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Background: One of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells., Methods: We added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles., Results: iPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time., Conclusion: Our preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The repair of damaged articular cartilage using currently available implantation techniques is not sufficient for the full recovery of patients. Pluripotent stem cells (iPSC)-based therapies could bring new perspectives in the treatment of joint diseases. A number of protocols of in vitro differentiation of iPSC in chondrocytes for regenerative purposes have been recently described. However, in order to use these cells in clinics, the elimination of animal serum and feeder cells is essential. In our study, a strictly defined and controllable protocol was designed for the differentiation of pluripotent stem cells (BG01V, ND 41658*H, GPCCi001-A) in chondrocyte-like cells in serum- and a feeder cell-free system, using the embryoid bodies step. The extension of the protocol and culture conditions (monolayer versus 3D culture) was also tested after the initial 21 days of chondrogenic differentiation. Promotion of the chondrogenic differentiation in 3D culture via the elevated expression of genes related to chondrogenesis was achieved. Using immunofluorescence and immunohistochemistry staining techniques, the increased deposition of the specific extracellular matrix was indicated. As a result, chondrocyte-like cells in the early stages of their differentiation using pellet culture under fully controlled and defined conditions were obtained., Competing Interests: The authors declare no conflict of interest.

A human induced pluripotent stem cell line (GPCCi001-A) created by our group was differentiated towards chondrocyte-like cells (ChiPS) via monolayer culturing with growth factors. ChiPS are promising because they have the potential to be used in tissue engineering to regenerate articular cartilage. However, their safety must be confirmed before they can be routinely used in regenerative medicine. Using microarray analysis, we compared the ChiPS to both GPCCi001-A cells and chondrocytes. The analysis showed that, compared to both GPCCi001-A cells and chondrocytes, the expression of genes engaged in DNA damage and in the tumor protein p53 signalling pathways was significantly higher in the ChiPS. The significant amount of DNA double strand breaks and increased DNA damage response may lead to incomplete DNA repair and the accumulation of mutations and, ultimately, to genetic instability. These findings provide evidence indicating that the differentiation process in vitro places stress on human induced pluripotent stem cells (hiPSCs). The results of this study raise doubts about the use of stem cell-derived components given the negative effects of the differentiation process in vitro on hiPSCs., Competing Interests: The authors have declared that no competing interests exist.

Human induced pluripotent stem cells (hiPSCs) constitute an important breakthrough in regenerative medicine, particularly in orthopedics, where more effective treatments are urgently needed. Despite the promise of hiPSCs only limited data on in vitro chondrogenic differentiation of hiPSCs are available. Therefore, we compared the gene expression profile of pluripotent genes in hiPSC-derived chondrocytes (ChiPS) to that of an hiPSC cell line created by our group (GPCCi001-A). The results are shown on heatmaps and plots and confirmed by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analysis. Unlike the ChiPS, our GPCCi001-A cells maintained their pluripotency state during long-term culture, thus demonstrating that this cell line was comprised of stable, fully pluripotent hiPSCs. Moreover, these chondrocyte-like cells not only presented features that are characteristic of chondrocytes, but they also lost their pluripotency, which is an important advantage in favor of using this cell line in future clinical studies., Competing Interests: The authors declare no conflict of interest.

Introduction: Human articular cartilage has a poor regenerative capacity. This often results in the serious joint disease- osteoarthritis (OA) that is characterized by cartilage degradation. An inability to self-repair provided extensive studies on AC regeneration. The cell-based cartilage tissue engineering is a promising approach for cartilage regeneration. So far, numerous cell types have been reported to show chondrogenic potential, among others human embryonic stem cells (hESCs)., Materials and Methods: However, the currently used methods for directed differentiation of human ESCs into chondrocyte-like cells via embryoid body (EB) formation, micromass culture (MC) and pellet culture (PC) are not highly efficient and require further improvement. In the present study, these three methods for hESCs differentiation into chondrocyte-like cells in the presence of chondrogenic medium supplemented with diverse combination of growth factors (GFs) were evaluated and modified., Results: The protocols established here allow highly efficient, simple and inexpensive production of a large number of chondrocyte-like cells suitable for transplantation into the sites of cartilage injury. The most crucial issue is the selection of appropriate GFs in defined concentration. The obtained stem-derived cells reveal the presence of chondrogenic markers such as type II collagen, Sox6 and Sox9 as well as the lack or significantly lower level of pluripotency markers including Nanog and Oct3/4., Discussion: The most efficient method is the differentiation throughout embryoid bodies. In turn, chondrogenic differentiation via pellet culture is the most promising method for implementation on clinical scale. The most useful GFs are TGF-β1, -3 and BMP-2 that possess the most chondrogenic potential. These methods can also be used to obtain chondrocyte-like cells from differentiating induced pluripotent stem cells (iPSCs).

