In this study, we aimed at investigating the interactions between primary chondrocytes and mesenchymal stem/stromal cells (MSC) accounting for improved chondrogenesis in coculture systems. Expanded MSC from human bone marrow (BM-MSC) or adipose tissue (AT-MSC) were cultured in pellets alone (monoculture) or with primary human chondrocytes from articular (AC) or nasal (NC) cartilage (coculture). In order to determine the reached cell number and phenotype, selected pellets were generated by combining: (i) human BM-MSC with bovine AC, (ii) BM-MSC from HLA-A2+ with AC from HLA-A2- donors, or (iii) human green fluorescent protein transduced BM-MSC with AC. Human BM-MSC and AC were also cultured separately in transwells. Resulting tissues and/or isolated cells were assessed immunohistologically, biochemically, cytofluorimetrically, and by RT-PCR. Coculture of NC or AC (25%) with BM-MSC or AT-MSC (75%) in pellets resulted in up to 1.6-fold higher glycosaminoglycan content than what would be expected based on the relative percentages of the different cell types. This effect was not observed in the transwell model. BM-MSC decreased in number (about fivefold) over time and, if cocultured with chondrocytes, increased type II collagen and decreased type X collagen expression. Instead, AC increased in number (4.2-fold) if cocultured with BM-MSC and maintained a differentiated phenotype. Chondro-induction in MSC-chondrocyte coculture is a robust process mediated by two concomitant effects: MSC-induced chondrocyte proliferation and chondrocyte-enhanced MSC chondrogenesis. The identified interactions between progenitor and mature cell populations may lead to the efficient use of freshly harvested chondrocytes for ex vivo cartilage engineering or in situ cartilage repair.

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In embryonic models and stem cell systems, mesenchymal cells derived from the neuroectoderm can be distinguished from mesoderm-derived cells by their Hox-negative profile--a phenotype associated with enhanced capacity of tissue regeneration. We investigated whether developmental origin and Hox negativity correlated with self-renewal and environmental plasticity also in differentiated cells from adults. Using hyaline cartilage as a model, we showed that adult human neuroectoderm-derived nasal chondrocytes (NCs) can be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes, including HOXC4 and HOXD8. In contrast to ACs, serially cloned NCs could be continuously reverted from differentiated to dedifferentiated states, conserving the ability to form cartilage tissue in vitro and in vivo. NCs could also be reprogrammed to stably express Hox genes typical of ACs upon implantation into goat articular cartilage defects, directly contributing to cartilage repair. Our findings identify previously unrecognized regenerative properties of HOX-negative differentiated neuroectoderm cells in adults, implying a role for NCs in the unmet clinical challenge of articular cartilage repair. An ongoing phase 1 clinical trial preliminarily indicated the safety and feasibility of autologous NC-based engineered tissues for the treatment of traumatic articular cartilage lesions.

Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1β (IL-1β) on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC), namely cell proliferation, differentiation and maturation/ remodelling of the resulting hypertrophic cartilage. Low doses of IL-1β (50 pg/mL) enhanced colony-forming unitsfibroblastic (CFU-f) and-osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1β finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.

Fractures of the humeral head are frequent and will further increase due to demographic changes. Prior to operative fracture treatment, the regional differences of bone quality, especially of elderly people, have to be carefully considered to assure stable implant fixation. However, conclusive data concerning the variation of histomorphometric parameters are still lacking. Consequently, the purpose of this study was to analyze the age-and sex-related changes in bone microarchitecture. For that reason, 60 proximal humeri were harvested from patients at autopsy. Twelve regions of interest (ROI) were defined for each centered coronar humeral head slice and the specimens were subjected to radiographic, histological, and histomorphometric analyses. We could demonstrate that in contrast to men, women over 60 years of age had a significant age-related decrease in bone mass. The most prominent decrease was observed in the region of the greater tuberosity, which represents an osteoporotic fracture site. The most superior and medially located part of the centered coronar humeral head slice showed, independent from age and sex, the highest bone mass and can therefore be considered as the best location for subchondral screw placement. Taken together, our study revealed distinct sex-related changes of the humeral head bone microarchitecture with aging, which should be considered in implant positioning. ß

