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.

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.

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.

Virtual microscopy as a teaching-learning tool for histology in a competency-based medical curriculum.

Background: Histology forms an important component of first-year medical education. Unfortunately, it is limited to the practical laboratory due to the need for a microscope and good quality slides. Virtual microscopy is a recent advancement, which uses computers as an alternative to microscopes. The aim of the study was to compare virtual microscopy (VM)-based practical classes with traditional microscopy (TM)-based practical classes for two cohorts of first-year medical students, by comparing learning achieved using two different test scores as well as a qualitative assessment of student and faculty perspectives regarding the feasibility and usefulness of VM. Methods: Each cohort of students was divided into two equal batches and each batch underwent eight histology modules of which, four utilised traditional microscopes and four utilised virtual microscopes. Quantitative analysis was performed using a theory test (which assessed preparation, theory knowledge and understanding) as well as a spotter test (which assessed identification skills, reasoning, and recall). Qualitative analysis was performed using a structured questionnaire and focus group discussions. Results: Modules using VM were better when compared with those using TM, showing statistically significant and better grades. Qualitative analysis performed, yielded important information as to how this technology can serve as a good adjunct to traditional histology classes in the competency-based curriculum by increasing student interest, enabling self-study, and reducing students dependence on the tutor. Conclusions: VM forms a good adjunct as well as a standalone modality of learning to TM, as it improves accessibility to slides and promotes self-learning.

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.

Deplastination: Preservation of Histological Structures and its Anticipated Role in the Field of Histopathology.

Deplastination is the process of reversing plastination such that a plastinated specimen can be reverted to its raw nature. This would enable its use in the field of histopathology. The present study aims to ascertain if deplastinates can be used for histopathological studies after a time period. Tissue samples were taken from patients undergoing maxillofacial surgeries for oral carcinomas after obtaining written informed consent. The 12 specimens obtained were divided into two groups. One set of tissues was processed for paraffin embedding after 10% formalin fixation. The other set was plastinated by S10 silicon plastination. After 3 months, the plastinates were deplastinated using sodium methoxide and processed for routine hematoxylin and eosin staining, similar to the formalin fixed specimens. The slides were quantitatively assessed on parameters like tissue architecture, staining property, and intracellular structure. In addition, the slides were qualitatively evaluated by a pathologist who was blinded to the mode of preservation to see if identification of pathological features was possible on a deplastinated slide. The formalin preserved specimens and deplastinated tissue slides compared closely in all three parameters tested with the need to identify the endpoint of deplastination. Qualitatively, deplastination did not hamper identification of tissue pathology. Deplastination increases the scope of a stored plastinate by allowing histological studies in the future without the need for any preservatives or special storage equipment. It preserves structure and maintains tissue pathology. An improved method of ensuring the endpoint of deplastination needs to be identified. Clin. Anat. 32:108-112, 2019. © 2019 Wiley Periodicals, Inc.