Stem cells (SCs) are a promising approach to regenerative medicine, with the potential to treat numerous orthopedic disorders, including osteo-degenerative diseases. The development of human-induced pluripotent stem cells (hiPSCs) has increased the potential of SCs for new treatments. However, current methods of differentiating hiPSCs into chondrocyte-like cells are suboptimal and better methods are needed. The aim of the present study was to assess four different chondrogenic differentiation protocols to identify the most efficient method of generating hiPSC-derived chondrocytes. For this study, hiPSCs were obtained from primary human dermal fibroblasts (PHDFs) and differentiated into chondrocyte-like cells using four different protocols: 1) monolayer culture with defined growth factors (GF); 2) embryoid bodies (EBs) in a chondrogenic medium with TGF-β3 cells; 3) EBs in chondrogenic medium conditioned with human chondrocytes (HC-402-05a cell line) and 4) EBs in chondrogenic medium conditioned with human chondrocytes and supplemented with TGF-β3. The cells obtained through these four protocols were evaluated and compared at the mRNA and protein levels. Although chondrogenic differentiation of hiPSCs was successfully achieved with all of these protocols, the two fastest and most cost-effective methods were the monolayer culture with GFs and the medium conditioned with human chondrocytes. Both of these methods are superior to other available techniques. The main advantage of the conditioned medium is that the technique is relatively simple and inexpensive while the directed method (i.e., monolayer culture with GFs) is faster than any protocol described to date because it is does not require additional steps such as EB formation.

To improve the recovery of damaged cartilage tissue, pluripotent stem cell-based therapies are being intensively explored. A number of techniques exist that enable monitoring of stem cell differentiation, including immunofluorescence staining. This simple and fast method enables changes to be observed during the differentiation process. Here, two protocols for the differentiation of human embryonic stem cells into chondrocytes were used (monolayer cell culture and embryoid body formation). Cells were labeled for markers expressed during the differentiation process at different time points (pluripotent: NANOG, SOX2, OCT3/4, E-cadherin; prochondrogenic: SOX6, SOX9, Collagen type II; extracellular matrix components: chondroitin sulfate, heparan sulfate; beta-catenin, CXCR4, and Brachyury). Comparison of the signal intensity of differentiated cells to control cell populations (articular cartilage chondrocytes and human embryonic stem cells) showed decreased signal intensities of pluripotent markers, E-cadherin and beta-catenin. Increased signal intensities of prochondrogenic markers and extracellular matrix components were observed. The changes during chondrogenic differentiation monitored by evaluation of pluripotent and chondrogenic markers signal intensity were described. The changes were similar to several studies over chondrogenesis. These results were confirmed by semi-quantitative analysis of IF signals. In this research we indicate a bioimaging as a useful tool to monitor and semi-quantify the IF pictures during the differentiation of hES into chondrocyte-like.

Magnetic resonance imaging is gold standard for noninvasive evaluation of articular cartilage damage and has been also used for monitoring cartilage repair. The aim of this study was to find correlation between histological microscopy and microscopic MR in evaluation of the repair of osteochondral defects in articular cartilage. Study was based animal model (rabbit). The cartilage repair process was evaluated histology and micro MR. Most of the defects were filled with fibrocartilage and fibrous tissue formed. Both methods were equally efficient to show repair tissue thickness, subchondral bone reconstruction and disintegration. Result of observation by both histological and MR microscopy and showed good correlation. Micro MR is promising evaluation tool for cartilage repair monitoring. Results of micro MR correlate well with standard microscopy.

