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Background There is a substantial need for ex vivo cartilage damage models to assess new emerging cartilage repair strategies. Ex vivo cartilage explant models have the advantages of achieving standardized and reproducible experimental conditions while maintaining the cells in their native tissue environment. This study aimed to establish a bovine cartilage damage model to evaluate the safety and efficacy of novel cartilage repair therapies. We hypothesized that combining transient exposure to matrix-degrading enzymes with mechanical loading on bovine cartilage would simulate cartilage damage. Methods Prior to mechanical load, bovine osteochondral plugs underwent a brief 5-minutes treatment with collagenase to induce mild cartilage damage by disrupting the collagen network. To induce a moderate cartilage damage, aggrecanase 1 and aggrecanase 2 were additionally applied to the cartilage for 40 min post-collagenase treatment to degrade aggrecan. Data was analyzed using ANOVA or the Friedman test. Results Observations revealed a statistically significant loss of sulphated glycosaminoglycan (sGAG) using both Collagenase Treatment (CT) and Collagenase and Aggrecanase Treatment (CAT), while chondrocytes viability was maintained. Both treatments resulted in a significantly elevated release of inflammation markers during the initial two days, including IL6 and nitric oxide. Collagenase treatment also significantly increased neo-epitopes of aggrecan compared to the untreated plugs at day 7, suggesting endogenous aggrecanase activation upon collagen network disruption. The additional effect of mechanical loading on cartilage degeneration was also explored in the CT group. Mildly damaged cartilage treated solely with collagenase could withstand 1 h per day of cyclical load, at 10-20% compression of cartilage thickness combined with interfacial shear at 25 degrees. However, higher compression levels (20-40% of cartilage thickness) with the same shear stress regimen led to a significant increase in surface chondrocyte death, with no evidence of TUNEL staining. Conclusions This study establishes a promising model for evaluating cartilage repair strategies, and screening anti-catabolic drugs, particularly overload-related cartilage damage.

Articular cartilage, as a mechanosensitive tissue, supports and distributes various mechanical forces—including compression, shear, hydrostatic pressure, and tensile strain—during joint loading and motion. These external forces deform not only the chondrocytes but also their pericellular matrix and the surrounding extracellular matrix (ECM). Those mechanical cues are detected by mechanosensors on the plasma membrane (e.g., integrins) and transmitted through the cytoskeleton, ultimately being converted into biochemical signals. These signals activate key mechanoresponsive intracellular pathways—including TGF-β-induced SMAD, Rho-GTPase, MAPKs (ERK, JNK, p38), PI3K/AKT/mTOR, MRTF-SRF, and YAP/TAZ—that regulate chondrogenic differentiation and cartilage-specific matrix synthesis. This field of study is known as mechanobiology. Over the past decades, it has gained increasing recognition, particularly with the emergence of tissue-engineering constructs as a novel strategy for cartilage repair. However, progress in chondrogenic mechanobiology has primarily centred on intrinsic substrate- or matrix-derived cues, while overlooking the role of extrinsic mechanical forces. This review therefore provides an updated perspective on chondrogenic mechanobiology, with a particular focus on the cellular responses to external mechanical stimuli. It also emphasizes the therapeutic potential of incorporating mechanical stimulation into tissue-engineering strategies for cartilage repair, an emerging filed referred to as Regenerative Rehabilitation (RR). Since this concept has so far been investigated mainly in vitro, we highlight only those studies and refer to it as In vitro Regenerative Rehabilitation. Moreover, this review also addresses post-traumatic osteoarthritis (PTOA), a common joint disorder that frequently results from traumatic cartilage damage. It explores the mechanobiological mechanisms underlying OA and discusses in vitro regenerative rehabilitation studies, highlighting how external forces could serve as an alternative to conventional biochemical treatments for preventing OA progression.

Fracture non-unions affect many patients worldwide, however, known risk factors alone do not predict individual risk. The identification of novel biomarkers is crucial for early diagnosis and timely patient treatment. This study focused on the identification of microRNA (miRNA) related to the process of fracture healing. Serum of fracture patients and healthy volunteers was screened by RNA sequencing to identify differentially expressed miRNA at various times after injury. The results were correlated to miRNA in the conditioned medium of human bone marrow mesenchymal stromal cells (BMSCs) during in vitro osteogenic differentiation. hsa-miR-1246, hsa-miR-335-5p, and miR-193a-5p were identified both in vitro and in fracture patients and their functional role in direct BMSC osteogenic differentiation was assessed. The results showed no influence of the downregulation of the three miRNAs during in vitro osteogenesis. However, miR-1246 may be involved in cell proliferation and recruitment of progenitor cells. Further studies should be performed to assess the role of these miRNA in other processes relevant to fracture healing.

