Comparison of methods for the isolation and culture of Migratory chondroprogenitors from Human articular cartilage.

PURPOSE: Resident articular stem cells isolated using a migratory assay called Migratory Chondroprogenitors (MCPs) have emerged as a promising cellular therapeutic for the treatment of cartilage pathologies. In-vivo studies using MCPs report their superiority over bone-marrow mesenchymal stem cells and chondrocytes for treating chondral defects. However, there is no consensus on their isolation protocol. This study aimed to compare four reported isolation methods of MCPs and identify the optimal and feasible protocol for future translational work. METHODS: Human MCPs isolated from osteoarthritic cartilage (n = 3) were divided into four groups: a) MCP1: 8-15 mm cartilage explants, b) MCP2: 8-10 mm explants digested in 0.1% collagenase for 2 hrs. and cultured c) MCP3: 1 mm cartilage explants and d) MCP 4: 25 mm explants with a X tear, 7-day culture, and trypsinization to release migrated cells. The MCPs were subjected to the following analysis: growth kinetics, surface marker expression, mRNA gene expression for markers of chondrogenesis and hypertrophy, and trilineage differentiation. RESULTS: MCPs isolated via the four methods showed similar surface marker profiles, chondrogenic (SOX-9, ACAN, COL2A1) and hypertrophic (COL1, RUNX2) gene expression. The migration time for the MCP3 group was the longest. The MCP1, MCP2, and MCP4 groups produced MCPs with comparable cellular expansion feasibility. CONCLUSIONS: MCPs can be preferably isolated by the any of the three above methods based on the investigator's discretion. In the case of small cartilage samples similar to the MCP3 group, the isolation of MCP is plausible, keeping in mind the additional time required.

Supplementation of articular cartilage-derived chondroprogenitors with bone morphogenic protein-9 enhances chondrogenesis without affecting hypertrophy.

INTRODUCTION: Chondroprogenitors (CPCs) have emerged as a promising cellular therapy for cartilage-related pathologies due to their inherent primed chondrogenic potential. Studies report that the addition of growth factors such as parathyroid hormone (PTH) and Bone Morphogenic Protein (BMP) enhance the chondroinducive potential in chondrocytes and mesenchymal stem cells. This study evaluated if supplementation of the standard culture medium for cell expansion with 1-34 PTH and BMP-9 would enhance the chondrogenic potential of CPCs and reduce their hypertrophic tendency. METHODS: Human chondrocytes were isolated from patients undergoing total knee replacement for osteoarthritis (n = 3). Following fibronectin adhesion assay, passage 1 CPCs were divided and further expanded under three culture conditions (a) control, i.e., cells continued under standard culture conditions, (b) 1-34 PTH group, additional intermittent 6 h exposure with 1-34 PTH and (c) BMP-9 group, additional BMP-9 during culture expansion. All the groups were evaluated for population-doubling, cell cycle analysis, surface marker and gene expression for chondrogenesis, hypertrophy, multilineage differentiation and GAG (glycosaminoglycan)/DNA following chondrogenic differentiation. RESULTS: Concerning growth kinetics, the BMP-9 group exhibited a significantly lower S-phase and population-doubling when compared to the other two groups. Qualitative analysis for chondrogenic potential (Alcian blue, Safranin O staining and Toluidine blue for GAG) revealed that the BMP-9 group exhibited the highest uptake. The BMP-9 group also showed significantly higher COL2A1 expression than the control group, with no change in the hypertrophy marker expression. CONCLUSION: BMP-9 can potentially be used as an additive for CPCs expansion, to enhance their chondrogenic potential without affecting their low hypertrophic tendency. The mitigating effects of 1-34PTH on hypertrophy would benefit further investigation when used in combination with BMP-9 to enhance chondrogenesis whilst reducing hypertrophy.

Similar regeneration of articular cartilage defects with autologous & allogenic chondrocytes in a rabbit model.

