Differentiation Of Mesenchymal Stromal Cells Research Articles

Inhibition of PGE2 in Subchondral Bone Attenuates Osteoarthritis.

Aberrant subchondral bone architecture is a crucial driver of the pathological progression of osteoarthritis, coupled with increased sensory innervation. The sensory PGE2/EP4 pathway is involved in the regulation of bone mass accrual by the induction of differentiation of mesenchymal stromal cells. This study aimed to clarify whether the sensory PGE2/EP4 pathway induces aberrant structural alteration of subchondral bone in osteoarthritis. Destabilization of the medial meniscus (DMM) using a mouse model was combined with three approaches: the treatment of celecoxib, capsaicin, and sensory nerve-specific prostaglandin E2 receptor 4 (EP4)-knockout mice. Cartilage degeneration, subchondral bone architecture, PGE2 levels, distribution of sensory nerves, the number of osteoprogenitors, and pain-related behavior in DMM mice were assessed. Serum and tissue PGE2 levels and subchondral bone architecture in a human sample were measured. Increased PGE2 is closely related to subchondral bone’s abnormal microstructure in humans and mice. Elevated PGE2 concentration in subchondral bone that is mainly derived from osteoblasts occurs in early-stage osteoarthritis, preceding articular cartilage degeneration in mice. The decreased PGE2 levels by the celecoxib or sensory denervation by capsaicin attenuate the aberrant alteration of subchondral bone architecture, joint degeneration, and pain. Selective EP4 receptor knockout of the sensory nerve attenuates the aberrant formation of subchondral bone and facilitates the prevention of cartilage degeneration in DMM mice. Excessive PGE2 in subchondral bone caused a pathological alteration to subchondral bone in osteoarthritis and maintaining the physiological level of PGE2 could potentially be used as an osteoarthritis treatment.

A STAT5-Smad3 dyad regulates adipogenic plasticity of visceral adipose mesenchymal stromal cells during chronic inflammation

Adipogenic differentiation of visceral adipose tissue-resident multipotent mesenchymal stromal cells (VA-MSC) into adipocytes is metabolically protective. Under chronic inflammatory stress, this neoadipogenesis process is suppressed by various pro-inflammatory cytokines and growth factors. However, the underlying mechanism(s) regulating VA-MSC plasticity remains largely unexplored. Using an adipogenic differentiation screen, we identified IFNγ and TGFβ as key inhibitors of primary human VA-MSC differentiation. Further studies using human and mouse VA-MSCs and a chronic high-fat diet-fed murine model revealed that IFNγ/JAK2-activated STAT5 transcription factor is a central regulator of VA-MSC differentiation under chronic inflammatory conditions. Furthermore, our results indicate that under such conditions, IFNγ-activated STAT5 and TGFβ-activated Smad3 physically interact via Smad4. This STAT5–Smad4-Smad3 complex plays a crucial role in preventing the early adipogenic commitment of VA-MSCs by suppressing key pro-adipogenic transcription factors, including CEBPδ, CEBPα, and PPARγ. Genetic or pharmacological disruption of IFNγ-TGFβ synergy by inhibiting either STAT5 or Smad3 rescued adipogenesis under chronic inflammatory stress. Overall, our study delineates a central mechanism of MSC plasticity regulation by the convergence of multiple inflammatory signaling pathways.

CAMP Response Element-Binding Protein Controls the Appearance of Neuron-Like Traits in Chorion Mesenchymal Cells.

Mesenchymal stromal cells (MSC) from bone marrow have been reported to undergo the initial phases of neural differentiation in response to an increase of intracellular cAMP. We investigated the possibility that a similar effect applies to chorion-derived MSC. The intracellular concentration of cAMP was increased either by forskolin, to promote its synthesis, or by inhibitors of its degradation. The consequent reduction in the expression of mesenchymal markers was associated with the appearance of neuron-like morphology in a subset of cells. The effect was measured and characterized using biomarkers and an inhibitor of cAMP response element-binding protein (CREB). The dramatic morphological change induced by all the treatments that promoted intracellular cAMP was transient and peaked on the third day. After that, cells returned to the typical fibroblast-like appearance within 24 hours. The distinctive morphology was associated to the expression of neuregulin 1, doublecortin, neuron-specific class III β-tubulin, and required cAMP response element-binding protein activity. Basic-fibroblast growth factor (b-FGF) treatment increased both the timeframe and number of cells undergoing the morphological change induced by the effect of forskolin. As opposite, arginine-vasopressin (AVP) and sphingosine-1-phosphate (S1P) reduced it. We conclude that cAMP and the ensuing CREB activation trigger a preliminary step towards neuronal differentiation of chorion-derived MSC. However, likewise other MSC, the stimulus is not sufficient to promote stable differentiation.

