Tissue Engineering-based Approaches Research Articles

Progression Course in the Birth Parturition Waste Materials Toward Application in Tissue Engineering-Based Approaches

Today, the biological waste products produced during gestation and after childbirth, referred to as postpartum waste materials attracted the attention of scientists in different subjects of tissue engineering. As the goal of tissue engineering is to develop biological substitutes that can repair or replace damaged tissues and organs in the human body, the application of these waste materials after childbirth has become popular to achieve these goals, resulting from their positive aspects, such as having a valuable source of cells. In this review, we attempted to introduce the structural aspects and application of these waste materials in this emerging science.

The Long and Winding Road to Cardiac Regeneration

Cardiac regeneration is a critical endeavor in the treatment of heart diseases, aimed at repairing and enhancing the structure and function of damaged myocardium. This review offers a comprehensive overview of current advancements and strategies in cardiac regeneration, with a specific focus on regenerative medicine and tissue engineering-based approaches. Stem cell-based therapies, which involve the utilization of adult stem cells and pluripotent stem cells hold immense potential for replenishing lost cardiomyocytes and facilitating cardiac tissue repair and regeneration. Tissue engineering also plays a prominent role employing synthetic or natural biomaterials, engineering cardiac patches and grafts with suitable properties, and fabricating upscale bioreactors to create functional constructs for cardiac recovery. These constructs can be transplanted into the heart to provide mechanical support and facilitate tissue healing. Additionally, the production of organoids and chips that accurately replicate the structure and function of the whole organ is an area of extensive research. Despite significant progress, several challenges persist in the field of cardiac regeneration. These include enhancing cell survival and engraftment, achieving proper vascularization, and ensuring the long-term functionality of engineered constructs. Overcoming these obstacles and offering effective therapies to restore cardiac function could improve the quality of life for individuals with heart diseases.

Mussel-Based Biomimetic Strategies in Musculoskeletal Disorder Treatment: From Synthesis Principles to Diverse Applications

Musculoskeletal disorders are the second leading cause of disability worldwide, posing a huge global burden to the public sanitation system. Currently, tissue engineering-based approaches act as effective strategies, which are, however, challenging in limited application scenarios. Mussel-based biomimetic materials, exhibit numerous unique properties such as intense adhesion, biocompatibility, moisture resistance, and injectability, to name only a few, and have attracted extensive research interest. In particular, featuring state-of-the-art properties, mussel-inspired biomaterials have been widely explored in innumerable musculoskeletal disorder treatments including osteochondral defects, osteosarcoma, osteoarthritis, ligament rupture, and osteoporosis. Nevertheless, a comprehensive and timely discussion of their applications in musculoskeletal disorders is insufficient. In this review, we emphasize on (1) the main categories and characteristics of mussel foot proteins and their fundamental mechanisms for the spectacular adhesion in mussels; (2) the diverse synthetic methods and modification of various polymers; and (3) the emerging applications of mussel-biomimetic materials, the future perspectives, and challenges, especially in the area of musculoskeletal disorder. We envision that this review will provide a unique and insightful perspective to improve the development of a new generation of mussel biomimetic strategies.

Recent advances in tailoring stimuli-responsive hybrid scaffolds for cardiac tissue engineering and allied applications.

To recapitulate bio-physical properties and functional behaviour of native heart tissues, recent tissue engineering-based approaches are focused on developing smart/stimuli-responsive materials for interfacing cardiac cells. Overcoming the drawbacks of the traditionally used biomaterials, these smart materials portray outstanding mechanical and conductive properties while promoting cell-cell interaction and cell-matrix transduction cues in such excitable tissues. To date, a large number of stimuli-responsive materials have been employed for interfacing cardiac tissues alone or in combination with natural/synthetic materials for cardiac tissue engineering. However, their comprehensive classification and a comparative analysis of the role played by these materials in regulating cardiac cell behaviour and in vivo metabolism are much less discussed. In an attempt to cover the recent advances in fabricating stimuli-responsive biomaterials for engineering cardiac tissues, this review details the role of these materials in modulating cardiomyocyte behaviour, functionality and surrounding matrix properties. Furthermore, concerns and challenges regarding the clinical translation of these materials and the possibility of using such materials for the fabrication of bio-actuators and bioelectronic devices are discussed.