Introduction: Restricted potential of cartilage to regeneration following trauma induced search for new techniques of articular cartilage repair. Present study aimed at defining in experimental conditions of autologous osteochondral paste value by evaluation of cellularity and presence of necrosis in the defect-filling tissue., Material and Methods: Full thickness defect (IV(o)--ICRS scale) on distal rabbit femur joint surface was made. Three groups were specified: A--defect with paste graft (cartilage and contiguous bone collected from joint surface, crushed into homogenous paste; B--defect with the paste graft covered with periosteum; C--defect left unfilled. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale., Results: In evaluation of cellularity in the developed repair tissue, in long-term observations best results were obtained in the group of osteochondral paste (A) and in the group of unfilled defect (C). Following 12 weeks of observation cellularity of the repair tissue in the groups amounted to around 95%, as compared to the surrounding healthy cartilage. In evaluation of necrotic traits in the newly developed tissue, their highest intensity after 4 weeks of observation was noted in the group with periosteum covered osteochondral paste (B), and lower but still significant in the group in which paste alone was applied (A). This resulted from massive dying out of the grafted bone elements, and in the case of group B, also of the periosteal cells in the covering flap and of certain number of chondrocytes composing the graft. The trait was more objectively evaluated in long-term observations, when necrotic foci involved only elements of the proper repair tissue. After 12 weeks of observation degenerative changes showed the least pronounced intensity in the group with the paste alone (A). Following 4 weeks the intensity was least pronounced in the group with the unfilled defect (C), i.a. due to the absence of graft with the decomposing bone elements. However, with elapsing time degenerative lesions intensified in the group, pointing to the low stability of the developed tissue. The results indicated that application of the osteochondral paste in treatment of cartilage defects yields a stable, highly cellular tissue, resistant to time and trauma.

Introduction: A limited ability of the cartilage to heal after trauma was the reason to start research on new methods concerning better cartilage reconstruction. The aim of the study was evaluation of repair tissue thickness and surface regularity after osteochondral paste transplantation., Material and Methods: Full thickness defect (IV(o)--ICRS scale) on distal rabbit femur joint surface was made. Three groups were specified: A--defect with paste graft (cartilage and contiguous bone collected from joint surface, crushed into homogenous paste; B--defect with the paste graft covered with periosteum; C--defect left unfilled. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale., Results: Newly formed tissue was well integrated with surrounding cartilage in group A (paste graft). That trait of repair tissue in group A was much better than in other groups, especially in late observations. Structural integrity of tissue filling the defect was similar to integrity of normal cartilage in groups A and C, but tissue formed in group C didn't represent a hyaline-like cartilage character. In all the examined groups reconstruction of subchondral bone exhibited similar rate. 12 weeks from the procedure, around 80% of subchondral bone was rebuilt. The obtained results indicate, that osteochondral paste autologous transplantation in cartilage defects treatment effects with forming well integrated (structurally and with surrounding cartilage) cartilage tissue, of almost complete subchondral bone rebuilding.

Unlabelled: Articular cartilage cells are immersed in semi-solid matrix and isolated from the rest of the body because of lack of nervous fibres, and blood and lymphatic vessels. Trauma and aging processes result in cartilage ultrastructure disorders. Those changes leads to progressive decreasing of durability and rigidity of cartilage. Research on articular cartilage reconstruction focuses on, among other things, reaching by newly formed tissue optimal amount of vital cells. The aim of this part of the study was microscopic evaluation of repair tissue degenerative changes (cellularity and signs of necrosis)., Material and Methods: Repair of partial thickness cartilage defect (III tertiary grade) on distal femur joint surface was evaluated (25 adolescent rabbits). Procedures were performed in two groups: I--autologous chondrocyte transplantation under periosteal flap, II--periosteal graft. Chondrocytes were isolated from the cartilage specimens by enzymatic digestion and cultured in vitro. The follow-up periods were established at 4, 8 and 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale., Results: Repair tissue cellularity. In group I (with chondrocytes), 4 weeks after the procedure tissue of high cellularity was formed, corresponding in amount of cells to the structure of early differentiated hyaline-like cartilage. Amount of cells slightly decreased with time, as it occurs in maturing cartilage. In group II (without chondrocytes), 4 weeks after the procedure the repair tissue was characterized by small amount of cells, which was decreasing with time. Signs of necrosis. In group I, 4, 8 and 12 weeks after the procedure moderate intensity of necrotic signs was observed. In group II, significant intensity of necrosis signs in all observation periods was observed., Conclusion: Obtained results indicate, that autologous chondrocyte transplantation in treatment of partial thickness cartilage defects effects with forming tissue of high cellularity, not degenerating with time, much better as compared to untreated defect.

