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.

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.

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.

Evaluation of Chondral Defect Repair Using Human Fibronectin Adhesion Assay-Derived Chondroprogenitors Suspended in Lyophilized Fetal Collagen Scaffold: An Ex Vivo Osteochondral Unit Model Study.

Introduction: Chondral defect repair is challenging due to a scarcity of reparative cells and the need to fill a large surface area, compounded by the absence of self-healing mechanisms. Fibronectin adhesion assay-derived chondroprogenitors (FAA-CPs) have emerged as a promising alternative with enhanced chondrogenic ability and reduced hypertrophy. De-cellularized bio-scaffolds are reported to act as extracellular matrix, mimicking the structural and functional characteristics of native tissue, thereby facilitating cell attachment and differentiation. This study primarily assessed the synergistic effect of FAA-CPs suspended in fetal cartilage-derived collagen-containing scaffolds in repairing chondral defects. Methodology: The de-cellularized and lyophilized fetal collagen was prepared from the tibio-femoral joint of a 36 + 4-week gestational age fetus. FAA-CPs were isolated from osteoarthritic cartilage samples (n = 3) and characterized. In ex vivo analysis, FAA-CPs at a density of 1 × 106 cells were suspended in the lyophilized scaffold and placed into the chondral defects created in the Osteochondral Units and harvested on the 35th day for histological examination. Results: The lyophilized scaffold of de-cellularized fetal cartilage with FAA-CPs demonstrated effective healing of the critical size chondral defect. This was evidenced by a uniform distribution of cells, a well-organized collagen-fibrillar network, complete filling of the defect with alignment to the surface, and favorable integration with the adjacent cartilage. However, these effects were less pronounced in the plain scaffold control group and no demonstrable repair observed in the empty defect group. Conclusion: This study suggests the synergistic potential of FAA-CPs and collagen scaffold for chondral repair which needs to be further explored for clinical therapy. Supplementary Information: The online version contains supplementary material available at 10.1007/s43465-024-01192-6.

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.

Immortalised chronic myeloid leukemia (CML) derived mesenchymal stromal cells (MSCs) line retains the immunomodulatory and chemoprotective properties of CML patient-derived MSCs.

Despite the success of Tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML), leukemic stem cells (LSCs) persist, contributing to relapse and resistance. CML Mesenchymal Stromal Cells (MSCs) help in LSC maintenance and protection from TKIs. However, the limited passage and self-differentiation abilities of primary CML MSCs hinder extensive research. To overcome this, we generated and characterized an immortalised CML patient-derived MSC (iCML MSC) line and assessed its role in LSC maintenance. We also compared the immunophenotype and differentiation potential between primary CML MSCs at diagnosis, post-treatment, and with normal bone marrow MSCs. Notably, CML MSCs exhibited enhanced chondrogenic differentiation potential compared to normal MSCs. The iCML MSC line retained the trilineage differentiation potential and was genetically stable, enabling long-term investigations. Functional studies demonstrated that iCML MSCs protected CML CD34+ cells from imatinib-induced apoptosis, recapitulating the bone marrow microenvironment-mediated resistance observed in patients. iCML MSC-conditioned media enabled CML CD34+ and AML blast cells to proliferate rapidly, with no impact on healthy donor CD34+ cells. Gene expression profiling revealed dysregulated genes associated with calcium metabolism in CML CD34+ cells cocultured with iCML MSCs, providing insights into potential therapeutic targets. Further, cytokine profiling revealed that the primary CML MSC lines abundantly secreted 25 cytokines involved in immune regulation, supporting the hypothesis that CML MSCs create an immune modulatory microenvironment that promotes growth and protects against TKIs. Our study establishes the utility of iCML MSCs as a valuable model to investigate leukemic-stromal interactions and study candidate genes involved in mediating TKI resistance in CML LSCs.

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.

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.

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.

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.

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.

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.