Recent Advances in Animal Models, Diagnosis, and Treatment of Temporomandibular Joint Osteoarthritis.

Temporomandibular joint osteoarthritis (TMJOA) is a gradual degenerative jaw joint condition. Until recent years, TMJOA is still relatively unrecognized and ineffective to be treated. Appropriate animal models with reliable detection methods can help researchers understand the pathophysiology of TMJOA and find therapeutic options. In this study, we summarized common animal models of TMJOA created by chemical, surgical, mechanical, and genetical approaches. The relevant pathological symptoms and induction mechanisms were outlined. In addition, different pathological indicators, furthermore, emerging therapeutic regimens, such as intra-articular drug delivery and tissue engineering-based approaches to treat TMJOA based on these animal models, were summarized and updated. Understanding the physiology and pathogenesis of the TMJOA, together using various ways to diagnose the TMJOA, were elaborated, including imaging techniques, molecular techniques for detecting inflammatory cytokines, histochemical staining, and histomorphometry measures. A more reliable diagnosis will enable the development of new prevention and more effective treatment strategies and thereby improve the quality of life of TMJOA patients. Impact statement Temporomandibular joint osteoarthritis (TMJOA) affects 8 to 16 percent of the population worldwide. However, TMJOA is still relatively unrecognized and ineffective to be treated in the clinic. Appropriate animal models with reliable diagnostic methods can help researchers understand the pathophysiology of TMJOA and find therapeutic options. We herein summarized common animal models of TMJOA and various ways to diagnose the TMJOA. More importantly, emerging therapeutic regimens to treat TMJOA based on these animal models were summarized. With the aid of strategies listed, more effective treatment strategies will be developed and thereby improve the life quality of TMJOA patients.

Mechanical regulation of signal transduction in angiogenesis.

Biophysical and biochemical cues work in concert to regulate angiogenesis. These cues guide angiogenesis during development and wound healing. Abnormal cues contribute to pathological angiogenesis during tumor progression. In this review, we summarize the known signaling pathways involved in mechanotransduction important to angiogenesis. We discuss how variation in the mechanical microenvironment, in terms of stiffness, ligand availability, and topography, can modulate the angiogenesis process. We also present an integrated view on how mechanical perturbations, such as stretching and fluid shearing, alter angiogenesis-related signal transduction acutely, leading to downstream gene expression. Tissue engineering-based approaches to study angiogenesis are reviewed too. Future directions to aid the efforts in unveiling the comprehensive picture of angiogenesis are proposed.

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

Designing Cardiovascular Implants Taking in View the Endothelial Basement Membrane.

Insufficient endothelialization of cardiovascular grafts is a major hurdle in vascular surgery and regenerative medicine, bearing a risk for early graft thrombosis. Neither of the numerous strategies pursued to solve these problems were conclusive. Endothelialization is regulated by the endothelial basement membrane (EBM), a highly specialized part of the vascular extracellular matrix. Thus, a detailed understanding of the structure–function interrelations of the EBM components is fundamental for designing biomimetic materials aiming to mimic EBM functions. In this review, a detailed description of the structure and functions of the EBM are provided, including the luminal and abluminal interactions with adjacent cell types, such as vascular smooth muscle cells. Moreover, in vivo as well as in vitro strategies to build or renew EBM are summarized and critically discussed. The spectrum of methods includes vessel decellularization and implant biofunctionalization strategies as well as tissue engineering-based approaches and bioprinting. Finally, the limitations of these methods are highlighted, and future directions are suggested to help improve future design strategies for EBM-inspired materials in the cardiovascular field.

Non-viral Gene Delivery Methods for Bone and Joints

Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.

Tissue Engineering for Musculoskeletal Regeneration and Disease Modeling.