Unlabelled: Experimental and clinical tests proved that cultured, autologous chondrocytes retain their properties and have ability to reconstruct hyaline-like cartilage, which represents chemical composition and biomechanical characteristics similar to normal hyaline cartilage. The aim of this part of the study was microscopic evaluation of repair tissue structural integrity and surface regularity after autologous chondrocyte transplantation., Material and Methods: Repair of partial thickness cartilage defect (ICRS III(o) grade) on distal femur joint surface was evaluated (25 adolescent rabbits). Procedures were performed in two groups: I--autologous chondrocyte transplantation under periosteal flap, II--periosteal graft. Chondrocytes were isolated from the cartilage specimens by enzymatic digestion and cultured in vitro. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale., Results: In group I, 4 weeks after the procedure surface of the reconstructed tissue was irregular. 8- and 12-week observation found the surface regular and plain, and repair tissue exhibited complete structural integrity. In group II, in all follow-up periods regenerate surface was irregular, there was many fissures and cracks in graft tissue, and in several cases--regenerate disintegration., Conclusion: Obtained results indicate, that hyaline-like cartilage reconstructed after autologous chondrocyte transplantation was characterized by regular, plain surface and complete structural integrity.

Unlabelled: Lack of cartilage vascularization is the reason of its low regenerative potential. The aim of this part of the study was microscopic evaluation of repair tissue thickness and its integration with surrounding cartilage, after autologous chondrocyte transplantation., Material and Methods: Repair of partial thickness cartilage defect (ICRS III(o) grade) on distal femur joint surface was evaluated (25 adolescent rabbits). Procedures were performed in two groups: I--autologous chondrocyte transplantation under periosteal flap, II--periosteal graft. Chondrocytes were isolated from the cartilage specimens by enzymatic digestion and cultured in vitro. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale., Results: In group I, 8 weeks after the procedure most of defects were filled with the newly formed tissue almost completely. Regenerate thickness after 4 and 12 weeks usually exceeded 1/2 of surrounding cartilage. In group II, 8 weeks after the procedure regenerate thickness amounted to at least 1/2 of surrounding cartilage, but 4- and 12-week observation revealed the decreased repair tissue thickness. In group I, 4 weeks after the procedure regenerative tissue was well integrated with surrounding cartilage, and this trait still gradually increased with time. In group II, there was partial integration or no integration of repair tissue with surrounding cartilage., Conclusion: Obtained results indicate, that tissue formed after autologous chondrocyte transplantation with use of periosteal flap was better in its thickness and integration with surrounding cartilage, as compared to tissue formed after use of periosteum alone. Autologous chondrocyte transplantation can not guarantee complete filling of the cartilage defect with the graft tissue and full integration with surrounding cartilage, without three-dimensional scaffold application.

Introduction: A limited ability of the cartilage to heal after trauma was the reason to start research on new methods concerning better cartilage reconstruction. The aim of the study was evaluation of repair tissue integration with surrounding cartilage, its structural integrity and subchondral bone reconstruction after osteo-chondral paste transplantation., Material and Methods: Full thickness defect (IV degree--ICRS scale) on distal rabbit femur joint surface was made. Three groups were specified: A--defect with paste graft (cartilage and contiguous bone collected from joint surface, crushed into homogenous paste; B--defect with the paste graft covered with periosteum; C--defect left unfilled. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale., Results: Newly formed tissue was well integrated with surrounding cartilage in group A (paste graft). That trade of repair tissue in group A was much better than in other groups, especially in late observations. Structural integrity of tissue filling the defect was similar to integrity of normal cartilage in groups A and C, but tissue formed in group C didn't represent a hyaline-like cartilage character. In all the examined groups reconstruction of subchondral bone exhibited similar rate. 12 weeks from the procedure, around 80% of subchondral bone was rebuilt. The obtained results indicate, that osteo-chondral paste autologous transplantation in cartilage defects treatment effects with forming well integrated (structurally and with surrounding cartilage) cartilage tissue, of almost complete subchondral bone rebuilding.

