difference. Although positive control and PEMF study arms demonstrated notably better staining than negative control, the level of latent TGF-ß1 was seen to be significantly high in supernatant from positive control (P < 0.05) when compared to other groups. CONCLUSION: Our results indicate that PEMF induced chondrogenesis might involve other signalling molecules, which require further evaluation.

An in vitro analysis of the effect of hyperosmolarity on the chondrogenic potential of human articular cartilage derived chondroprogenitors.

PURPOSE: Chondroprogenitors display promise for articular cartilage regeneration. It is imperative to standardize culture conditions, to further enhance chondrogenicity and reduce tendency for hypertrophy. Cartilage matrix provides a unique hyperosmolar microenvironment that enables native cells to resist compressive stress. However, commonly used culture media have osmolarities relatively hypoosmotic when compared to in-vivo conditions. Previous reports involving chondrocytes demonstrated enhanced chondrogenic potential secondary to utilization of hyperosmolar culture conditions. The study aimed to assess the effect of hyperosmolarity (either mimicking normal joint conditions or short-term hyperosmotic stress) on chondroprogenitor phenotype. MATERIALS AND METHODS: Fibronectin adhesion assay derived human articular chondroprogenitors (n = 3) were divided into 3 groups: a) Control: cells grown in standard culture conditions (320 mOsm/L), b) Test A: cells grown in hyperosmolar media mimicking joint conditions (409 mOsm/L) and c) Test B: cells exposed to short-term hyperosmotic stress (504 mOsm/L) for 24 h, prior to assessment. Evaluation parameters included population doubling, cell size, surface marker expression, mRNA expression (markers of chondrogenesis, dedifferentiation and hypertrophy) and multilineage potential. RESULTS: Subjecting these cells to increased osmolarity in culture did not demonstrably favor chondrogenesis (control vs Test A: comparable COL2A1) while hyperosmotic stress further increased the tendency for hypertrophy and terminal differentiation (high COL1A1 and low COL2A1, P = 0.006). Additionally, growth kinetics, surface marker expression and multilineage potential were comparable across groups. CONCLUSION: Chondroprogenitors displayed sensitivity to increase in osmolarity as chondrogenic phenotype did not improve, while hypertrophic propensity was heightened, although further analysis of culture and phenotypic parameters will aid in optimizing chondroprogenitor use in cartilage regeneration.

Articular chondroprogenitors in platelet rich plasma for treatment of osteoarthritis and osteochondral defects in a rabbit knee model.

BACKGROUND: Articular chondroprogenitors are a promising contender for cartilage repair due to their inherent nature which stands primed for chondrogenesis and minimal hypertrophic preponderance. Platelet rich plasma (PRP) has been extensively used for treating cartilage defects and osteoarthritis (OA), due to its chondro-inductive properties and abundant pool of growth factors. The aim of this study was to assess the efficacy of chondroprogenitors injected with PRP versus PRP alone in the healing of experimentally created early OA and osteochondral defects (OCD) in a rabbit model. METHODS: Adult New Zealand White male rabbits were used for cell and PRP isolation. Chondroprogenitors were isolated by fibronectin adhesion assay, labelled with iron oxide, characterized for surface markers, differential potential and expanded. PRP was isolated by double spin centrifugation using a TriCell kit. Study groups included (a) Monosodium iodoacetate induced early OA and (b) critical OCD. Following intervention (test arm: PRP+ chondroprogenitors and control arm: PRP), assessment was performed at 6- and 12-weeks which included histopathological examination and scoring (OARSI and Modified Wakitani score), immunohistochemistry analysis (Collagen type II and X) and synovial fluid S100A12 levels. RESULTS AND CONCLUSION: Comparable, evident healing was noticed in both test and control arms when the OA group samples were assessed at both time points. In the OCD group, PRP alone exhibited significantly better results than the test arm, although repair was notable in both interventions. Further evaluation of chondroprogenitors is required to assess their role as a standalone therapy and in combination with PRP to further cartilage regeneration.

Comparison of Human Platelet Lysate versus Fetal Bovine Serum for Expansion of Human Articular Cartilage-Derived Chondroprogenitors.