Musculoskeletal injuries and associated conditions are the leading cause of physical disability worldwide. The concept of tissue engineering has opened up novel approaches to repair musculoskeletal defects in a fast and/or efficient manner. Biomaterials, cells, and signaling molecules constitute the tissue engineering triad. In the past 40years, significant progress has been made in developing and optimizing all three components, but only a very limited number of technologies have been successfully translated into clinical applications. A major limiting factor of this barrier to translation is the insufficiency of two-dimensional cell cultures and traditional animal models in informing the safety and efficacy of in-human applications. In recent years, microphysiological systems, often referred to as organ or tissue chips, generated according to tissue engineering principles, have been proposed as the next-generation drug testing models. This chapter aims to first review the current tissue engineering-based approaches that are being applied to fabricate and develop the individual critical elements involved in musculoskeletal organ/tissue chips. We next highlight the general strategy of generating musculoskeletal tissue chips and their potential in future regenerative medicine research. Exemplary microphysiological systems mimicking musculoskeletal tissues are described. With sufficient physiological accuracy and relevance, the human cell-derived, three-dimensional, multi-tissue systems have been used to model a number of orthopedic disorders and to test new treatments. We anticipate that the novel emerging tissue chip technology will continually reshape and improve our understanding of human musculoskeletal pathophysiology, ultimately accelerating the development of advanced pharmaceutics and regenerative therapies.

Tissue engineering-based approaches to enhance physiological relevancy of cell-based assays

Animal-free and mechanism-based understanding of human body responses is the ultimate goal of alternative to animal experiments. To achieve this goal, integration of advanced cell-based assays using iPS/ES cell technologies with various numerical methods are required. In this review, from the standpoint of tissue engineering, we focused first on the enhancement of physiological relevance of tissue culture models by overcoming the problem between 3D cellular organization and oxygen/nutrient supply. Second, we summarized the concept and actual systems of microfluidic-based body/organ on-a-chip systems, also called as microphysiological system, MPS, particularly for liver on-a-chip systems. Finally, remaining issues were discussed to realize better physiological relevance in vitro.

Could a functional artificial skeletal muscle be useful in muscle wasting?

Regardless of the underlying cause, skeletal muscle wasting is detrimental for a person's life quality, leading to impaired strength, locomotion, and physiological activity. Here, we propose a series of studies presenting tissue engineering-based approaches to reconstruct artificial muscle in vitro and in vivo. Skeletal muscle tissue engineering is attracting more and more attention from scientists, clinicians, patients, and media, thanks to the promising results obtained in the last decade with animal models of muscle wasting. The use of novel and refined biomimetic scaffolds mimicking three-dimensional muscle environment, thus supporting cell survival and differentiation, in combination with well characterized myogenic stem/progenitor cells, revealed the noteworthy potential of these technologies for creating artificial skeletal muscle tissue. In vitro, the production of three-dimensional muscle structures offer the possibility to generate a drug-screening platform for patient-specific pharmacological treatment, opening new frontiers in the development of new compounds with specific therapeutic actions. In vivo, three-dimensional artificial muscle biomimetic constructs offer the possibility to replace, in part or entirely, wasted muscle by means of straight reconstruction and/or by enhancing endogenous regeneration. Reports of tissue engineering approaches for artificial muscle building appeared in large numbers in the specialized press lately, advocating the suitability of this technology for human application upon scaling up and a near future applicability for medical care of muscle wasting. http://links.lww.com/COCN/A9

Engineering natural heart valves: possibilities and challenges.

Heart valve replacement is considered to be the most prevalent treatment approach for cardiac valve-related diseases. Among current solutions for heart valve replacement, e.g. mechanical and bioprosthetic valves, the main shortcoming is the lack of growth capability, repair and remodelling of the substitute valve. During the past three decades, tissue engineering-based approaches have shown tremendous potential to overcome these limitations by the development of a biodegradable scaffold, which provides biomechanical and biochemical properties of the native tissue. Among various scaffolds employed for tissue engineering, the decellularized heart valve (DHV) has attracted much attention, due to its native structure as well as comparable haemodynamic characteristics. Although the human DHV has shown optimal properties for valve replacement, the limitation of valve donors in terms of time and size is their main clinical issue. In this regard, xenogenic DHV can be a promising candidate for heart valve replacement. Xenogenic DHVs have similar composition to human valves, which will overcome the need for human DHVs. The main concern regarding xenogeneic DHV replacement is the immunological reaction and calcification following implantation, weak mechanical properties and insufficient recellularization capacity. In this review, we describe the essential steps required to address these impediments through novel engineering approaches. Copyright © 2016 John Wiley & Sons, Ltd.