Background Mechanical stimuli are of crucial importance for the development and maintenance of articular cartilage. For conditioning of cartilaginous tissues, various bioreactor systems have been developed that have mainly aimed to produce cartilaginous grafts for tissue engineering applications. Emphasis has been on in vitro preconditioning, whereas the same devices could be used to attempt to predict the response of the cells in vivo or as a prescreening method before animal studies. As a result of the complexity of the load and motion patterns within an articulating joint, no bioreactor can completely recreate the in vivo situation. Questions/purposes This article aims to classify the various loading bioreactors into logical categories, highlight the response of mesenchymal stem cells and chondrocytes to the various stimuli applied, and determine which data could be used within a clinical setting. Methods We performed a Medline search using specific search terms, then selectively reviewed relevant research relating to physical stimulation of chondrogenic cells in vitro, focusing on cellular responses to the specific load applied. Results There is much data pertaining to increases in chondrogenic gene expression as a result of controlled loading protocols. Uniaxial loading leads to selective upregulation of genes normally associated with a chondrogenic phenotype, whereas multiaxial loading results in a broader pattern of chondrogenic gene upregulation. The potential for the body to be used as an in vivo bioreactor is being increasingly explored. Conclusions Bioreactors are important tools for understanding the potential response of chondrogenic cells within the joint environment. However, to replicate the natural in vivo situation, more complex motion patterns are required to induce more physiological chondrogenic gene upregulation.

Pneumonia, always a major malady, became the main public health and economic disaster of historical proportions with the COVID-19 pandemic. This study was based on a premise that pathology of lung metabolism in inflammation may have features invariant to the nature of the underlying cause. Amino acid uptake by the lungs was measured from plasma samples collected pre-terminally from a carotid artery and vena cava in mice with bleomycin-induced lung inflammation (N = 10) and compared to controls treated with saline instillation (N = 6). In the control group, the difference in concentrations between the arterial and venous blood of the 19 amino acids measured reached the level of statistical significance only for arginine (-10.7%, p = 0.0372) and phenylalanine (+5.5%, p = 0.0266). In the bleomycin group, 11 amino acids had significantly lower concentrations in the arterial blood. Arginine concentration was decreased by 21.1% (p<0.0001) and only that of citrulline was significantly increased (by 20.1%, p = 0.0002). Global Arginine Bioavailability Ratio was decreased in arterial blood by 19.5% (p = 0.0305) in the saline group and by 30.4% (p<0.0001) in the bleomycin group. Production of nitric oxide (NO) and citrulline from arginine by the inducible nitric oxide synthase (iNOS) is greatly increased in the immune system’s response to lung injury. Deprived of arginine, the endothelial cells downstream may fail to provide enough NO to prevent the activation of thrombocytes. Thrombotic-related vascular dysfunction is a defining characteristic of pneumonia, including COVID-19. This experiment lends further support to arginine replacement as adjuvant therapy in pneumonia.

In the field of regenerative medicine, considerable advances have been made from the technological and biological point of view. However, there are still large gaps to be filled regarding translation and application of mesenchymal stromal cell (MSC)-based therapies into clinical practice. Indeed, variables such as cell type, unpredictable donor variation, and expansion/differentiation methods lead to inconsistencies. Most protocols use bovine serum (FBS) derivatives during MSC expansion. However, the xenogeneic risks associated with FBS limits the use of MSC-based products in clinical practice. Herein we compare a chemically defined, xenogeneic-free commercial growth medium with a conventional medium containing 10% FBS and 5 ng/ml FGF2. Furthermore, the effect of a fibronectin-coated growth surface was investigated. The effect of the different culture conditions on chondrogenic commitment was assessed by analyzing matrix deposition and gene expression of common chondrogenic markers. Chondrogenic differentiation potential was similar between the FBS-containing αMEM and the chemically defined medium with fibronectin coating. On the contrary, the use of fibronectin coating with FBS-containing medium appeared to reduce the differentiation potential of MSCs. Moreover, cells that were poorly responsive to in vitro chondrogenic stimuli were shown to improve their differentiation potential after expansion in a TGF-β1 containing medium. In conclusion, the use of a xenogeneic-free medium provides a suitable alternative for human bone marrow MSC expansion, due the capability to maintain cell characteristic and potency. To further improve chondrogenic potential of BMSCs, priming the cells with TGF-β1 during expansion is a promising strategy.