PURPOSE: Articular chondroprogenitors, a suitable contender for cell-based therapy in cartilage repair, routinely employ fetal bovine serum (FBS) for expansion and differentiation. The possibility of transplant rejections or zoonoses transmissions raise a need for xeno-free alternatives. Use of human platelet lysate (hPL), a nutrient supplement abundant in growth factors, has not been reported for human chondroprogenitor expansion thus far. Our aim was to compare the biological profile of chondroprogenitors grown in hPL versus FBS. METHODS: Chondroprogenitors were isolated from 3 osteoarthritic knee joints. Following differential fibronectin adhesion assay, passage 0 cells grown in (a) 10% FBS and (b) 10% hPL were considered for assessment of growth kinetics, surface marker expression, gene expression, and trilineage differentiation. Latent transforming growth factor-ß1 (TGFß1) levels were also measured for each culture medium used. RESULTS: Cellular proliferation was significantly higher in cells grown with hPL (P < 0.01). Surface marker expression was comparable except in CD-146 where hPL group had significantly higher values (P = 0.03). Comparison of mRNA expression revealed notably low values of collagen I, collagen X, aggrecan, and collagen II (P < 0.05). Trilineage differentiation was seen in both groups with higher alizarin red uptake noted in hPL. There were also significantly higher levels of latent TGFß1 in the medium containing hPL as compared to FBS. CONCLUSIONS: This is the first in vitro xeno-free study to affirm that hPL can serve as an optimal growth supplement for expansion of articular chondroprogenitors, although an in-depth assessment of resident growth factors and evaluation of different dilutions of hPL is required to assess suitability for use in translational research.

Prospective Isolation and Characterization of Chondroprogenitors from Human Chondrocytes Based on CD166/CD34/CD146 Surface Markers.

PURPOSE: Chondrocytes, isolated from articular cartilage, are routinely utilized in cell-based therapeutics for the treatment of cartilage pathologies. However, restoration of the biological tissue faces hindrance due to the formation of primarily fibrocartilaginous repair tissue. Chondroprogenitors have been reported to display superiority in terms of their chondrogenic potential and lesser proclivity for hypertrophy. In line with our recent results, comparing chondroprogenitors and chondrocytes, we undertook isolation of progenitors from the general pool of chondrocytes, based on surface marker expression, namely, CD166, CD34, and CD146, to eliminate off-target differentiation and generate cells of stronger chondrogenic potential. This study aimed to compare chondrocytes, chondroprogenitors, CD34-CD166+CD146+ sorted chondrocytes, and CD34-CD166+CD146- sorted chondrocytes. METHODS: Chondrocytes obtained from 3 human osteoarthritic knee joints were subjected to sorting, to isolate CD166+ and CD34- subsets, and then were further sorted to obtain CD146+ and CD146- cells. Chondrocytes and fibronectin adhesion-derived chondroprogenitors served as controls. Assessment parameters included reverse transcriptase polymerase chain reaction for markers of chondrogenesis and hypertrophy, trilineage differentiation, and total GAG/DNA content. RESULTS: Based on gene expression analysis, CD34-CD166+CD146+ sorted chondrocytes and chondroprogenitors displayed comparability and significantly higher chondrogenesis with a lower tendency for hypertrophy when compared to chondrocytes and CD34-CD166+CD146- sorted chondrocytes. The findings were also reiterated in multilineage potential differentiation with the 146+ subset and chondroprogenitors displaying lower calcification and chondroprogenitors displaying higher total GAG/DNA content compared to chondrocytes and 146- cells. CONCLUSION: This unique progenitor-like population based on CD34-CD166+CD146+ sorting from chondrocytes exhibits efficient potential for cartilage repair and merits further evaluation for its therapeutic application.

Comparative analysis of gene expression between articular cartilage-derived cells to assess suitability of fibronectin adhesion assay to enrich chondroprogenitors.