Secretome Analysis of Human Mesenchymal Stem Cells Undergoing Chondrogenic Differentiation

Human mesenchymal stem cells (hMSCs) can be triggered to differentiate toward chondrocytes and thus harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we followed a double-stable isotope labeling by amino acids in cell culture (SILAC) strategy to evaluate the quantitative modulation of the secretome of stem cells isolated from bone marrow (hBMSCs) during the first steps of their chondrogenic differentiation. Analysis by LC-ESI-MS/MS led to the identification of 221 proteins with a reported extracellular localization. Most of them were characteristic of cartilage extracellular matrix, and 34 showed statistically significant quantitative alterations during chondrogenesis. These include, among others, cartilage markers such as Proteoglycan 4 or COMP, anticatabolic markers (TIMP1), reported markers of cartilage development (Versican), and a suggested marker of chondrogenesis, CRAC1. Altogether, our work demonstrates the usefulness of secretome analysis for understanding the mechanisms responsible for cartilage matrix formation, and it reports a panel of extracellular markers potentially useful for the evaluation of tissue development in cell therapy- or tissue engineering-based approaches for cartilage repair.

Functional Tissue Engineering of the Liver and Islets

Cell-based therapies by using hepatocytes and islets have recently been evaluated as a new therapeutic modality for patients with many forms of liver diseases and insulin-deficient diabetes mellitus. In most of the recently conducted clinical trials, cells have been delivered into liver vasculatures by infusing them through the portal circulation. More recently, tissue engineering-based approaches have spurred significant interests, using hepatocytes and islets in which small but functional new tissues would be created in vivo. Under circumstances in which a higher level of cell engraftment could be obtained, these approaches could provide therapeutic effects. Considerable efforts have been given to sustaining engineered tissues and maintaining their therapeutic effects. This review highlights several strategies that can achieve a higher level of cell survival for creating new functional liver and islet tissues.

Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.

Hyaluronic acid-human blood hydrogels for stem cell transplantation

Tissue engineering-based approaches have the potential to improve stem cell engraftment by increasing cell delivery to the myocardium. Our objective was to develop and characterize a naturally-derived, autologous, biodegradable hydrogel in order to improve acute stem cell retention in the myocardium. HA-blood hydrogels (HA-BL) were synthesized by mixing in a 1:1(v/v) ratio, lysed whole blood and hyaluronic acid (HA), whose carboxyl groups were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA–NHS). We performed physical characterization and measured survival/proliferation of cardiosphere-derived cells (CDCs) encapsulated in the hydrogels. Hydrogels were injected intra-myocardially or applied epicardially in rats. NHS-activated carboxyl groups in HA react with primary amines present in blood and myocardium to form amide bonds, resulting in a 3D hydrogel bound to tissue. HA-blood hydrogels had a gelation time of 58 ± 12 s, swelling ratio of 10 ± 0.5, compressive and elastic modulus of 14 ± 3 and 1.75 ± 0.6 kPa respectively. These hydrogels were not degraded at 4wks by hydrolysis alone. CDC encapsulation promoted their survival and proliferation. Intra-myocardial injection of CDCs encapsulated in these hydrogels greatly increased acute myocardial retention (p = 0.001). Epicardial application of HA-blood hydrogels improved left ventricular ejection fraction following myocardial infarction (p = 0.01). HA-blood hydrogels are highly adhesive, biodegradable, promote CDC survival and increase cardiac function following epicardial application after myocardial infarction.