Background & objectives: Articular cartilage defects in the knee have a very poor capacity for repair due to avascularity. Autologous chondrocyte transplantation (ACT) is an established treatment for articular cartilage defects. Animal studies have shown promising results with allogenic chondrocyte transplantation since articular cartilage is non-immunogenic. In addition to being economical, allogenic transplantation has less morbidity compared to ACT. This study was undertaken to compare ACT with allogenic chondrocyte transplantation in the treatment of experimentally created articular cartilage defects in rabbit knee joints. Methods: Cartilage was harvested from the left knee joints of six New Zealand white rabbits (R1-R6). The harvested chondrocytes were cultured to confluence and transplanted onto a 3.5 mm chondral defect in the right knees of 12 rabbits [autologous in 6 rabbits (R1-R6) and allogenic in 6 rabbits (R7-R12)]. After 12 wk, the rabbits were euthanized and histological evaluation of the right knee joints were done with hematoxylin and eosin and safranin O staining. Quality of the repair tissue was assessed by the modified Wakitani histological grading scale. Results: Both autologous and allogenic chondrocyte transplantation resulted in the regeneration of hyaline/mixed hyaline cartilage. The total histological scores between the two groups showed no significant difference. Interpretation & conclusions: Allogenic chondrocyte transplantation seems to be as effective as ACT in cartilage regeneration, with the added advantages of increased cell availability and reduced morbidity of a single surgery.

Self-directed learning readiness of Indian medical students: a mixed method study.

BACKGROUND: Self-directed learning (SDL) is defined as learning on one's own initiative, with the learner having primary responsibility for planning, implementing, and evaluating the effort. Medical education institutions promote SDL, since physicians need to be self-directed learners to maintain lifelong learning in the ever-changing world of medicine and to obtain essential knowledge for professional growth. The purpose of the study was to measure the self-directed learning readiness of medical students across the training years, to determine the perceptions of students and faculty on factors that promote and deter SDL and to identify the role of culture and curriculum on SDL at the Christian Medical College, Vellore, India. METHODS: Guglielmino's SDL Readiness Scale (SDLRS) was administered in 2015 to six student cohorts (452 students) at admission, end of 1st, 2nd, 3rd and 4th year of training, and at the beginning of internship in the undergraduate medicine (MBBS) program. Analysis of variance (ANOVA) was used to compare SDL scores between years of training. 5 student focus groups and 7 interviews with instructors captured perceptions of self-direction. Transcripts were coded and analyzed thematically. RESULTS: The overall mean SDLRS score was 212.91. There was no significant effect of gender and age on SDLR scores. There was a significant drop in SDLRS scores on comparing students at admission with students at subsequent years of training. Qualitative analysis showed the prominent role of culture and curriculum on SDL readiness. CONCLUSIONS: Given the importance of SDL in medicine, the current curriculum may require an increase in learning activities that promote SDL. Strategies to change the learning environment that facilitates SDL have to be considered.

An ultrastructural report of human articular cartilage resident cells in correlation with their phenotypic characteristics.

The recent discovery of progenitors based on their differential fibronectin-adhesion (FAA-CPs) and migratory-based (MCPs) assay has evoked interest due to their superiority in terms of their efficient chondrogenesis and reduced hypertrophic propensity. This study aims to isolate and enrich three articular cartilage subsets, chondrocytes, FAA-CPs, and MCPs, and compare their undifferentiated and chondrogenic differentiated status, using in-vitro phenotypical characterization in correlation with ultrastructural analysis using Transmission Electron Microscopy (TEM). Following informed consent, cartilage shavings were procured from a non-diseased human ankle joint and cultured to obtain the three subsets. Chondrocytes exhibited higher CD106 and lower CD49b and CD146 levels. Following chondrogenic differentiation, corroborative results were seen, with the MCP group showing the highest GAG/DNA ratio levels and uptake of extracellular matrix stain as compared to the FAA-CP group. TEM analysis of the chondrocytes revealed the presence of more autolytic cells with disintegrated cytoplasm and plasma membrane. The differentiated FAA-CPs and MCPs displayed higher collagen and rough endoplasmic reticulum. The results presented in this study provide novel information on the ultrastructural characteristics of cartilage resident cells, with the chondrocyte group displaying features of terminal differentiation. Both progenitor subtypes showed superiority in varied contexts, with greater collagen fibrils and greater GAG content in MCPs. The display of preferential and differentiation traits sheds insight on the necessity to enrich progenitors and coculturing them with the general pool of constituent cells to combine their advantages and reduce their drawbacks to achieve a regenerative tissue displaying genuine hyaline-like repair while limiting their terminal differentiation.

Comparative assessment of chondral defect repair using migratory chondroprogenitors suspended in either gelled or freeze-dried platelet-rich plasma: An in vitro and ex vivo human osteochondral unit model study.

