Search Results (278)

Background: Transforming growth factor-β (TGF-β) and interleukin 1β (IL-1β) are key regulators of the chondrogenic differentiation, physiology and pathology of cartilage tissue, with TGF-β promoting chondrogenesis and matrix formation, while IL-1β exerts catabolic effects, inhibiting chondrogenesis and contributing to cartilage degradation. Both cytokines alter the intracellular calcium ion (iCa²⁺) levels; however, the exact pathways are not known. Objectives: This study aimed to evaluate the impact of TGF-β3 and IL-1β on calcium homeostasis in human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and chondrocytes during chondrogenesis. Results: TGF-β3 increased iCa²⁺ levels in both hBM-MSCs and chondrocytes. Furthermore, TGF-β3 increased the functional activity of L-type voltage-operated calcium channels (L-VOCCs) in hBM-MSCs but not in chondrocytes. TGF-β3 and IL-1β reduced L-VOCCs subunit CaV1.2 (CACNA1C) gene expression in chondrocytes. In hBM-MSCs, TGF-β3 and IL-1β increased SERCA pump (ATP2A2) gene expression, while in chondrocytes, this effect was observed only with TGF-β3. Conclusions: TGF-β3 increases iCa²⁺ both in osteoarthritic chondrocytes and hBM-MSCs during chondrogenesis. In hBM-MSCs, TGF-β3-mediated elevation in iCa²⁺ is related to the increased functional activity of L-VOCCs. IL-1β does not change iCa²⁺ in osteoarthritic chondrocytes and hBM-MSCs; however, it initiates the mechanisms leading to further downregulation of iCa²⁺ in both types of cells. The differential and cell-specific roles of TGF-β3 and IL-1β in the calcium homeostasis of osteoarthritic chondrocytes and hBM-MSCs during chondrogenesis may provide a new insight into future strategies for cartilage repair and osteoarthritis treatment. Full article

(1) Background: Knee cartilage injury is at the top of the rising concerns among bone and joint disorder patients. Autologous chondrocyte implantation (ACI) is widely used to approach knee cartilage deterioration. Integrating autologous chondrocytes and periosteal patches aids in forming new cartilage-like tissue at the lesion area. This study uses a novel cell source from one-day-old porcine cartilage to fabricate a biomembrane as a substitute for periosteal membranes in cell implantation techniques for treating knee cartilage injuries. (2) Methods: Cells isolated from one-day-old porcine cartilage tissue were identified and assessed for their proliferation capability, differentiation ability, and membrane formation potential. The protein component of the biomembrane was also defined by proteomics. The cartilage repair ability was also confirmed using an in vitro transplantation model. (3) Results: Negative results for porcine infectious diseases are pivotal in selecting suitable piglets to provide cartilage tissue. The cells successfully obtained from one-day-old porcine cartilage exhibited stem-cell-like characteristics (CD34-, CD45-, CD90+, CD105+), including a high proliferation to 20 passages (doubling time: 1–2 days) and a capacity to differentiate into various cell types (osteogenesis, adipogenesis, and chondrogenesis). The stem cells were successfully applied in the fabrication of the biomembranes. The protein components of the biomembrane included an extracellular matrix and growth factors. The in vitro transplantation model showed that the biomembrane induced the repair ability of cartilage defects. (4) Conclusions: This study is the first to successfully harvest stem cells from one-day-old porcine cartilage for biomembrane fabrication for a knee cartilage injury therapeutic application. Full article