MiR-100-5p inhibits osteogenic differentiation of human bone mesenchymal stromal cells by targeting TMEM135.

Osteoporosis is a disorder characterized by reduced bone mass, disruption of bone microarchitecture, and a propensity to fracture. The osteogenic differentiation of human bone mesenchymal stromal cells (hBMSCs) exerts a critical effect on preventing bone loss during osteoporosis. Herein, the study recognized miR-100-5p as a deregulated miRNA during osteoporosis (upregulated) and BMSC osteogenic differentiation (downregulated). miR-100-5p was upregulated in osteoporosis patients-isolated BMSCs compared to non-osteoporosis trauma patients-isolated BMSCs. hBMSCs, overexpression inhibited hBMSC proliferation and osteogenic differentiation, whereas miR-100-5p inhibition exerted opposite effects. TMEM135 was downregulated in osteoporosis and upregulated in differentiated osteoblasts, as well as downregulated upon the overexpression of miR-100-5p. MiR-100-5p directly targeted and inhibited TMEM135. In hBMSCs, TMEM135 silencing also inhibited hBMSC osteogenic differentiation. When co-transfected to hBMSCs, antagomir-100-5p promoted, whereas TMEM135 silencing inhibited hBMSC osteogenic differentiation; TMEM135 knockdown dramatically attenuated the effects of miR-100-5p inhibition. Taken together, miR-100-5p forms a regulatory axis with TMEM135 by direct binding. The miR-100-5p/TMEM135 axis modulates hBMSC differentiation into osteoblast. Considering the critical effect of BMSC osteogenesis on osteoporosis, this axis might play a role in osteoporosis, and further in vivo and clinical investigations are required.

Abstract P3081: Glypican 3 Regulates Diabetes Induced Mesenchymal Stromal Cells Dysfunctions

Mesenchymal stromal cells (MSC) based therapies are considered as an ideal stem cell-based treatment for cardiovascular diseases due to their immunosuppressive characteristics, anti-inflammatory properties, and differentiation potential. Diabetic microenvironment encompassing high glucose, inflammation, hypoxic conditions, and reactive oxygen species content has deleterious effects on the functionality of MSCs. The purpose of this study was to understand the molecular basis of diabetes-induced MSC dysfunction and potentially reversing these dysfunctions to enhance cell-based therapeutics for myocardial repair in diabetic patients. we isolated MSCs from bone marrow of mice from db/+ non-diabetic (WT-MSC) and diabetic mice (db/db-MSC) and examined the effect of diabetes on MSC differentiation, proliferation, angiogenesis, and immunomodulation in vitro, and their reparative functions on myocardial injury repair post-MI in mice. Compared to WT-MSC, MSCs isolated from bone marrow of diabetic mice showed impaired differentiation, decreased proliferation, reduced immunomodulatory activities and impairment to promote endothelial cell tubulogenesis. Our data also shows diminished functional activity of diabetic MSCs to improve post-MI cardiac functions. Furthermore, we found that glypican-3 (GPC3), a heparan sulfate proteoglycan, is highly upregulated in diabetic MSCs when compared to WT-MSC. GPC3 overexpression in WT-MSC showed decreased proliferation, lowers the immunosuppression activity, and reduced vascular tube formation. Interestingly, GPC3 knockdown in WT-MSC showed increased proliferation, improved the immunosuppression activity, and enhanced vascular tube formation. Our data also showed that, knockdown of GPC3 in diabetic MSCs restore their functions. In conclusion, these data suggest that diabetes increases GPC3 expression in MSC thereby impairs MSC functions under diabetic condition and knockdown of GPC3 in diabetic MSC rescued diabetes-induced dysfunctions. Ongoing studies are testing whether ex vivo GPC3 knockdown in db/db MSCs will rescue their defective cardiac reparative activities, post-MI.

