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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.
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Background: Gamma-H2AX immunofluorescence assay has gained popularity as a DNA double strand break marker. In this work, we have investigated the potential use of gamma H2AX immunofluorescence assay as a biological dosimeter for estimation of dose in our institution. Materials and methods: Seven healthy individuals were selected for the study and the blood samples collected from the first five individuals were irradiated to low doses (0-10 cGy) and high doses (50-500 cGy) in a telecobalt unit. All the samples were processed for gamma-H2AX immunofluorescence assay and the dose-response calibration curves for low and high doses were determined. In order to validate the determined dose-response calibration curves, the blood samples obtained from the sixth and seventh subjects were delivered a test dose of 7.5 cGy and 250 cGy. In addition, time and cost required to complete the assay were also reported. Results: The goodness of fit (R2) values was found to be 0.9829 and 0.9766 for low and high dose-response calibration curves. The time required to perform the gamma-H2AX immunofluorescence assay was found to be 7 hours and 30 minutes and the estimated cost per sample was 5000 rupees (~ 60 USD). Conclusion: Based on this study we conclude that the individual dose-response calibration curves determined with gamma-H2AX immunofluorescence assay for both low and high dose ranges of gamma radiation can be used for biological dosimetry. Further, the gamma-H2AX immunofluorescence assay can be used as a rapid cost-effective biodosimetric tool for institutions with an existing confocal microscope facility.
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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.
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Cartílago Articular , Liofilización , Plasma Rico en Plaquetas , Humanos , Condrocitos , Condrogénesis/fisiología , Movimiento Celular , Diferenciación Celular , Andamios del Tejido , Células CultivadasRESUMEN
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.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Células Madre Mesenquimatosas , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Médula Ósea/metabolismo , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Microambiente TumoralRESUMEN
BACKGROUND: In vitro studies with human fetal islets of different gestational ages (GA) would be a great tool to generate information on the developmental process of the islets as this would help to recontextualize diabetes research and clinical practice. Pancreatic islets from human cadavers and other animal species are extensively researched to explore their suitability for islet transplantation procedure, one of the upcoming treatment strategies for insulin-dependent diabetes mellitus. Although human fetal islets are also considered for islet transplantation, ethical issues and limited knowledge constraints their use. The fetal islets could be explored to address the information lacunae on the maturity process of pancreatic islets and the endocrine-exocrine signaling mechanisms. AIM: This study aimed to assess the feasibility of isolating viable islets and study the cytoarchitecture of the fetal pancreas of GA 22-29 weeks, not reported otherwise. METHODOLOGY: Pancreas obtained from the aborted fetuses of GA 22-29 weeks were subjected to collagenase digestion and were further cultured to determine the viability in vitro. Parameters assessed were expression of markers for endocrine cell lineages and insulin release to glucose challenge. RESULTS: Islets were viable in vitro and islets were shown to maintain cues for post-digestion re-aggregation and expansion in culture. The immunofluorescent staining showed islets of varying sizes, homogenous cell clusters aggregating to form heterogenous cell clusters, otherwise not reported for this GA. On stimulation with different concentrations of glucose (2.8 and 28 mM), the fetal islets in the culture exhibited insulin release, and this response confirmed their viability in vitro. CONCLUSION: Our findings showed that viable islets could be isolated and cultured in vitro for further in-depth studies to explore their proliferative potential as well as for the identification of pancreatic progenitors, a good strategy to take forward.
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BACKGROUND: Cleistanthus collinus is a poisonous shrub commonly used for deliberate self-harm in rural south India. Boiled decoction or a paste made from its leaves is used for suicide. Cleistanthoside A and Cleistanthin A are the major toxins identified from this plant. In this study, we disclose the mechanism of Cleistanthin A toxicity and concentrations of the two toxins in different extracts of Cleistanthus collinus. METHODS: The effect of Cleistanthin A was studied on isolated goat leg arteries using two different preparations namely transverse cylinder and longitudinal strip. The influence of Cleistanthin A on peripheral vascular resistance and myocardial contractility was evaluated by rat hind limb and isolated rat heart experiments, respectively. For the quantification of toxins, five different extracts of C. collinus leaves were prepared. The extracts were subjected to analytical HPLC to quantify Cleistanthoside A and Cleistanthin A. RESULTS AND CONCLUSION: Cleistanthin A increased vascular tension in transverse cylinder preparation and increased peak, trough and mean aortic pressures in the rat hind limb preparations. In isolated rat heart experiments, there was an increase in diastolic and mean ventricular pressure with a significant decrease in ventricular pulse pressure. These observations suggest that the hypotension in C. collinus poisoning patients may be due to cardiotoxicity and not due to vasodilation as is currently believed. Quantification of different extracts showed that boiled extracts had higher quantities of Cleistanthoside A whereas crushed leaf extracts yielded significantly higher amounts of Cleistanthin A.