Ear reconstruction surgeries for congenital deformities and trauma are common, highlighting the need for improved cartilage regeneration. Lactoferrin (LF), a natural and cost-effective protein, is promising due to its anti-inflammatory, antimicrobial, and prochondrogenic properties. This study investigates the effects of LF on the viability, proliferation, and chondrogenesis of rabbit auricular chondrocytes. For in vitro studies, auricular chondrocytes were cultured for three passages, after which 3D pellets were formed. LF significantly increased chondrocyte metabolic activity by 1.5 times at doses of 10 and 500 μg/mL. At passage 3, LF at concentrations of 10 and 100 μg/mL increased cell proliferation rates by 2- and 1.5-fold, respectively. Immunohistochemical staining of the pellets demonstrated that LF at 10 μg/mL increased the amount of sex-determining region Y-Box Transcription Factor 9 (Sox9)+ cells by 30%, while at 100 μg/mL, it doubled the type II collagen deposits. For in vivo studies, a rabbit ear defect model was utilized. On post-operative day 60, the LF-treated group exhibited more mature cartilage regeneration, with a higher density of elastic fibers. By day 90 post-surgery, LF application led to the restoration of normal elastic cartilage throughout the defect. These findings suggest that LF promotes auricular chondrocytes chondrogenesis and could be beneficial for tissue engineering of the elastic cartilage. Full article

Background/Objectives: Treatment of knee osteoarthritis (OA) with autologous stem cells from microfragmented adipose tissue (MFAT) has shown promising but varying results. Multiple stem cell types, including CD34⁺, CD146⁺, and CD271⁺ stem cells, have been identified within MFAT. Patient-specific heterogeneity in stem cell populations and the content of highly potent cells may be determining factors for a successful treatment outcome. The current study aimed to identify the most promising stem cell type in MFAT to treat OA, focusing on their chondrogenic and osteogenic differentiation performance. Methods: CD34⁺, CD146⁺, and CD271⁺ stem cells from the MFAT of eight patients with knee OA were separated using magnetic-activated cell sorting (MACS) and analyzed as subtypes. Unsorted cells were used as a control. Chondrogenic and osteogenic in vitro differentiation were assessed through Safranin-O and H&E staining, pellet size, and qPCR for chondrogenesis, as well as Alizarin Red S staining and qPCR for osteogenesis. Results: CD34⁺, CD146⁺, and CD271⁺ stem cells were doubled using MACS. All subtypes were able to undergo osteogenic differentiation with Alizarin Red S staining, revealing a significant increase in calcium deposits of induced cells compared to non-induced controls. CD146⁺ stem cells showed higher calcium deposition compared to CD34⁺, CD271⁺, and unsorted stem cells. All cell types could form chondrogenic pellets. CD271⁺ stem cells produced more proteoglycans, as shown by Safranin-O staining, than CD34⁺ and CD146⁺ stem cells, but not more than the unsorted stem cells. After differentiation induction, all cell types showed an upregulation of most chondrogenic and osteogenic biomarkers. Conclusions: CD146⁺ stem cells showed the highest osteogenic differentiation performance for calcium deposition, while CD271⁺ stem cells showed the greatest chondrogenic differentiation performance for proteoglycan formation. The prevalence of these stem cell types may play a critical role in the clinical effectiveness when treating OA. Full article

Extracellular vesicles (EVs), particularly exosomes (EXOs) of various skeletal and stem cells, were shown to delay osteoarthritis (OA) progression, and apoptotic bodies (ABs), another EV subtype, of osteoclasts showed osteoanabolic actions and were involved in the osteoclastic-regulation of local bone formation. Moreover, this study demonstrates that microvesicles (MVs) released by osteoclasts displayed potent pro-chondrogenic, pro-osteogenic, and anti-inflammatory activities. These activities were unique to osteoclastic MVs and were not shared by osteoclastic ABs and EXOs or MVs of other cell types. Because chronic synovial inflammation, progressive articular cartilage erosion, abnormal subchondral bone remodeling, and inability to regenerate articular cartilage are key etiologies of OA, we postulate that the foregoing activities of osteoclastic MVs could simultaneously target multiple etiologies of OA and could thereby be an effective therapy for OA. Accordingly, this study sought to assess the feasibility of an osteoclastic MV-based strategy for OA with a mouse tibial plateau injury model of OA. Briefly, tibial plateau injuries were created on the right knees of adult C57BL/6J mice, MVs were intraarticularly injected into the injured joints biweekly, and the OA progression was monitored histologically at five weeks post-injury. The MV treatment reduced the OA-induced losses of articular cartilage area and thickness, decreased irregularity in the articular cartilage surface, reduced loss of gliding/intermediate zone of articular cartilage, reduced osteophyte formation, suppressed synovial inflammation, and decreased the OARSI OA score. In summary, treatment with osteoclastic MVs delayed or reversed OA progression. Thus, this study supports the feasibility of an osteoclastic MV-based therapy for OA. Full article