Mast4 determines the cell fate of MSCs for bone and cartilage development

Mesenchymal stromal cells (MSCs) differentiation into different lineages is precisely controlled by signaling pathways. Given that protein kinases play a crucial role in signal transduction, here we show that Microtubule Associated Serine/Threonine Kinase Family Member 4 (Mast4) serves as an important mediator of TGF-β and Wnt signal transduction in regulating chondro-osteogenic differentiation of MSCs. Suppression of Mast4 by TGF-β1 led to increased Sox9 stability by blocking Mast4-induced Sox9 serine 494 phosphorylation and subsequent proteasomal degradation, ultimately enhancing chondrogenesis of MSCs. On the other hand, Mast4 protein, which stability was enhanced by Wnt-mediated inhibition of GSK-3β and subsequent Smurf1 recruitment, promoted β-catenin nuclear localization and Runx2 activity, increasing osteogenesis of MSCs. Consistently, Mast4−/− mice demonstrated excessive cartilage synthesis, while exhibiting osteoporotic phenotype. Interestingly, Mast4 depletion in MSCs facilitated cartilage formation and regeneration in vivo. Altogether, our findings uncover essential roles of Mast4 in determining the fate of MSC development into cartilage or bone.

Human Platelet Lysate as Alternative of Fetal Bovine Serum for Enhanced Human In Vitro Bone Resorption and Remodeling.

IntroductionTo study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells.MethodsWe compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs.Results and DiscussionSupplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations.ConclusionWe conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models.

Long noncoding RNA ZBTB40-IT1 regulates bone mass by directing the differentiation of human bone marrow mesenchymal stromal cells via the microRNA-514a-3p/FOXO4 axis.

This study intended to clarify the mechanism of long noncoding RNA ZBTB40-IT1 in directing human bone marrow-derived mesenchymal stromal cell (hBMSC) differentiation. hBMSCs underwent osteogenic and adipogenic induction, and an osteoporosis mouse model was established via ovariectomy (OVX). Gain- and loss-of-function approaches were utilized in hBMSCs and mice to investigate the function of ZBTB40-IT1, microRNA (miR)-514a-3p, and forkhead box O4 (FOXO4). Dual-luciferase reporter and RNA pulldown assays were applied to evaluate the binding of miR-514a-3p to ZBTB40-IT1 or FOXO4. The femur of the OVX mice had upregulated ZBTB40-IT1 and FOXO4 expression and downregulated miR-514a-3p expression. The bone mass was increased in OVX mice through ZBTB40-IT1 or FOXO4 knockdown. ZBTB40-IT1 and FOXO4 were downregulated, whereas miR-514a-3p was upregulated in osteogenesis-induced hBMSCs, which was the opposite in adipogenesis-induced hBMSCs. ZBTB40-IT1 or FOXO4 knockdown or miR-514a-3p overexpression increased ARS/ALP absorbance and RUNX2 and OCN levels but decreased fat density and PPARγ and FABP4 levels in hBMSCs. Mechanistically, ZBTB40-IT1 elevated FOXO4 expression by binding to miR-514a-3p. miR-514a-3p inhibition annulled the effects of ZBTB40-IT1 downregulation on hBMSC osteogenesis and adipogenesis, and FOXO4 overexpression abolished the impacts of miR-514a-3p upregulation on hBMSC osteogenesis and adipogenesis. Conclusively, ZBTB40-IT1 inhibition promotes the osteogenic differentiation of hBMSCs via the miR-514a-3p/FOXO4 axis, thereby increasing bone mass.

Serum-Free Cultures: Could They Be a Future Direction to Improve Neuronal Differentiation of Mesenchymal Stromal Cells?

Mesenchymal stem/stromal cells (MSCs) are undifferentiated cells with multilinear potential, known for their immunomodulatory and regenerative properties. Although the scientific community is working to improve their application, concerns limit their use to repair tissues following neurological damage. One of these obstacles is represented by the use of culture media supplemented with fetal bovine serum (FBS), which, due to its xenogenic nature and the risk of contamination, has increased scientific, ethical and safety problems. Therefore, the use of serum-free media could improve MSC culture methods, avoiding infectious and immunogenic transmission problems as well as MSC bioprocesses, without the use of animal components. The purpose of our review is to provide an overview of experimental studies that demonstrate that serum-free cultures, along with the supplementation of growth factors or chemicals, can lead to a more defined and controlled environment, enhancing the proliferation and neuronal differentiation of MSCs.