BACKGROUND: Enhanced chondrogenesis and reduction in hypertrophy are essential pre-requisites for cell-based therapy in regenerative research for cartilage loss. Chondroprogenitors, isolated by fibronectin adhesion assay (FAA), have shown promising results in various preclinical studies due to their inherent characteristics. However, the need for monolayer culture and the effect of expansion on cell phenotype render differentiation between chondroprogenitors and chondrocytes (native cartilage cells) difficult. This is further complicated due to reported de-differentiation of chondrocytes in culture. Thus, the aim of our study was to harvest cells from articular cartilage and compare their gene expression to cells demonstrating adherence and non-adherence to fibronectin. METHOD: Fresh-cells (FC) were isolated from human osteoarthritic knee joints(n = 3) and subjected to FAA. Cells unbound to fibronectin (20 min after plating) were termed as FAA-ve. Attached cells were further cultured for five population doublings and designated FAA+ve. RNA from all three cell groups was assessed for SOX-9, ACAN, COL2A1, COL1A1, RUNX2 and COL10A1. RESULTS: All three groups exhibited moderate to high expression of markers of chondrogenesis and marker of chondrocyte hypertrophy. FAA+ve group exhibited significantly lower levels of hypertrophy markers: RUNX2 (vs FC and FAA-ve, P = 0.018) and COL10A1(vs FAA-ve, P = 0.005). CONCLUSIONS: Our results demonstrated that fibronectin effectively isolated cells distinct from mature chondrocytes in terms of reduced hypertrophic tendency. This is noteworthy as cells isolated by FAA, retaining their inherent progenitor phenotype, with upregulation of chondrogenic markers may be used successfully for cartilage repair in future translational work.

Evaluation of CD49e as a distinguishing marker for human articular cartilage derived chondroprogenitors.

BACKGROUND: Cell-based therapy in cartilage repair can benefit from the use of chondroprogenitors; a cell type classified as mesenchymal stem cells, demonstrating reduced hypertrophy. Fibronectin, routinely used to isolate chondroprogenitors, classically binds to α5ß1 integrins (CD49e + CD29), of which CD49e is said to be highly expressed in progenitors. The aim of our study was to assess the specificity of CD49e as a distinguishing marker for chondroprogenitors; because studies report low expression in fresh chondrocytes (FCs), but recent conflicting data has exhibited incremental expression of CD49e in cultured chondrocytes. METHODS: FCs were isolated from three human osteoarthritic knee joints and CD49e- cells (sorted by flow cytometry) were cultured in adherent and non-adherent conditions and reassessed for CD49e and CD29 at multiple time points. Colony-forming efficiency (CFE) following fibronectin adhesion assay was calculated for FC, CD49e+ and CD49e- cells. RESULTS: A statistically significant increase in CD49e and CD29 expression was seen in both adherent and non-adherent cultures of CD49e- cells (P < 0.01), as early as 24 h. All groups grew clonally and CFE was similar without any significant difference. CD49e- chondrocytes turned positive when cultured, possibly due to an inherent phenotypic drift, seen after release from cartilage and not because of plastic adherence or chondroprogenitor overgrowth, as non-adherent cultures also showed high expression. CONCLUSIONS: As the specificity of CD49e is questionable, there is a pressing need for a specific differentiating marker, to isolate a pure population of chondroprogenitors, as this cell type shows inherent chondrogenesis and reduced hypertrophy, both requisites for cartilage repair.

Comparative analysis of fresh chondrocytes, cultured chondrocytes and chondroprogenitors derived from human articular cartilage.

INTRODUCTION: Interest in chondroprogenitors arose due to their inherent stem cell like properties, and their initial characterization was based on identification of a small percentage of CD49e positive cells in cultured chondrocytes (CC). It was further noted that when fresh chondrocytes (FC; reported to express low CD49e) were subjected to fibronectin adhesion assay, an isolate of chondroprogenitors was obtained, which was highly positive for CD49e, thus making it a distinguishing marker for this cell population. However, this notion was challenged when reports demonstrated high CD49e expression in CC as well. Therefore, our aim was to compare CD49e expression in FC, CC and chondroprogenitors. METHODS: Chondrocytes and chondroprogenitors were isolated from articular cartilage of osteoarthritic joints from three patients. Assessment of classic fibronectin receptor (CD49e, CD29), positive (CD105, CD73, CD90) and negative (CD45, CD34) mesenchymal stem cell marker expression in all groups was performed, as chondroprogenitors fulfill the minimal criteria laid down by International Society for Cellular Therapy. Following this, adipogenic, osteogenic and chondrogenic differentiation was assessed by Oil red O, Alizarin Red and Alcian Blue staining respectively. RESULTS AND CONCLUSION: Our observations indicate that FC show significantly low surface marker expression as compared to CC and chondroprogenitors, whereas no significant difference was seen in values when CC and chondroprogenitors were compared. Moreover, comparable results were exhibited when trilineage differentiation potential was compared across groups. Since CC and chondroprogenitors show similar characteristics, there is a pressing need for a specific differentiating marker to isolate a pure population of chondroprogenitors.