In the context of bone fractures, the influence of the mechanical environment on the healing outcome is widely accepted, while its influence at the cellular level is still poorly understood. This study explores the influence of mechanical load on naïve mesenchymal stem cell (MSC) differentiation, focusing on hypertrophic chondrocyte differentiation. Unlike primary bone healing, which involves the direct differentiation of MSCs into bone-forming cells, endochondral ossification uses an intermediate cartilage template that remodels into bone. A high-throughput uniaxial bioreactor system (StrainBot) was used to apply varying percentages of strain on naïve MSCs encapsulated in GelMa hydrogels. This research shows that cyclic uniaxial compression alone directs naïve MSCs towards a hypertrophic chondrocyte phenotype. This was demonstrated by increased cell volumes and reduced glycosaminoglycan (GAG) production, along with an elevated expression of hypertrophic markers such as MMP13 and Type X collagen. In contrast, Type II collagen, typically associated with resting chondrocytes, was poorly detected under mechanical loading alone conditions. The addition of chondrogenic factor TGFβ1 in the culture medium altered these outcomes. TGFβ1 induced chondrogenic differentiation, as indicated by higher GAG/DNA production and Type II collagen expression, overshadowing the effect of mechanical loading. This suggests that, under mechanical strain, hypertrophic differentiation is hindered by TGFβ1, while chondrogenesis is promoted. Biochemical analyses further confirmed these findings. Mechanical deformation alone led to a larger cell size and a more rounded cell morphology characteristic of hypertrophic chondrocytes, while lower GAG and proteoglycan production was observed. Immunohistology staining corroborated the gene expression data, showing increased Type X collagen with mechanical strain. Overall, this study indicates that mechanical loading alone drives naïve MSCs towards a hypertrophic chondrocyte differentiation path. These insights underscore the critical role of mechanical forces in MSC differentiation and have significant implications for bone healing, regenerative medicine strategies and rehabilitation protocols. Full article

The present study investigates the influence of nitrosamines and etoposide on mesenchymal stromal cells (MSCs) in a differentiation state- and biological age-dependent manner. The genotoxic effects of the agents on both neonatal and adult stem cell populations after treatment, before, or during the course of differentiation, and the sensitivity of the different MSC types to different concentrations of MNU or etoposide were assessed. Hereby, the multipotent differentiation capacity of MSCs into osteoblasts, adipocytes, and chondrocytes was analyzed. Our findings reveal that while all cell types exhibit DNA damage upon exposure, neonatal CB-USSCs demonstrate enhanced resistance to genotoxic damage compared with their adult counterparts. Moreover, the osteogenic differentiation of MSCs was more susceptible to genotoxic damage, whereas the adipogenic and chondrogenic differentiation potentials did not show any significant changes upon treatment with genotoxin. Furthermore, we emphasize the cell-specific variability in responses to genotoxic damage and the differences in sensitivity and reaction across different cell types, thus advocating the consideration of these variabilities during drug testing and developmental biological research. Full article

Nanotechnology, delving into the realm of nanometric structures, stands as a transformative force in orthopedics, reshaping diagnostics, and numerous regenerative interventions. Commencing with diagnostics, this scientific discipline empowers accurate analyses of various diseases and implant stability, heralding an era of unparalleled precision. Acting as carriers for medications, nanomaterials introduce novel therapeutic possibilities, propelling the field towards more targeted and effective treatments. In arthroplasty, nanostructural modifications to implant surfaces not only enhance mechanical properties but also promote superior osteointegration and durability. Simultaneously, nanotechnology propels tissue regeneration, with nanostructured dressings emerging as pivotal elements in accelerating wound healing. As we navigate the frontiers of nanotechnology, ongoing research illuminates promising avenues for further advancements, assuring a future where orthopedic practices are not only personalized but also highly efficient, promising a captivating journey through groundbreaking innovations and tailored patient care. Full article