Craniofacial sutures: Signaling centres integrating mechanosensation, cell signaling, and cell differentiation

Cranial sutures are dynamic structures in which stem cell biology, bone formation, and mechanical forces interface, influencing the shape of the skull throughout development and beyond. Over the past decade, there has been significant progress in understanding mesenchymal stromal cell (MSC) differentiation in the context of suture development and genetic control of suture pathologies, such as craniosynostosis. More recently, the mechanosensory function of sutures and the influence of mechanical signals on craniofacial development have come to the forefront. There is currently a gap in understanding of how mechanical signals integrate with MSC differentiation and ossification to ensure appropriate bone development and mediate postnatal growth surrounding sutures. In this review, we discuss the role of mechanosensation in the context of cranial sutures, and how mechanical stimuli are converted to biochemical signals influencing bone growth, suture patency, and fusion through mediation of cell differentiation. We integrate key knowledge from other paradigms where mechanosensation forms a critical component, such as bone remodeling and orthodontic tooth movement. The current state of the field regarding genetic, cellular, and physiological mechanisms of mechanotransduction will be contextualized within suture biology.

Effect of high content nanohydroxyapatite composite scaffolds prepared via melt extrusion additive manufacturing on the osteogenic differentiation of human mesenchymal stromal cells.

The field of bone tissue engineering seeks to mimic the bone extracellular matrix composition, balancing the organic and inorganic components. In this regard, additive manufacturing (AM) of high content calcium phosphate (CaP)-polymer composites holds great promise towards the design of bioactive scaffolds. Yet, the biological performance of such scaffolds is still poorly characterized. In this study, melt extrusion AM (ME-AM) was used to fabricate poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT)-nanohydroxyapatite (nHA) scaffolds with up to 45wt% nHA, which presented significantly enhanced compressive mechanical properties, to evaluate their in vitro osteogenic potential as a function of nHA content. While osteogenic gene upregulation and matrix mineralization were observed on all scaffold types when cultured in osteogenic media, human mesenchymal stromal cells did not present an explicitly clear osteogenic phenotype, within the evaluated timeframe, in basic media cultures (i.e. without osteogenic factors). Yet, due to the adsorption of calcium and inorganic phosphate ions from cell culture media and simulated body fluid, the formation of a CaP layer was observed on PEOT/PBT-nHA 45wt% scaffolds, which is hypothesized to account for their bone forming ability in the long term in vitro, and osteoconductivity in vivo.

Notoginsenoside R1 Promotes Migration, Adhesin, Spreading, and Osteogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stromal Cells.

Cellular activities, such as attachment, spreading, proliferation, migration, and differentiation are indispensable for the success of bone tissue engineering. Mesenchymal stromal cells (MSCs) are the key precursor cells to regenerate bone. Bioactive compounds from natural products had shown bone regenerative potential. Notoginsenoside R1 (NGR1) is a primary bioactive natural compound that regulates various biological activities, including cardiovascular protection, neuro-protection, and anti-cancer effects. However, the effect of NGR1 on migration, adhesion, spreading, and osteogenic differentiation of MSCs required for bone tissue engineering application has not been tested properly. In this study, we aimed to analyze the effect of NGR1 on the cellular activities of MSCs. Since human adipose-derived stromal cells (hASCs) are commonly used MSCs for bone tissue engineering, we used hASCs as a model of MSCs. The optimal concentration of 0.05 μg/mL NGR1 was biocompatible and promoted migration and osteogenic differentiation of hASCs. Pro-angiogenic factor VEGF expression was upregulated in NGR1-treated hASCs. NGR1 enhanced the adhesion and spreading of hASCs on the bio-inert glass surface. NGR1 robustly promoted hASCs adhesion and survival in 3D-printed TCP scaffold both in vitro and in vivo. NGR1 mitigated LPS-induced expression of inflammatory markers IL-1β, IL-6, and TNF-α in hASCs as well as inhibited the RANKL/OPG expression ratio. In conclusion, the biocompatible NGR1 promoted the migration, adhesion, spreading, osteogenic differentiation, and anti-inflammatory properties of hASCs.