Tissue accumulation of p16INK4a-positive senescent cells is associated with age-related disorders, such as osteoarthritis (OA). These cell-cycle arrested cells affect tissue function through a specific secretory phenotype. The links between OA onset and senescence remain poorly described. Using experimental OA protocol and transgenic Cdkn2a+/luc and Cdkn2aluc/luc mice, we found that the senescence-driving p16INK4a is a marker of the disease, expressed by the synovial tissue, but is also an actor: its somatic deletion partially protects against cartilage degeneration. We test whether by becoming senescent, the mesenchymal stromal/stem cells (MSCs), found in the synovial tissue and sub-chondral bone marrow, can contribute to OA development. We established an in vitro p16INK4a-positive senescence model on human MSCs. Upon senescence induction, their intrinsic stem cell properties are altered. When co-cultured with OA chondrocytes, senescent MSC show also a seno-suppressive properties impairment favoring tissue degeneration. To evaluate in vivo the effects of p16INK4a-senescent MSC on healthy cartilage, we rely on the SAMP8 mouse model of accelerated senescence that develops spontaneous OA. MSCs isolated from these mice expressed p16INK4a. Intra-articular injection in 2-month-old C57BL/6JRj male mice of SAMP8-derived MSCs was sufficient to induce articular cartilage breakdown. Our findings reveal that senescent p16INK4a-positive MSCs contribute to joint alteration.

Bone structure and quality are an important parameter in the propensity of bone to fracture. Although the calcaneus is used as diagnostic reference site for osteoporosis by ultrasound, its structure has never been analyzed in detail. The purpose of this study was therefore to histomorphometrically analyze the trabecular microarchitecture of the calcaneus, and to determine whether the calcaneal bone structure is changing with age. Sixty complete human calcanei were harvested from thirty age-and gender-matched patients at autopsy. Each of the three different age groups (group I: 20 to 40, group II: 41 to 60, group III: 61 to 80 years of age) was represented by 20 specimens. The specimens were subjected to radiographic, mCT, and histologic analysis. Bone structure and bone mass of the calcaneus were quantified for three different regions of interest: the anterior ROI, the superior ROI (the subtalar region under the posterior facet), and the posterior ROI. An iliac crest biopsy was obtained from all patients to exclude any metabolic bone disease. Histomorphometric analysis revealed significant differences in bone volume within the calcaneus with highest values in the superior ROI: age group I: 31.3% (27.8-34.8%); II: 25.5% (22.1-28.9%); III: 18.9% (16.6-21.2%) and lowest bone volumes in the anterior ROI; age group I: 6.2% (4.8-7.6%); II: 3.6% (2.1-5.1%); III: 3.9% (2.9-4.9%). There was a significant age-related decrease in bone volume (BV/TV) in aging. Interestingly, this bone loss was most prominent in the superior ROI, with a 39% decrease in BV/TV between age group I and III ( p < 0.001). Qualitative and structural analysis of trabecular number, thickness, and spacing demonstrated that the bone loss in the thalamic portion of the calcaneus was due to the transition of plate-like trabecular elements into a rod-like structure. In conclusion, our study demonstrated that the calcaneus displayed age-related changes in its microarchitecture that are known to reduce the biomechanical stability of trabecular bone, and that the age-related bone loss was most prominent in the region adjacent to the posterior facet (superior ROI). These results suggest that bone mass and structure are risk factors in respect to the occurrence and severity of calcaneal fractures, and indicate that calcaneal fractures are at least in part osteoporotic fractures. ß

Cells deriving from neural crest are generally acknowledged during embryonic development for their multipotency and plasticity, accounting for their capacity to generate various cell and tissue types even across germ layers. At least partial preservation of some of these properties in adulthood makes neural crest derived cells of large interest for regenerative purposes. Chondrocytes from fully mature nasal septum cartilage in adults are also derivatives of neural crest cells and were recently demonstrated to be able not only to maintain functionality across serial cloning, as surrogate self-renewal test, but also to respond and adapt to heterotopic transplantation sites. Based on these findings, cartilage grafts engineered by nasal chondrocytes were clinically used to reconstitute the nasal alar lobule and to repair articular cartilage defects. This article discusses further perspectives of potential clinical utility for nasal chondrocytes in musculoskeletal regeneration. It then highlights the need to derive deeper understanding of their biological properties in order to inform on possible therapeutic modes of action. This acquired knowledge will help to optimise manufacturing conditions to guarantee defined functional traits associated with safety and therapeutic potency of nasal chondrocytes in regenerative medicine.