Background/Objectives: Cuminum cyminum L. has been utilized as a medicinal plant for centuries. This research sought to examine the effects of cumin methanolic extract (CMT) on the chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. Methods: Spheroids were generated using human stem cells and cultured with CMT at concentrations between 0 and 1 µg/mL. Morphological assessments and cell viability tests were conducted on days 1 and 3. Chondrogenic differentiation expression was evaluated through quantitative polymerase chain reaction, Western blot, and RNA sequencing. SOX9, FAM20B, COL2A1, and COL1A1 mRNA expression levels were determined using real-time polymerase chain reaction. Protein expression was analyzed via Western blot. Results: Throughout this study, the spheroids maintained their integrity and shape. No significant variations in spheroid diameter were observed among the groups. CMT treatment enhanced the expression of SOX9 and FAM20B. Conclusions: The methanolic extract of Cuminum cyminum facilitated chondrogenic differentiation in human bone marrow-derived mesenchymal stem cells by modulating SOX9 and FAM20B expression. This indicates its potential application in cartilage tissue engineering. Full article

Protein kinase C (PKC) plays an essential role during many biological processes including development from early embryonic stages until the terminal differentiation of specialized cells. This review summarizes the current knowledge about the involvement of PKC in molecular processes during the differentiation of stem/precursor cells into tissue cells with a particular focus on osteogenic, adipogenic, chondrogenic and neuronal differentiation by using a comprehensive approach. Interestingly, studies examining the overall role of PKC, or one of its three isoform groups (classical, novel and atypical PKCs), often showed controversial results. A discrete observation of distinct isoforms demonstrated that the impact on differentiation differs highly between the isoforms, and that during a certain process, the influence of only some isoforms is crucial, while others are less important. In particular, PKCβ inhibits, and PKCδ strongly supports osteogenesis, whereas it is the other way around for adipogenesis. PKCε is another isoform that overwhelmingly supports adipogenic differentiation. In addition, PKCα plays an important role in chondrogenesis, while neuronal differentiation has been positively associated with numerous isoforms including classical, novel and atypical PKCs. In a cellular context, various upstream mediators, like the canonical and non-canonical Wnt pathways, endogenously control PKC activity and thus, their activity interferes with the influence of PKC on differentiation. Downstream of PKC, several proteins and pathways build the molecular bridge between the enzyme and the control of differentiation, of which only a few have been well characterized so far. In this context, PKC also cooperates with other kinases like Akt or protein kinase A (PKA). Furthermore, PKC is capable of directly phosphorylating transcription factors with pivotal function for a certain developmental process. Ultimately, profound knowledge about the role of distinct PKC isoforms and the involved signaling pathways during differentiation constitutes a promising tool to improve the use of stem cells in regenerative therapies by precisely manipulating the activity of PKC or downstream effectors. Full article

Background/Objectives: Three-dimensional bioprinting technology has enabled great advances in the treatment of articular cartilage (AC) defects by the biofabrication of biomimetic constructs that restore and/or regenerate damaged tissue. In this sense, the selection of suitable cells and biomaterials to bioprint constructs that mimic the architecture, composition, and functionality of the natural extracellular matrix (ECM) of the native tissue is crucial. In the present study, a novel cartilage-like biomimetic hybrid construct (CBC) was developed by 3D bioprinting to facilitate and promote AC regeneration. Methods: The CBC was biofabricated by the co-bioprinting of a bioink based on hyaluronic acid (HA) and alginate (AL) loaded with human mesenchymal stromal cells (hMSCs), with polylactic acid supporting the biomaterial, in order to mimic the microenvironment and structural properties of native AC, respectively. The CBC was biologically in vitro characterized. In addition, its physiochemical characteristics were evaluated in order to determine if the presence of hMSCs modified its properties. Results: Results from biological analysis demonstrated that CBC supported the high viability and proliferation of hMSCs, facilitating chondrogenesis after 5 weeks in vitro. The evaluation of physicochemical properties in the CBCs confirmed that the CBC developed could be suitable for use in cartilage tissue engineering. Conclusions: The results demonstrated that the use of bioprinted CBCs based on hMSC-AL/HA-bioink for AC repair could enhance the regeneration and/or formation of hyaline cartilaginous tissue. Full article