Calcium-magnesium phosphate biphasic microspheres with nutrient microchannels promote angiogenesis and osteogenic differentiation

Vascularisation and mineralisation are indispensable conditions for bone repair materials. The introduction of magnesium ions into calcium phosphate-based materials can give them angiogenic and mineralising capabilities. However, the optimum calcium to magnesium ratio has yet to be determined. Microspheres have received much attention from researchers due to their flexibility, non-agglomeration and other advantages. We prepared calcium-magnesium phosphate biphasic microspheres (CMBS) using the water-in-oil emulsion cross-linking method to achieve osteogenic differentiation and vascularisation. The simultaneous use of the sacrificial template method and heat treatment endow the microspheres with a rich micro-pore structure. In addition, we investigated the compounding ratio of calcium phosphate to magnesium phosphate and determined the optimal compounding ratio (Ca/Mg = 3/1–1/1) for this material. The prepared microspheres promoted the attachment, proliferation and directed differentiation of bone marrow mesenchymal stromal cells (BMSCs) and human umbilical vein cells (HUVECs). CMBS-I (Ca/Mg = 3/1) and CMBS-II (Ca/Mg = 1/1) microspheres showed the best promotion effect among all groups. These microspheres were implanted under the skin of Sprague- Dawley (SD) rats, and the composite microspheres significantly induced vascular regeneration compared to pure phase microspheres.

Fabrication and Effect of Strontium-Substituted Calcium Silicate/Silk Fibroin on Bone Regeneration In Vitro and In Vivo.

Achieving rapid osteogenesis and angiogenesis was the key factor for bone regeneration. In the present study, the strontium-substituted calcium silicate (SrCS)/silk fibroin (SF) composite materials have been constructed by combining the different functional component ratios of SrCS (12.5 wt%, 25 wt%) and SF. Then, the effects of SrCS/SF materials on proliferation, osteogenic differentiation, and angiogenic factor secretion of rat bone marrow-derived mesenchymal stromal cells (rBMSCs) were first evaluated in vitro. Moreover, the in vivo effect of osteogenesis was evaluated in a critical-sized rat calvarial defect model. In vitro studies showed that SrCS/SF significantly enhanced the cell proliferation, alkaline phosphatase (ALP) activity, and the expression of osteogenic and angiogenic factors of rBMSCs as compared with the SF and CS/SF, and the optimum proportion ratio was 25 wt%. Besides, the results also showed that CS/SF achieved enhanced effects on rBMSCs as compared with SF. The in vivo results showed that 25 wt% SrCS/SF could obviously promote new bone formation more than SF and CS/SF. The present study revealed that SrCS could significantly promote the osteogenic and angiogenic activities of SF, and SrCS/SF might be a good scaffold material for bone regeneration.

LncMIR181A1HG is a novel chromatin-bound epigenetic suppressor of early stage osteogenic lineage commitment

Bone formation requires osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) and lineage progression of committed osteoblast precursors. Osteogenic phenotype commitment is epigenetically controlled by genomic (chromatin) and non-genomic (non-coding RNA) mechanisms. Control of osteogenesis by long non-coding RNAs remains a largely unexplored molecular frontier. Here, we performed comprehensive transcriptome analysis at early stages of osteogenic cell fate determination in human MSCs, focusing on expression of lncRNAs. We identified a chromatin-bound lncRNA (MIR181A1HG) that is highly expressed in self-renewing MSCs. MIR181A1HG is down-regulated when MSCs become osteogenic lineage committed and is retained during adipogenic differentiation, suggesting lineage-related molecular functions. Consistent with a key role in human MSC proliferation and survival, we demonstrate that knockdown of MIR181A1HG in the absence of osteogenic stimuli impedes cell cycle progression. Loss of MIR181A1HG enhances differentiation into osteo-chondroprogenitors that produce multiple extracellular matrix proteins. RNA-seq analysis shows that loss of chromatin-bound MIR181A1HG alters expression and BMP2 responsiveness of skeletal gene networks (e.g., SOX5 and DLX5). We propose that MIR181A1HG is a novel epigenetic regulator of early stages of mesenchymal lineage commitment towards osteo-chondroprogenitors. This discovery permits consideration of MIR181A1HG and its associated regulatory pathways as targets for promoting new bone formation in skeletal disorders.