BACKGROUND: Chondroprogenitors, with enhanced chondrogenic potential, have emerged to be a promising alternative for cell-based therapy in cartilage repair. Platelet-rich plasma (PRP), widely used for intra-articular treatment, has a short half-life. Freeze-dried PRP (FD-PRP), with an extended half-life and retained growth factors, is gaining attention. This study compares the efficacy of Migratory Chondroprogenitors (MCPs) in gelled PRP and FD-PRP using in-vitro and ex-vivo models, assessing FD-PRP as a potential off-the-shelf option for effective cartilage repair. METHODOLOGY: MCPs were isolated from osteoarthritic cartilage samples (n = 3), characterized through FACS and RT-PCR. For in-vitro analysis, cells were loaded into gelled PRP and FD-PRP scaffolds at a density of 1x106 cells per scaffold. Trilineage differentiation studies and live-dead assays were conducted on MCPs using Calcein AM/Propidium Homodimer-1. In ex-vivo analysis, MCPs of the same density were added to Osteochondral Units (OCU) with chondral defects containing PRP gel and FD-PRP scaffolds, harvested on the 15th and 35th days for histological examination. Controls included cell-free scaffolds. RESULTS: Our in-vitro analysis demonstrates the robust viability of MCPs in both scaffolds, with no discernible impact on their differentiation capacity. Ex-vivo analysis of the OCU for cartilage repair showed that the chondrogenic potential characterized by the accumulation of extracellular matrix containing glycosaminoglycans and collagen type II production (with no alteration in collagen type X), was observed to be better with the gel PRP and the gel PRP containing MCP groups. CONCLUSIONS: These findings support the preference for gel PRP as a superior synergistic scaffold for chondroprogenitor delivery.

Systematic review of articular cartilage derived chondroprogenitors for cartilage repair in animal models.

Purpose of research: The potential for cartilage repair using articular cartilage derived chondroprogenitors has recently gained popularity due to promising results from in-vitro and in-vivo studies. Translation of results from in-vitro to a clinical setting requires a sufficient number of animal studies displaying significant positive outcomes. Thus, this systematic review comprehensively discusses the available literature (January 2000-March 2022) on animal models employing chondroprogenitors for cartilage regeneration, highlighting the results and limitations associated with their use.As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a web-based search of PubMed and SCOPUS databases was performed for the following terminologies: "chondroprogenitors", "cartilage-progenitors", and "chondrogenic-progenitors", which yielded 528 studies. A total of 12 studies met the standardized inclusion criteria, which included chondroprogenitors derived from hyaline cartilage isolated using fibronectin adhesion assay (FAA) or migratory assay from explant cultures, further analyzing the role of chondroprogenitors using in-vivo animal models. Principal results: Analysis revealed that FAA chondroprogenitors demonstrated the ability to attenuate osteoarthritis, repair chondral defects and form stable cartilage in animal models. They displayed better outcomes than bone marrow-derived mesenchymal stem cells but were comparable to chondrocytes. Migratory chondroprogenitors also demonstrated superiority to BM-MSCs in terms of higher chondrogenesis and lower hypertrophy, although a direct comparison to FAA-CPs and other cell types is warranted. Major conclusions: Chondroprogenitors exhibit superior properties for chondrogenic repair; however, limited data on animal studies necessitates further studies to optimize their use before clinical translation for neo-cartilage formation.

Evaluation of ghrelin as a distinguishing marker for human articular cartilage-derived chondrocytes and chondroprogenitors.

Purpose of the study: Cell-based therapeutics for articular cartilage repair primarily employed bone marrow-derived mesenchymal stem cells and chondrocytes. Research to overcome their limitation of formation of a functionally poor fibro-hyaline type of repair tissue led to the discovery of chondroprogenitors (CPCs), cartilage resident stem cells. These cells isolated by adhesion assay using fibronectin (FAA-CPs) and migration of progenitors from explants (MCPs) display higher chondrogenic and lower terminal differentiation potential. During in-vitro culture, chondrocytes tend to de-differentiate and acquire characteristics similar to stem cells, thus making it challenging to distinguish them from other cell groups. Ghrelin, a cytoplasmic growth hormone secretagogue, has been proposed to play a vital role in chondrogenesis, with reports of its higher expression in chondrocytes than BM-MSCs. The aim of this study was to compare the mRNA expression of Ghrelin between BM-MSCs, chondrocytes, FAA-CPs and MCP and the possibility of it serving as a distinguishing marker. Methods: The four populations isolated from three human osteoarthritic knee joints were characterised by CD marker expression for positive (CD 90, CD73 and CD105) and negative (HLA-DR, CD34 and CD45) MSC markers and trilineage differentiation (adipogenic, osteogenic and chondrogenic) and subjected to qRT-PCR to assess Ghrelin's gene expression. Results: This study showed that all groups exhibited similar expression of CD markers and multilineage potential. Though chondrocytes showed greater expression of Ghrelin, it was not statistically significant to classify it as a distinguishing marker between these cell populations. Conclusion: Ghrelin does not serve to differentiate the subpopulations in terms of their mRNA expression. Further evaluation using their associated enzymes and receptors could provide valuable information to uncover their potential as unequivocal biomarkers.

