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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. [ABSTRACT FROM AUTHOR]

Background and Objectives: The objective of this study was to evaluate the effects of bisphosphonate (BP) administration on tooth growth, using CT-data of a minipig animal model investigation. Materials and Methods: Tooth growth was evaluated in minipigs, with eight animals receiving weekly zoledronate (ZOL) and three animals serving as the control group. Tooth growth was evaluated at the right 2nd molar (M2) in the maxilla. A computed tomography-based measuring method was applied to evaluate tooth growth in the coronal-apical, buccal-oral and mesial-distal axis. Results: ZOL-administration was found to impact tooth growth in all evaluated measuring axes, with the highest effect observed in the coronal-apical axis. Conclusions: Detrimental effects of BP administration on growing teeth have been reported by a number of investigators. The results of this investigation demonstrate that intravenous ZOL affects the growth of the whole tooth within a short period of administration. With BPs being administered to a growing number of pediatric patients, further studies should be conducted to qualify and quantify the effects of BPs on developing teeth. [ABSTRACT FROM AUTHOR]

Articular cartilage injury and repair is an issue of growing importance. Although common, defects of articular cartilage present a unique clinical challenge due to its poor self-healing capacity, which is largely due to its avascular nature. There is a critical need to better study and understand cellular healing mechanisms to achieve more effective therapies for cartilage regeneration. This article aims to describe the key features of cartilage which is being modelled using tissue engineered cartilage constructs and ex vivo systems. These models have been used to investigate chondrogenic differentiation and to study the mechanisms of cartilage integration into the surrounding tissue. The review highlights the key regeneration principles of articular cartilage repair in healthy and diseased joints. Using co-culture models and novel bioreactor designs, the basis of regeneration is aligned with recent efforts for optimal therapeutic interventions. [ABSTRACT FROM AUTHOR]

In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFβ1 or BMP2, as well as low (2% O2) or high (20% O2) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O2 significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O2 tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O2; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O2 tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population. [ABSTRACT FROM AUTHOR]

Despite the huge body of research on osteogenic differentiation and bone tissue engineering, the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocorticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dexamethasone do not represent a good model, and further research is warranted in this field. [ABSTRACT FROM AUTHOR]

The goal of the present study is to identify the differential expression of circular RNA (circRNA), miRNA, and piwi-interacting RNA (piRNA) after lineage commitment towards osteo- and chondrogenesis of human bone marrow mesenchymal stromal cells (hMSCs). The cells were maintained for 7 days in either osteogenic or chondrogenic medium. RNA sequencing was performed to assess the expression of miRNA and piRNA, while RNA hybridization arrays were used to identify which circRNA were differentially expressed. qPCR validation of a selection of targets for both osteogenic and chondrogenic differentiation was carried out. The differential expression of several circRNA, miRNA, and piRNA was identified and validated. The expression of total and circular isoforms of FKBP5 was upregulated both in osteo- and chondrogenesis and it was influenced by the presence of dexamethasone. ZEB1, FADS2, and SMYD3 were also identified as regulated in differentiation and/or by dexamethasone. In conclusion, we have identified a set of different non-coding RNAs that are differentially regulated in early osteogenic and chondrogenic differentiation, paving the way for further investigation to understand how dexamethasone controls the expression of those genes and what their function is in MSC differentiation. [ABSTRACT FROM AUTHOR]

In this study, 34 Traditional Chinese Medicine (TCM) compounds were screened for potential anabolic and anti-inflammatory properties on human osteoarthritic (OA) chondrocytes. The anabolic effects were assessed by measuring the glycosaminoglycan (GAG) relative to the DNA content using a 3D pellet culture model. The most chondrogenic compounds were tested in an inflammatory model consisting of 3 days of treatment with cytokines (IL-1β/TNF-α) with or without supplementation of TCM compounds. The anti-inflammatory effects were assessed transcriptionally, biochemically and histologically. From the 34 compounds, Vanilic acid (VA), Epimedin A (Epi A) and C (Epi C), 2′′-O-rhamnosylicariside II (2-O-rhs II), Icariin, Psoralidin (PS), Protocatechuicaldehyde (PCA), 4-Hydroxybenzoic acid (4-HBA) and 5-Hydroxymethylfurfural (5-HMF) showed the most profound anabolic effects. After induction of inflammation, pro-inflammatory and catabolic genes were upregulated, and GAG/DNA was decreased. VA, Epi C, PS, PCA, 4-HBA and 5-HMF exhibited anti-catabolic and anti-inflammatory effects and prevented the up-regulation of pro-inflammatory markers including metalloproteinases and cyclooxygenase 2. After two weeks of treatment with TCM compounds, the GAG/DNA ratio was restored compared with the negative control group. Immunohistochemistry and Safranin-O staining confirmed superior amounts of cartilaginous matrix in treated pellets. In conclusion, VA, Epi C, PS, PCA, 4-HBA and 5-HMF showed promising anabolic and anti-inflammatory effects. [ABSTRACT FROM AUTHOR]

(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. [ABSTRACT FROM AUTHOR]

Recent studies highlighting mesenchymal stem cell (MSC) epigenetic memory suggest that a different differentiation medium may be required depending on the tissue of origin. As synovial-derived stem cells (SDSCs) attract interest we aimed to investigate the influence of TGF-β1, BMP-2 and dexamethasone on SDSC chondrogenesis in vitro. We demonstrate that dexamethasone-free medium led to enhanced chondrogenic differentiation at both the mRNA and matrix level. The greatest COL2A1/COL10A1 ratio was detected in cells exposed to a combination medium containing 10 ng/mL BMP-2 and 1 ng/mL TGF-β1 in the absence of dexamethasone, and this was reflected in the total amount of glycosaminoglycans produced. In summary, dexamethasone-free medium containing BMP-2 and TGF-β1 may be the most suitable when using SDSCs for cartilage tissue regeneration. [ABSTRACT FROM AUTHOR]

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair. [ABSTRACT FROM AUTHOR]

The osteocyte network inside the bone matrix is of functional importance and osteocyte cell death is a characteristic feature of pathological bone diseases. Osteocytes have emerged as key regulators of bone tissue maintenance, yet maintaining their phenotype during in vitro culture remains challenging. A 3D co-culture system for osteocytes with osteoblasts was recently presented, enabling the determination of more physiological effects of growth factors on cells in vitro. MLO-Y4 cells were embedded within a type I collagen gel and cultured in the presence of surface MG-63 cells. Co-culture was performed in the presence or absence of TGFβ₃. Gene expression by quantitative PCR, protein expression by fluorescent immunohistochemistry and cell viability tests were performed. The 3D co-culture induced cell differentiation of MG-63 cells seen by increased type I collagen and osteocalcin mRNA expression. TGFβ₃ maintained osteocyte differentiation of MLO-Y4 cells during co-culture as determined by stable E11 and osteocalcin mRNA expression till day 4. Interestingly, most of the effects of TGFβ₃ on co-cultured cells were serum-dependent. Also, TGFβ₃ reduced cell death of 3D co-cultured MLO-Y4 cells in a serum-dependent manner. This study shows that 3D co-culture upregulates differentiation of MG-63 cells to a more mature osteoblast-like phenotype; while the addition of TGFβ₃ maintained the characteristic MLO-Y4 osteocyte-like phenotype and viability in a serum-dependent manner.