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Depresión , Lignanos , Humanos , Ratas , Animales , Vasoconstricción , GlicósidosRESUMEN
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.
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Cartílago Articular , Humanos , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/metabolismo , Diferenciación Celular/genética , Células Madre/metabolismo , Hipertrofia/metabolismo , CondrogénesisRESUMEN
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.
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Cartílago Articular , Condrocitos , Humanos , Adulto , Condrocitos/metabolismo , Condrogénesis , Células Cultivadas , Cartílago Articular/metabolismo , Diferenciación Celular , ADN/metabolismoRESUMEN
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.
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Cartílago Articular , Condrogénesis , Diferenciación Celular , Células Cultivadas , Condrocitos/metabolismo , Suplementos Dietéticos , Factor 2 de Diferenciación de Crecimiento/metabolismo , Factor 2 de Diferenciación de Crecimiento/farmacología , Humanos , Hipertrofia/metabolismoRESUMEN
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.
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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.
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Regeneración Ósea , Cartílago Articular/fisiología , Condrogénesis , Fibronectinas/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Adipogénesis , Biomarcadores , Ciclo Celular , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Persona de Mediana EdadRESUMEN
Cleistanthus collinus leaf extracts are consumed for suicidal purposes in southern India. The boiled decoction is known to be more toxic than the fresh leaf juice. Although several compounds have been isolated and their toxicity tested, controversy remains as to which compounds are responsible for the high level of toxicity of C. collinus. We report herein that cleistanthoside A is the major toxin in the boiled aqueous extract of fresh leaves and causes death in rats in small doses. The toxicity of the boiled extract prepared in the manner described can be attributed entirely to cleistanthoside A. Cleistanthin A could also be isolated from the boiled extract, albeit in trace amounts. As hypotension not responding to vasoconstrictors is the cause of death in patients who have consumed the boiled extract, effects of cleistanthoside A on the determinants of blood pressure, namely, force of cardiac contraction and vascular resistance, were tested in isolated organ experiments. Cleistanthoside A has a direct vasoconstrictor effect; however, it inhibits ventricular contractility. Therefore, the notion that the shock in C. collinus poisoning is of vascular origin must be considered carefully, and the possibility of cardiogenic shock must be studied. We present the crystal structure of cleistanthin A and show the potency of fast NMR methods (NOAH4-BSCN-NUS) in the full spectral assignment of cleistanthoside A as a real-world sample of a natural product. We also compare the results of the NOAH4-BSCN-NUS NMR experiments with conventional NMR methods.
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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.
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Antígenos CD34/inmunología , Antígenos CD/inmunología , Cartílago Articular , Moléculas de Adhesión Celular Neuronal/inmunología , Condrocitos , Proteínas Fetales/inmunología , Antígeno CD146/metabolismo , Diferenciación Celular , Condrocitos/metabolismo , Condrogénesis/genética , HumanosRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0227316.].
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This sourcebook update describes a variation of a previous sourcebook experiment that used isolated extensor digitorum longus muscle from mouse to teach skeletal muscle properties (Head SI, Arber MS. Adv Physiol Educ 37: 405-414, 2013). Gastrocnemius-sciatic nerve preparation in an anaesthetized rat was developed and muscle contractions were recorded in a computerized data acquisition system using an isometric force transducer. Teachers and students in physiology or biology can use this preparation to demonstrate skeletal muscle properties like simple muscle twitch, quantal summation, wave summation, superposition, incomplete tetanus, complete tetanus, treppe, fatigue, and length-tension relationship.
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Contracción Muscular , Músculo Esquelético , Animales , Contracción Isométrica , Ratones , Ratas , Nervio CiáticoRESUMEN
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.
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Cartílago Articular/citología , Condrocitos/citología , Condrogénesis , Células Madre Mesenquimatosas/citología , Concentración Osmolar , Biomarcadores/metabolismo , Linaje de la Célula/genética , Proliferación Celular/genética , Tamaño de la Célula , Supervivencia Celular/genética , Condrocitos/metabolismo , Condrogénesis/genética , Regulación de la Expresión Génica , Humanos , Hipertrofia , Cinética , Células Madre Mesenquimatosas/metabolismoRESUMEN
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) CD49bâ¯+â¯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.
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Células de la Médula Ósea , Cartílago Articular , Condrocitos , Condrogénesis , Células Madre Mesenquimatosas , Osteoartritis de la Rodilla , Regeneración , Anciano , Antígenos CD/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Cartílago Articular/lesiones , Cartílago Articular/fisiología , Condrocitos/metabolismo , Condrocitos/patología , Femenino , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Persona de Mediana Edad , Osteoartritis de la Rodilla/metabolismo , Osteoartritis de la Rodilla/patologíaRESUMEN
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.