NELL-1 Promotes Cartilage Regeneration in an In Vivo Rabbit Model

Repair of cartilage due to joint trauma remains challenging due to the poor healing capacity of cartilage and adverse effects related to current growth factor-based strategies. NELL-1 (Nel-like molecule-1; Nel [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]), a protein first characterized in the context of premature cranial suture fusion, is believed to accelerate differentiation along the osteochondral lineage. We previously demonstrated the ability of NELL-1 protein to maintain the cartilaginous phenotype of explanted rabbit chondrocytes in vitro. Our objective in the current study is to determine whether NELL-1 can affect endogenous chondrocytes in an in vivo cartilage defect model. To generate the implant, NELL-1 was incorporated into chitosan nanoparticles and embedded into alginate hydrogels. These implants were press fit into 3-mm circular osteochondral defects created in the femoral condylar cartilage of 3-month-old New Zealand White rabbits (n=10). Controls included unfilled defects (n=8) and defects filled with phosphate-buffered saline-loaded chitosan nanoparticles embedded in alginate hydrogels (n=8). Rabbits were sacrificed 3 months postimplantation for histological analysis. Defects filled with alginate containing NELL-1 demonstrated significantly improved cartilage regeneration. Remarkably, histology of NELL-1-treated defects closely resembled that of native cartilage, including stronger Alcian blue and Safranin-O staining and increased deposition of type II collagen and absence of the bone markers type I collagen and Runt-related transcription factor 2 (Runx2) as demonstrated by immunohistochemistry. Our results suggest that NELL-1 may produce functional cartilage with properties similar to native cartilage, and is an exciting candidate for tissue engineering-based approaches for treating diverse pathologies of cartilage defects and degeneration.

In vitro Models for Adipose Tissue Engineering with Adipose-Derived Stem Cells Using Different Scaffolds of Natural Origin

Soft tissue regeneration with cell and tissue engineering-based approaches has numerous potential applications in plastic and reconstructive surgery. Adipose-derived stem cells (ASC) have been proved as a feasible source for adipose tissue engineering as they possess high proliferative and differentiation capacity. The purpose of our study was to evaluate adipogenic differentiation of human ASC in four different 3D scaffolds of natural origin, namely human platelet-poor plasma, alginate, fibrin gel and collagen sponge, to define their suitability for adipose tissue engineering and potential clinical applications. ASC were isolated from lipoaspirates of three adult female patients, seeded in the scaffolds, and adipogenic differentiation was induced. After two weeks of cultivation, the constructs were assessed for their mechanical and handling properties, cell viability and adipogenic differentiation. Additionally, the expression of vascular endothelial growth factor (VEGF) was analysed in different culture systems. The results indicate that the levels of specific adipogenic markers and VEGF expression were increased in 3D cultures, as compared to 2D culture. Among 3D scaffolds, fibrin gel showed optimal combination of mechanical characteristics and support of adipogenic differentiation; it was easy to handle, allowed high cell viability, and at the same time supported adipogenic differentiation and VEGF expression.

Are ankle chondrocytes from damaged fragments a suitable cell source for cartilage repair?

To characterize the post-expansion cartilage-forming capacity of chondrocytes harvested from detached fragments of osteochondral lesions (OCLs) of ankle joints (Damaged Ankle Cartilage Fragments, DACF), with normal ankle cartilage (NAC) as control. DACF were obtained from six patients (mean age: 35 years) with symptomatic OCLs of the talus, while NAC were from 10 autopsies (mean age: 55 years). Isolated chondrocytes were expanded for two passages and then cultured in pellets for 14 days or onto HYAFF-11 meshes (FAB, Italy) for up to 28 days. Resulting tissues were assessed histologically, biochemically [glycosaminoglycan (GAG), DNA and type II collagen (CII)] and biomechanically. As compared to NAC, DACF contained significantly lower amounts of DNA (3.0-fold), GAG (5.3-fold) and CII (1.5-fold) and higher amounts of type I collagen (6.2-fold). Following 14 days of culture in pellets, DACF-chondrocytes generated tissues less intensely stained for Safranin-O and CII, with significantly lower GAG contents (2.8-fold). After 28 days of culture onto HYAFF((R))-11, tissues generated by DACF-chondrocytes were less intensely stained for Safranin-O and CII, contained significantly lower amounts of GAG (1.9-fold) and CII (1.4-fold) and had lower equilibrium (1.7-fold) and dynamic pulsatile modulus (3.3-fold) than NAC-chondrocytes. We demonstrated that DACF-chondrocytes have inferior cartilage-forming capacity as compared to NAC-chondrocytes, possibly resulting from environmental changes associated with trauma/disease. The study opens some reservations on the use of DACF-derived cells for the repair of ankle cartilage defects, especially in the context of tissue engineering-based approaches.