Comparison of Electrophysiological Properties and Gene Expression between Human Chondrocytes and Chondroprogenitors Derived from Normal and Osteoarthritic Cartilage.

OBJECTIVES: Bone-marrow mesenchymal stem cells (MSCs) and chondrocytes are currently used for cell-based therapy in cartilage repair. Chondroprogenitors (CPs), resident cells of articular cartilage, demonstrate likeness to stem cells. Reports suggest that chondrocytes phenotype is altered in culture, thus making differentiation between the two cell populations difficult. Our objectives were to electrophysiologically assess chondrocytes and CPs, compare their mRNA expression with that of ionic channels already reported in MSCs, and to observe the effect of time in culture and osteoarthritic damage on cells. DESIGN AND RESULTS: Chondrocytes and CPs at passages 0 (p0) and 5 (p5) derived from normal and osteoarthritic (OA) knee joints were used. Ionic currents were recorded by subjecting cells to depolarizing voltage pulses, and reverse transcriptase-polymerase chain reaction (RT-PCR) was used for studying ion channel expression. Our results demonstrated that both chondrocytes and CPs showed the presence of similar currents belonging to voltage-gated potassium channel subfamily, with RT-PCR confirming high mRNA expression of Maxi K, HKv1.1, HKv1.4, HKv4.2, and hEAG1 channels. Our finding also suggested that CPs were comparatively more sensitive to increased time in culture and inflammatory processes as observed in OA, as was evidenced by the significant decrease in mean current density (p0 normal CP: 183.171 ± 50.80 pA/pF; p5 normal CP: 50.225 ± 17.63 pA/pF; P = 0.0280) and significant increase in cellular size (p0 normal CP: 21.564 ± 2.98 pF; p0 OA CP: 37.939 ± 3.55 pF; P = 0.0057). CONCLUSION: Both cell types appear to be optimal candidates for cell-based therapy although initial seeding density, cell source (normal vs. OA), and time in culture are matters of concern, prior to cell-type selection.

Characterization of human articular chondrocytes and chondroprogenitors derived from non-diseased and osteoarthritic knee joints to assess superiority for cell-based therapy.

PURPOSE: Cell based therapy is constantly underway since regeneration of genuine hyaline cartilage is under par. Much attention has been afforded to chondroprogenitors recently, as an alternative cell substitute for cartilage repair. Although single source derivation of chondrocytes and chondroprogenitors is advantageous, lack of a characteristic differentiating marker obscures clear identification, which is essential to create a biological profile and is also required to assess cell type superiority for cartilage repair. METHODS: Cells obtained from three non-diseased/osteoarthritic human knee joints each, were expanded in culture up to passage 10. Characterization studies were performed using flow cytometry; gene expression was studied using RT-PCR; growth kinetics and tri-lineage differentiation was also studied to construct a better profile of chondroprogenitors as well as chondrocytes. RESULTS AND CONCLUSION: Our results showed that both cell populations exhibited similar cell surface characteristics except for non-diseased chondroprogenitors, which showed markedly low expression of CD34 and high expression of CD166. Trilineage data was suggestive of multilineage potential for both cell types with chondroprogenitors showing notably higher glycosaminoglycan and lower calcified matrix deposition. Data acquired from this study aided in describing cellular behavior of human articular cartilage derived chondroprogenitors in conditions not reported earlier. Our comparative analysis suggests that sorting based on a combination of markers (CD34- and CD166+) would yield a population of cells with minimal contamination by chondrocytes, which may provide translatable results in terms of enhanced chondrogenesis and reduced hypertrophy; both indispensable for the field of cartilage regeneration.

Correlation between synovial fluid calcium containing crystal estimation and varying grades of osteoarthritis created using a rabbit model: Potential diagnostic tool.