Human fetal cartilage-derived chondrocytes and chondroprogenitors display a greater commitment to chondrogenesis than adult cartilage resident cells.

Obtaining regeneration-competent cells and generating high-quality neocartilage are still challenges in articular cartilage tissue engineering. Although chondroprogenitor cells are a resident subpopulation of native cartilage and possess a high capacity for proliferation and cartilage formation, their potential for regenerative medicine has not been adequately explored. Fetal cartilage, another potential source with greater cellularity and a higher cell-matrix ratio than adult tissue, has been evaluated for sourcing cells to treat articular disorders. This study aimed to compare cartilage resident cells, namely chondrocytes, fibronectin adhesion assay-derived chondroprogenitors (FAA-CPCs) and migratory chondroprogenitors (MCPs) isolated from fetal and adult cartilage, to evaluate differences in their biological properties and their potential for cartilage repair. Following informed consent, three human fetal and three adult osteoarthritic knee joints were used to harvest the cartilage samples, from which the three cell types a) chondrocytes, b) FAA-CPCs, and MCPs were isolated. Assessment parameters consisted of flow cytometry analysis for percentage expression of cell surface markers, population doubling time and cell cycle analyses, qRT-PCR for markers of chondrogenesis and hypertrophy, trilineage differentiation potential and biochemical analysis of differentiated chondrogenic pellets for total GAG/DNA content. Compared to their adult counterparts, fetal cartilage-derived cells displayed significantly lower CD106 and higher levels of CD146 expression, indicative of their superior chondrogenic capacity. Moreover, all fetal groups demonstrated significantly higher levels of GAG/DNA ratio with enhanced uptake of collagen type 2 and GAG stains on histology. It was also noted that fetal FAA CPCs had a greater proliferative ability with significantly higher levels of the primary transcription factor SOX-9. Fetal chondrocytes and chondroprogenitors displayed a superior propensity for chondrogenesis when compared to their adult counterparts. To understand their therapeutic potential and provide an important solution to long-standing challenges in cartilage tissue engineering, focused research into its regenerative properties using in-vivo models is warranted.

Assessment of the inherent chondrogenic potential of human articular cartilage-derived chondroprogenitors in pellet culture using a novel whole pellet processing approach.

Purpose: Cartilage-derived chondroprogenitors have been reported to possess the biological potential for cartilage repair. However, its inherent chondrogenic potential in pellet culture needs evaluation. In-vitro cartilage regeneration models based on pellet cultures have been employed to evaluate the chondrogenic potential of stem cells. Evaluation of the degree of differentiation routinely involves paraffin embedding, sectioning, and immunohistochemical staining of the pellet. However, since chondrogenic differentiation is commonly non-uniform, processing random sections could lead to inaccurate conclusions. The study aimed at assessing the inherent lineage bias of chondroprogenitors with and without chondrogenic induction, using a novel whole pellet processing technique. Methods: Human chondroprogenitors (n=3) were evaluated for MSC markers and processed in pellet cultures either with stromal medium (uninduced) or chondrogenic differentiation medium (induced) for 28 days. The whole pellets and the conventional paraffin-embedded sectioned pellets were subjected to Collagen type II immunostaining and assessed using confocal laser microscopy. The staining intensities of the whole pellet were compared to the paraffin sections and revalidated using qRT-PCR for COL2A1 expression. Results: Uninduced and induced pellets displayed Collagen type II in all the layers with comparable fluorescence intensities. COL2A1 expression in both pellets was comparable to confocal results. The study demonstrated that uninduced chondroprogenitors in pellet culture possess promising inherent chondrogenic potential. Confocal imaging of whole pellets displayed different degrees of chondrogenic differentiation in the entire pellet, thus its probable in-vivo behavior. Conclusion: The novel approach presented in this study could serve as an efficient in-vitro alternative for understanding translational application for cartilage repair.

