Musculoskeletal diseases represent a major part of medical costs in the Western society. Cartilage defects in the articular cartilage (AC) of the knee joint (KJ) leads to localized osteoarthritis and degeneration of the intervertebral disc (IVD) [1, 2] and are difficult to treat clinically [3–6]. Cartilage in general is considered to lack or to have poor repair capacity because of the avascular tissue structure and poor nutrition of cartilaginous cells provided by diffusion mechanisms [7–9]. Furthermore, the chondrocytes or chondrocyte-like cells are entrapped in the extracellular matrix and have a slow cellular turnover, which contributes to less repopulation of the defect and subsequent regeneration and repair of an injury. In general, articular cartilage has been considered to be a tissue containing one cell type, terminally differentiated chondrocytes. The center part, nucleus pulposus (NP) of a human adult intervertebral disc, contains mainly chondrocyte-like cells surrounded by extracellular matrix, where the most common collagen type is collagen type II and minor fractions of collagen types VI and XI [8, 10]. The NP is enclosed by the annulus fibrous (AF), which consists of cells, microfibrillae, and ring-like structures; the lamellae (collagen type I-rich fibers) [11, 12]. In addition, previous studies detected the presence of cells expressing progenitor/stem cell markers in adult cartilage [13–16], and cells isolated from articular cartilage of KJs have been demonstrated to possess clone-formation capacity in vitro[17, 18].
Limited knowledge exists about the normal regeneration processes in cartilaginous tissues, and increased knowledge in this area is of great importance from clinical and tissue-engineering perspectives. The homeostasis of many tissues is regulated by a balance of proliferation and differentiation of adult stem cells [19–21]. In different tissues, stem cells are located in special anatomic structured microenvironments termed niches. The location and nature of these stem cell niches (SNs) can vary, depending on the tissue type [20, 22]. SNs have been identified in, for example, skin, bone, intestine, and brain (hippocampus) [23–26]. In the niche, stem cells undergo mitosis in a hypothetical asymmetric cell-division pattern, in which one daughter cell gives rise to a more-differentiated progeny, whereas the other cell maintains the original stem cell stage [19, 27, 28].
SNs have been proposed to exist in the intervertebral disc region, located adjacent to the epiphyseal plate (EP) and in the outer zone of the AF . In the knee joint, progenitor cells have been detected in the superficial zone of articular cartilage , and potential stem cell niches (SNs) have been demonstrated in the synovial membrane [30, 31] and in the zone of the Ranvier groove, (adjacent to the EP of the knee joint), which anatomically corresponds to the identified area in the IVD [14, 32, 33].
Currently, limited knowledge exists about the in vivo potential cellular activities, chemoattractants for stem cell recruitment, and migration patterns of progenitor cells originating from these niches (SNs). However, recently in a lapine model, cells that expressed the prechondrocytic markers growth- and differentiation factor-5; GDF5 (GDF5 also termed cartilage-derived morphogenic protein-1); CDMP1 and bone-morphogenic-protein-14; BMP14), SOX9 (sex-determining region Y-box) and, for example, Snail homolog 1, a cytosol protein that is involved in cellular migration, were reported around these niches (SNs) in the IVD region . The chondrogenic lineage marker GDF5 was chosen for the explant studies in the present study because of its role in joint-development and cartilage-regeneration mechanisms [34–36]. In previous studies, it was demonstrated that injection of GDF5 into injured/degenerated IVDs resulted in some positive effects on disc height, improvement in magnetic resonance imaging scores  (bovine and lapine models), and extracellular matrix accumulation  (in vitro study, human cells), as well as in in vivo studies (murine model) .
Detection of cell-proliferation rates as well as slow-cycling cells (for example, stem cells) within different tissues can be performed with a commonly used in vivo labeling technique by using 5-bromo-2-deoxyuridine (BrdU) [22, 40]. BrdU is a thymidine analogue, incorporated into proliferating cells during mitosis [22, 27, 40, 41] when administered, and can thereafter be detected by using antibodies directed toward the BrdU molecule. Cells that do not undergo mitosis during the BrdU exposure period incorporate no BrdU into their DNA. Hence, after the exposure period, the amount of DNA-incorporated BrdU decreases in the labeled cells with each mitotic event, until it decreases below detection level. Thus, in tissues that have rapid cell turnover, BrdU will be detected solely at early time points after labeling. Label-retaining (slow-cycling) cells retain BrdU for a longer period in, for example, the stem cell niches [14, 22, 42], as well as during migration from these niches. These cells can be traced with BrdU-labeling methods [26, 29, 43]. Previously, such migration was reported (lapine model) in the IVD region by using BrdU method, in which a shift of the BrdU+ labeled fraction of cells was observed in different locations, indicating a gradual cellular migration toward the IVD. Sparse BrdU was observed within the AF and NP [29, 42].
Epithelial-mesenchymal transition (EMT) is an evolutionarily well-conserved process, present in many types of organisms, and means that cells dissolve from a certain tissue region and migrate to different locations [44, 45]. EMT is common in many processes, such as activation of immunoreactive cells (for example, in macrophages and in tumor cells that are migrating and developing tumor metastases [46, 47]). During the EMT process, the cytoskeleton of the cells is rearranged to a adapt a more flattened migratory phenotype [48, 49], and Snail homolog 1, a member of the Snail superfamily proteins [50, 51], is one of the key intermediators involved in rearranging events in the cytoskeleton. Different types of fluorochrome compounds can further be used as cell tracers in studies of cellular migration (for example, carboxyfluorescein diacetate or succinimidyl ester (CDFA-SE)). CDFA-SE passively diffuses over the cell membrane and is nonfluorescent until acetate groups of CDFA-SE are cleaved by intracellular esterases to form fluorescent, amine-reactive carboxyfluorescein succinimidyl esters. The succinimidyl ester group reacts with intracellular amines, and fluorescent conjugates are created and well retained within the cytosol.
For tracing of migrating cells, nontoxic iron particles (for example, super-paramagnetic nanoparticles (SPIOs, Endorem (Guerbet, Villepinte, France))) can be used for the detection of tumor metastases in the lymphatic system and for labeling of cells in in vitro and in vivo experiments [52–54].
The aim of the present study was to address the hypothesis of possible cellular migration and migration directions of cells originating from niches potentially involved in tissue maintenance and regeneration of cartilaginous tissues in the intervertebral disc and in the knee-joint regions in adult mammals.