Oxytocin (OXT) is a neurohypophysial nonapeptide that exerts its effects mainly through the oxytocin receptor (OXTR). Several studies have pointed out the role of OXT in the modulation of stem cell (SC) fate and properties. SCs are undifferentiated cells characterized by a remarkable ability to self-renew and differentiate into various cell types of the body. In this review, we focused on the role of OXT in SC differentiation. Specifically, we summarize and discuss the scientific research examining the effects of OXT on mesodermal SC-derived lineages, including cardiac, myogenic, adipogenic, osteogenic, and chondrogenic differentiation. The available studies related to the effects of OXT on SC differentiation provide little insights about the molecular mechanism mediated by the OXT–OXTR pathway. Further research is needed to fully elucidate these pathways to effectively modulate SC differentiation and develop potential therapeutic applications in regenerative medicine. Full article

Understanding the molecular mechanisms of differentiation is important for regenerative medicine and developmental biology. This study aims to characterise the role of the glycolysis/oxidative phosphorylation balance as a driver of mesenchymal stem cell (MSC) differentiation. Cells were maintained in normal conditions or stimulated towards the MSC trilineage cell types over 21 days. Multispectral imaging of cell autofluorescence was applied as a non-invasive methodology to continuously image cultures in situ. Spectral signals for collagen, NAD(P)H, and flavins were unmixed. MSCs cultured under chondrogenic conditions exhibited increased collagen levels relative to controls. Following osteogenic induction, MSCs showed increased collagen levels relative to controls during the earlier stages of culture; however, control cells increased their collagen levels as they became confluent. MSCs cultured under adipogenic conditions exhibited lower levels of collagen than controls. The redox ratio (RR; NAD(P)H/flavins) immediately decreased during chondrogenesis, with this early effect persisting throughout the culture compared to control cells, which appeared to increase their RR, similar to osteogenesis. Adipogenesis resulted in a small increase in RR on day 2 relative to control cells, followed by a persistent decrease. Chondrogenic and adipogenic differentiation favoured oxidative phosphorylation, whereas osteogenesis and MSC overgrowth resulted in a glycolytic metabolism. Following consideration of these findings, as well as the diverse reports in the literature, it is concluded that neither enhanced oxidative phosphorylation nor glycolysis are fundamental to the canonical modes of differentiation, and researchers should avoid interpreting shifts as indicating differentiation. Full article

Osteoarthritis is a joint disease that causes pain, stiffness, and reduced joint function because the protective cushioning inside the joints, called cartilage, gradually wears away. This condition is caused by various factors and complex processes in the joint’s environment, involving different types of cells producing factors that can either maintain the joint health or contribute to osteoarthritis. This study aimed to understand the factors influencing both healthy and diseased joints in DDD strategies for the in vitro preconditioning of MSCs. An electronic search in the PubMed, Scopus, and Web of Science databases was carried out using the terms (cartilage OR chondr*) AND (repair OR regeneration OR healing) AND (niche OR microenvironment)) AND (“growth factor” OR GF OR cytokine). Researchers used various methods, including macroscopic examinations, histology, immunohistochemistry, and microCT. Molecules associated with joint inflammation were identified, like macrophage markers, MMP-13, TNF, apoptotic markers, and interleukins. Chondrogenesis-related factors such as aggrecan GAG, collagen type II, and TGF beta family were also identified. This study suggests that balancing certain molecules and ensuring the survival of joint chondrocytes could be crucial in improving the condition of osteoarthritic joints, emphasizing the importance of chondrocyte survival and activity. Future preconditioning methods for MSC- and EV-based therapies can find suitable strategies in the described microenvironments to explore co-culture systems and soluble or extracellular matrix factors. Full article

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium’s ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. Full article