Engineered Cell‐Secreted Extracellular Matrix Modulates Cell Spheroid Mechanosensing and Amplifies Their Response to Inductive Cues for the Formation of Mineralized Tissues (Adv. Healthcare Mater. 10/2022)

Mesenchymal Stromal Cells In article number 2102337 by J. Kent Leach and co-workers, an engineered cell-secreted extracellular matrix (ECM) is incorporated into mesenchymal stromal cell (MSC) spheroids to promote cell–cell and cell–matrix interactions and enhance MSC survival and differentiation. Activation of YAP through integrin binding to the incorporated ECM promotes MSC proliferation and osteogenic differentiation, highlighting the therapeutic potential of ECM-loaded spheroids as building blocks for bone repair.

NF\u03baB inhibition to lift the mechano-competence of mesenchymal stromal cell-derived neocartilage toward articular chondrocyte levels

BackgroundFully functional regeneration of skeletal defects by multipotent progenitor cells requires that differentiating cells gain the specific mechano-competence needed in the target tissue. Using cartilage neogenesis as an example, we asked whether proper phenotypic differentiation of mesenchymal stromal cells (MSC) into chondrocytes in vitro will install the adequate biological mechano-competence of native articular chondrocytes (AC).MethodsThe mechano-competence of human MSC- and AC-derived neocartilage was compared during differentiation for up to 35 days. The neocartilage layer was subjected to physiologic dynamic loading in a custom-designed bioreactor and assayed for mechano-sensitive gene and pathway activation, extracellular matrix (ECM) synthesis by radiolabel incorporation, nitric oxide (NO) and prostaglandin E2 (PGE2) production. Input from different pathways was tested by application of agonists or antagonists.ResultsMSC and AC formed neocartilage of similar proteoglycan content with a hardness close to native tissue. Mechano-stimulation on day 21 and 35 induced a similar upregulation of mechano-response genes, ERK phosphorylation, NO production and PGE2 release in both groups, indicating an overall similar transduction of external mechanical signals. However, while AC maintained or enhanced proteoglycan synthesis after loading dependent on tissue maturity, ECM synthesis was always significantly disturbed by loading in MSC-derived neocartilage. This was accompanied by significantly higher COX2 and BMP2 background expression, > 100-fold higher PGE2 production and a weaker SOX9 stimulation in response to loading in MSC-derived neocartilage. Anabolic BMP-pathway activity was not rate limiting for ECM synthesis after loading in both groups. However, NFκB activation mimicked the negative loading effects and enhanced PGE2 production while inhibition of catabolic NFκB signaling rescued the load-induced negative effects on ECM synthesis in MSC-derived neocartilage.ConclusionsMSC-derived chondrocytes showed a higher vulnerability to be disturbed by loading despite proper differentiation and did not acquire an AC-like mechano-competence to cope with the mechanical stress of a physiologic loading protocol. Managing catabolic NFκB influences was one important adaptation to install a mechano-resistance closer to AC-derived neocartilage. This new knowledge asks for a more functional adaptation of MSC chondrogenesis, novel pharmacologic co-treatment strategies for MSC-based clinical cartilage repair strategies and may aid a more rational design of physical rehabilitation therapy after AC- versus MSC-based surgical cartilage intervention.

Characterization of Induction and Targeting of Senescent Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) from older donors have limited potential for bone tissue formation compared with cells from younger donors, and cellular senescence has been postulated as an underlying cause. There is a critical need for methods to induce premature senescence to study this phenomenon efficiently and reproducibly. However, the field lacks consensus on the appropriate method to induce and characterize senescence. Moreover, we have a limited understanding of the effects of commonly used induction methods on senescent phenotype. To address this significant challenge, we assessed the effect of replicative, hydrogen peroxide, etoposide, and irradiation-induced senescence on human MSCs using a battery of senescent cell characteristics. All methods arrested proliferation and resulted in increased cell spreading compared with low passage controls. Etoposide and irradiation increased expression of senescence-related genes in MSCs at early time points, proinflammatory cytokine secretion, DNA damage, and production of senescence-associated β-galactosidase. We then evaluated the effect of fisetin, a flavonoid and candidate senolytic agent, to clear senescent cells and promote osteogenic differentiation of MSCs entrapped in gelatin methacryloyl (GelMA) hydrogels in vitro. When studying a mixture of nonsenescent and senescent MSCs, we did not observe decreases in senescent markers or increases in osteogenesis with fisetin treatment. However, the application of the same treatment toward a heterogeneous population of human bone marrow-derived cells entrapped in GelMA decreased senescent markers and increased osteogenesis after 14 days in culture. These results identify best practices for inducing prematurely senescent MSCs and motivate the need for further study of fisetin as a senolytic agent. Impact Statement The accumulation of senescent cells within the body has detrimental effects on tissue homeostasis. To study the role of senescent cells on tissue repair and regeneration, there is a need for effective means to induce premature cell senescence. Herein, we characterized the influence of common stressors to induce premature senescence in human mesenchymal stromal cells (MSCs). Irradiation of MSCs resulted in a phenotype most similar to quiescent, high-passage cells. These studies establish key biomarkers for evaluation when studying senescent cells in vitro.