Background Identification of biomarkers to predict the risk of healing delays are of huge clinical interest since 10% of fracture patients progress to delayed or non-union. During endochondral ossification, which takes place in mechanically unstable regions, the bone regenerates through a cartilage intermediate. We previously identified miR-1246, miR-335-5p and miR-193a-5p as fracture-related biomarkers in patient serum, but they appear not to have a functional role in an in vitro model of direct ossification. However, their involvement in other processes related to fracture healing cannot be ruled out and the most common healing process in fracture repair is secondary healing by way of endochondral ossification. Therefore, this study aims to explore the role of miR-1246, miR-335-5p and miR-193a-5p during in vitro endochondral differentiation of human bone marrow-derived mesenchymal stromal cells (BMSCs). Methods The activity of miR-1246, miR-335-5p, and miR-193a-5p was transiently inhibited just before pellet formation and the start of chondrogenic differentiation in human BMSCs ( n  = 5 donors), serving as a model for early endochondral ossification. The effect of miRNA inhibition was assessed by histology (Safranin O/Fast Green), immunohistochemistry (type II and type X collagen), and gene expression analysis by bulk RNA sequencing and RT-qPCR. Results Inhibition of miR-1246 and miR-335-5p enhanced chondrogenic and hypertrophic differentiation in BMSCs from three out of five donors, while miR-193a-5p inhibition had minimal effect. Donors were categorized as “responders” or “non-responders” based on histological and gene expression profiles. RNA sequencing and RT-qPCR identified differentially expressed genes, including a 1.6 and 1.5-fold upregulation of GDF5 and CCN5 respectively ( p  < 0.05) and downregulation of SKIL (fold change: 1.3, p  = 0.0563) after miR-335-5p inhibition, while the same genes were unaltered by miRNA inhibition in non-responders, suggesting donor-specific responses to miRNA inhibition during early chondrogenesis. Conclusions These results suggest that miR-335-5p and miR-1246 have a regulatory effect on endochondral ossification, and genes regulated by miR-335-5p are involved in TGF-β signalling. The function of these miRNAs in human bone formation and repair should be further investigated to validate their potential role as prognostic markers in fracture healing. Graphical Abstract

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14–91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45−CD34−CD73+, CD45−CD34−CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a potential alterative cell source for cartilage tissue engineering applications.

Monocytes and their derived macrophages are found at the site of remodeling tissue, such as fracture hematoma, that is exposed to mechanical forces and have been previously implicated in the reparative response. However, the mechanoresponsive of monocytes and macrophages to skeletal tissue-associated mechanical forces and their subsequent contribution to skeletal repair remains unclear. The aim of this study was to investigate the potential of skeletal tissue-associated loading conditions to modulate human monocyte activation and phenotype. Primary human monocytes or the human monocyte reporter cell line, THP1-Blue, were encapsulated in agarose and exposed to a combination of shear and compressive loading for 1 h a day for 3 consecutive days. Exposure of monocytes to mechanical loading conditions increased their pro-inflammatory gene and protein expression. Exposure of undifferentiated monocytes to mechanical loading conditions significantly upregulated gene expression levels of interleukin(IL)-6 and IL-8 compared to free swelling controls. Additionally, multiaxial loading of unstimulated monocytes resulted in increased protein secretion of TNF-α (17.1 ± 8.9 vs. 8 ± 7.4 pg/ml) and MIP-1α (636.8 ± 471.1 vs. 124.1 ± 40.1 pg/ml), as well as IL-13 (42.1 ± 19.8 vs. 21.7 ± 13.6) compared monocytes cultured under free-swelling conditions. This modulatory effect was observed irrespective of previous activation with the M1/pro-inflammatory differentiation stimuli lipopolysaccharide and interferon-γ or the M2/anti-inflammatory differentiation factor interleukin-4. Furthermore, mechanical shear and compression were found to differentially regulate nitric oxide synthase 2 (NOS2) and IL-12B gene expression as well as inflammatory protein production by THP1-Blue monocytes. The findings of this study indicate that human monocytes are responsive to mechanical stimuli, with a modulatory effect of shear and compressive loading observed toward pro-inflammatory mediator production. This may play a role in healing pathways that are mechanically regulated. An in depth understanding of the impact of skeletal tissue-associated mechanical loading on monocyte behavior may identify novel targets to maximize inflammation-mediated repair mechanisms.