OBJECTIVES: Accurate diagnosis of osteoarthritis (OA) is the first important step in ensuring appropriate management of the disease. A multitude of tests involving assessment of biomarkers help in assessment of severity and grading of osteoarthritic damage. However, most tests are time consuming and are limited by the paucity in synovial fluid volume. In majority of OA effusions, calcium containing crystals are found. The aim of our study was to evaluate whether a correlation existed between the amount of calcium containing crystals present in synovial fluid and severity scoring of OA to propose a quick and inexpensive technique for disease assessment. MATERIALS AND METHODS: Monosodium-iodoacetate was used to induce high- and low-grade knee OA in adult New Zealand white rabbits (n = 6 joint each group). At 16 weeks, synovial fluid and joints were harvested for histopathological analysis. OA grading was established based on OARSI scoring. Synovial fluid calcium crystal count was assessed by light microscopy (Alizarin red) and confirmed by Fluo-4, AM imaging and polarized microscopy. Statistical analysis was performed using unpaired Student t-test and Pearson correlation. RESULTS AND CONCLUSION: The clumps counted in low-grade OA were significantly lower than high-grade OA, in addition to showing a positive correlation (coefficient: 0.65; P=0.021) between calcium crystal count and the grade of OA created. Fluo-4, AM staining, and polarized microscopy were indicative of calcium pyrophosphate dihydrate crystals. This is the first study to suggest that Alizarin red could serve as an effective and rapid, bed-side method for screening and assessing disease progression.

Comparison of human articular chondrocyte and chondroprogenitor cocultures and monocultures: To assess chondrogenic potential and markers of hypertrophy.

BACKGROUND AND OBJECTIVE: In the field of cartilage repair, use of two or more cell populations such as mesenchymal stem cells with chondrocytes in an in-vitro co-culture synergistic environment has been attempted to evade limitations of monoculture systems and promote/induce chondrogenesis. Articular cartilage-derived chondroprogenitors (CPs), considered to have stem-cell like characteristics have been proposed as a potential contender for neocartilage development. Our objective was to assess whether co-cultures using different ratios of chondrocytes(C) and CPs would demonstrate better results in terms of growth kinetics, surface marker expression, chondrogenic potential, tendency for hypertrophy and glycosaminoglycan deposition than monocultures. STUDY DESIGN: Human chondrocytes and CPs (fibronectin adhesion assay) from the same cartilage source were isolated. Passage 2 cells were subjected to monolayer/pellet cultures and were grown as monocultures and cocultures at the following percentage ratios(C:CP) 80:20, 65:35, 50:50, 35:65 and 20:80. RESULTS: Analysis of data acquired from population doubling, flow cytometry, RT-PCR and Safranin O uptake demonstrated similar results in all monoculture and co-culture groups with no significant inter-group variation, even when reported specific markers of identification (CD54 and CD44:chondrocyte markers) and isolation (CD29 and CD49e: forming heterodimeric fibronectin receptor for CP sorting) were examined. CONCLUSION: In conclusion, this study suggests the need for improved sorting techniques based on a characteristic differentiating biomarker for selection of cells which are true representatives of CPs possessing properties of enhanced chondrogenesis and reduced hypertrophy.

Comparison of incremental concentrations of micron-sized superparamagnetic iron oxide for labelling articular cartilage derived chondroprogenitors.

INTRODUCTION: In vivo tracking of labelled cells can provide valuable information about cellular behavior in the microenvironment, migration and contribution of transplanted cells toward tissue regeneration. Articular cartilage derived chondroprogenitors (CPs) show promise as a candidate for cell-based therapy as they have been classified as mesenchymal stem cells with inherent chondrogenic potential. Iron oxide labelling is known to withstand harsh processing techniques known to be associated with staining of osteochondral specimens. AIM AND METHODS: The aim of our study was to investigate the feasibility of labelling CPs with micron-sized super paramagnetic iron oxide (M-SPIO) particles and to study the effects of this approach on the labelling efficiency, viability, maintenance of phenotype and potential for differentiation. Human CPs were isolated using fibronectin adhesion assay, passage 2 cells were labelled using three concentrations of M-SPIO (12.75 µg/ml, 25.5 µg/ml and 38.25 µg/ml). At sub confluence, cells were assessed for a) iron uptake by Prussian blue stain and colorimetry b) viability using 7-amino actinomycin D, c) MSC marker expression by flow cytometric analysis and d) trilineage differentiation potential. RESULTS AND CONCLUSION: Iron uptake was higher with increase in M-SPIO concentration whereas CD73, CD90 marker expression significantly decreased and chondrogenic potential appreciably reduced with increase in M-SPIO concentration. In conclusion, 12.75 µg/ml M-SPIO can successfully label human articular cartilage derived chondroprogenitors with minimal effect on cellular viability, MSC marker expression and potential for differentiation.