Storage and release of rare earth elements in microsphere-based scaffolds for enhancing osteogenesis

In osteoporosis and diabetes, it is essential to accelerate the bone repair and regeneration process. Trace rare earth elements such as lanthanum (La) ions (La3+) with appropriate concentrations are bioactive and can effectively regulate bone tissue performances. However, few well-established bone tissue engineering scaffolds can precisely and stably release La3+ to promote bone regeneration significantly. Based on the advantages of biodegradable microspheres and microsphere-based scaffolds for controlled drug release, we developed poly(lactide-co-glycolide) (PLGA)-based microsphere-based scaffolds as both three-dimensional (3D) porous scaffolds and La3+ storage and release systems for osteogenesis. So far, there is no study about microsphere-based scaffolds to release trace La3+ to induce osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs). PLGA microspheres co-embedded with La-doped mesoporous silica (LMS) with different amounts of doped La were sintered to prepare the LMS/PLGA (LMSP) microsphere-based scaffold. The La3+ release behavior of LMSP can be controlled by adjusting the doping amount of La in mesoporous silica (MS). All these scaffolds possessed a 3D network architecture. With the increase of La doping, LMSP can better compensate for the pH decrease caused by PLGA degradation. The combination of MS and PLGA can avoid the cytotoxicity of MS alone. All prepared LMSP scaffolds were non-cytotoxic. After BMSCs were implanted on scaffolds, LMSP could promote cells adhesion, proliferation, and osteogenic differentiation. Among these microsphere-based scaffolds, LMSP-3 with stable and higher dose La3+ release behavior showed the strongest ability to enhance the osteogenesis of BMSCs. The results showed that microsphere-based scaffolds with the ability to store and stably control the release of La3+ could effectively improve osteogenic performance, which provides a new idea for the construction of bone tissue engineering scaffolds.

Decoupling the role of chemistry and microstructure in hMSCs response to an osteoinductive calcium phosphate ceramic.

Calcium phosphates (CaP) are widely used synthetic bone graft substitutes, having bioactivity that is regulated by a set of intertwined physico-chemical and structural properties. While some CaPs have shown to be as effective in regenerating large bone defects as autologous bone, there is still the need to understand the role of individual material properties in CaP performance. Here, we aimed to decouple the effects of chemical composition and surface-microstructure of a beta-tricalcium phosphate (TCP) ceramic, with proven osteoinductive potential, on human mesenchymal stromal cells (hMSCs) differentiation. To this end, we replicated the surface structure of the TCP ceramic into polylactic acid without inorganic additives, or containing the chemical constituents of the ceramic, i.e., a calcium salt, a phosphate salt, or TCP powder. The microstructure of the different materials was characterized by confocal laser profilometry. hMSCs were cultured on the materials, and the expression of a set of osteogenic genes was determined. The cell culture medium was collected and the levels of calcium and phosphate ions were quantified by inductively-coupled plasma mass spectrometry. The results revealed that none of the tested combinations of properties in polymer/composite replicas was as potent in supporting the osteogenic differentiation of hMSCs as the original ceramic. Nevertheless, we observed some effects of the surface structure in the absence of inorganics, as well as combined effects of surface structure and the added salts, in particular calcium, on osteogenic differentiation. The approach presented here can be used to study the role of independent properties in other CaP-based biomaterials.