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.

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.

Migratory chondroprogenitors retain superior intrinsic chondrogenic potential for regenerative cartilage repair as compared to human fibronectin derived chondroprogenitors.

Cell-based therapy for articular hyaline cartilage regeneration predominantly involves the use of mesenchymal stem cells and chondrocytes. However, the regenerated repair tissue is suboptimal due to the formation of mixed hyaline and fibrocartilage, resulting in inferior long-term functional outcomes. Current preclinical research points towards the potential use of cartilage-derived chondroprogenitors as a viable option for cartilage healing. Fibronectin adhesion assay-derived chondroprogenitors (FAA-CP) and migratory chondroprogenitors (MCP) exhibit features suitable for neocartilage formation but are isolated using distinct protocols. In order to assess superiority between the two cell groups, this study was the first attempt to compare human FAA-CPs with MCPs in normoxic and hypoxic culture conditions, investigating their growth characteristics, surface marker profile and trilineage potency. Their chondrogenic potential was assessed using mRNA expression for markers of chondrogenesis and hypertrophy, glycosaminoglycan content (GAG), and histological staining. MCPs displayed lower levels of hypertrophy markers (RUNX2 and COL1A1), with normoxia-MCP exhibiting significantly higher levels of chondrogenic markers (Aggrecan and COL2A1/COL1A1 ratio), thus showing superior potential towards cartilage repair. Upon chondrogenic induction, normoxia-MCPs also showed significantly higher levels of GAG/DNA with stronger staining. Focused research using MCPs is required as they can be suitable contenders for the generation of hyaline-like repair tissue.

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.

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.

Improving the effectiveness of physiology record books as a learning tool for first-year medical students in India.

In compliance with the Medical Council of India, preclinical medical students maintain a record of their laboratory work in physiology. The physiology record books also contain a set of questions to be answered by the students. Faculty members and students had indicated that responding to these questions did not serve the intended purpose of being an effective learning tool. The purpose of this study was to obtain the views of the medical students and faculty members at our institution concerning the usefulness of responding to the questions and to gather suggestions for possible improvement. Data were collected through focus groups and questionnaires to first-year medical students and faculty members in physiology and were analyzed using qualitative and quantitative methods. The students and faculty members viewed the physiology record books as a potentially useful learning aid, but lack of time led the students to write the answers without understanding the topic rather than generating their own responses to the questions. Faculty members and students recommended that the students should write the responses to the questions on site during the practical classes, using relevant on-site resources and interacting with faculty members. The findings of the present study may be of value to other medical colleges in India and outside India with modifications based on their specific needs to improve the effectiveness of physiology record books as a learning tool.

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.

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.

Allogeneic platelet rich plasma serves as a scaffold for articular cartilage derived chondroprogenitors.

Limited self-restorative ability of the cartilage has necessitated the use of cell and tissue engineering based therapies. Recent advances in the isolation, expansion and characterization of articular cartilage derived chondroprogenitors(CPs) has gained popularity in its role for cartilage repair. Platelet rich plasma (PRP) is a reliable biological scaffold for in-vitro and in-vivo studies with reported therapeutic applications in cartilage and bone pathologies. The aim of this study was to evaluate whether human allogeneic PRP could serve as a biological scaffold for chondroprogenitors (CPs) in cartilage repair. CPs were isolated from the superficial layer of three osteoarthritic knee joints by fibronectin adhesion assay and characterized using flow cytometric analysis. Allogeneic citrated blood was harvested from three subjects to obtain PRP. CPs at a concentration of one million cells per ml were gelled with PRP using calcium chloride. The PRP-CP scaffolds were subjected for adipogeneic, osteogenic, chondrogeneic differentiation and processed for post differentiation-staining studies (Oil Red O, Von Kossa, Alcian blue staining), immunofluorescence (collagen II) and live dead assays (Calcein AM-Ethidium Homodimer). We show that PRP was able to sustain CP cell viability and differentiate towards adipogenic, osteogenic and chondrogenic lineage under appropriate culture conditions. We also noted positive extracellular matrix production in PRP-CP scaffolds cultured without chondrogenic supplementation. Our results suggest that PRP could be a promising bio-active scaffold due to its synergistic effect in supporting cell proliferation, maintaining cell viability and favoring extracellular matrix production. PRP can be used as biological scaffold for the delivery of CPs in cartilage healing.