Articular cartilage defect is one of the main reasons of osteoarthritis. Currently, tissue engineering techniques are the methods concerning better cartilage reconstruction. The aim of this part of the study was macroscopic evaluation of degree of defect feeling, macroscopic appearance of repair tissue and microscopic analysis of predominant tissue after autologous chondrocytes transplantation. Repair of partial thickness cartilage defect on distal part of femur was evaluated (25 adolescent rabbits). Procedures were performed in II groups: I--autologous chondrocytes transplantation under periosteal flap, II--periosteal graft. Chondrocytes were isolated from the cartilage specimens by enzymatic digestion and cultured in vitro. The regenerates were inspected 4, 8 and 12 weeks after the operation. Macroscopic analysis in group I, in most cases revealed filling of the defect with tissue resembling surrounding cartilage. In group II the defect was partially filled, and there was many fissures and cracks in all regenerates. In microscopic analysis in group I, after 4 and 8 weeks following the transplantation the tissue similar to juvenile hyaline cartilage predominated. After 12 weeks it resembled mature hyaline cartilage. In group II, in all cases fibrous cartilage was observed after 4, 8, 12 weeks. Obtained results indicate, that macroscopic and microscopic characteristics of repair tissue after autologous chondrocytes transplantation more closely resembled hyaline cartilage, than in periosteal graft group. 12 weeks after autologous chondrocytes transplantation the repair tissue reached maturity, and demonstrated microscopic characteristics of hyaline-like cartilage. The method of autologous chondrocytes transplantation provides potential for clinical application.

One approach to cell differentiation is to use the natural capacity of pluripotent stem cells to form three germ layers via embryoid bodies (EB). However, unification of this process during in vitro culture remains challenging and many microenvironmental factors including the number of cells in the culture can influence differentiation patterns. The number of cells serves a crucial role as it determines access to nutrients, the distribution of oxygen concentration and cellular interactions, all of which influence the fate of the differentiated cells. The influence of EBs derived from human pluripotent cells on the chondrogenic potential of such cells is not well understood. For this reason, the present study sought to determine the effect of varying amounts of cells on the properties of EBs derived from human embryonic stem cells (BG01V cell line). In the present study, 500–2,000 cells per well were cultivated from 5 to 15 days in suspension cell culture. Expression of pluripotency genes and germ layer markers were evaluated in order to determine the EBs with the greatest and least mesodermal properties. Genes associated with pluripotency and chondrogenesis were also evaluated to assess the influence of suspension culture duration and EB size on chondrogenic differentiation. Immunofluorescence staining for pluripotent and chondrocyte-associated proteins confirmed successful differentiation into chondrocyte-like cells. Alcian blue staining confirmed deposition of proteoglycans. These results suggested that EBs formed in 500-cell wells possess the highest mesodermal and prochondrogenic properties. Differentiation of EBs into chondrocytes on day 5 in 500-cell wells was more efficient than in that observed in larger and older EBs. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cells (hiPSCs) offer promise in regenerative medicine, however more data are required to improve understanding of key aspects of the cell differentiation process, including how specific chondrogenic processes affect the gene expression profile of chondrocyte-like cells and the relative value of cell differentiation markers. The main aims of the present study were as follows: To determine the gene expression profile of chondrogenic-like cells derived from hiPSCs cultured in mediums conditioned with HC-402-05a cells or supplemented with transforming growth factor β3 (TGF-β3), and to assess the relative utility of the most commonly used chondrogenic markers as indicators of cell differentiation. These issues are relevant with regard to the use of human fibroblasts in the reprogramming process to obtain hiPSCs. Human fibroblasts are derived from the mesoderm and thus share a wide range of properties with chondrocytes, which also originate from the mesenchyme. Thus, the exclusion of dedifferentiation instead of chondrogenic differentiation is crucial. The hiPSCs were obtained from human primary dermal fibroblasts during a reprogramming process. Two methods, both involving embryoid bodies (EB), were used to obtain chondrocytes from the hiPSCs: EBs formed in a chondrogenic medium supplemented with TGF-β3 (10 ng/ml) and EBs formed in a medium conditioned with growth factors from HC-402-05a cells. Based on immunofluorescence and reverse transcription-quantiative polymerase chain reaction analysis, the results indicated that hiPSCs have the capacity for effective chondrogenic differentiation, in particular cells differentiated in the HC-402-05a-conditioned medium, which present morphological features and markers that are characteristic of mature human chondrocytes. By contrast, cells differentiated in the presence of TGF-β3 may demonstrate hypertrophic characteristics. Several genes [paired box 9, sex determining region Y-box (SOX) 5, SOX6, SOX9 and cartilage oligomeric matrix protein] were demonstrated to be good markers of early hiPSC chondrogenic differentiation: Insulin-like growth factor 1, Tenascin-C, and β-catenin were less valuable. These observations provide valuable data on the use of hiPSCs in cartilage tissue regeneration. [ABSTRACT FROM AUTHOR]