Osteogenic Differentiation Of Mesenchymal Stromal Cells Research Articles

Cigarette smoke and nicotine effect on human mesenchymal stromal cell wound healing and osteogenic differentiation capacity.

Mesenchymal stromal cells (MSCs) play a crucial role in promoting tissue regeneration and healing, particularly in bone tissue. Both smoking and nicotine use are known to delay and inhibit the healing process in patients. This study aims at delineating these cellular effects by comparing the impact of nicotine alone to cigarette smoke with equivalent nicotine content, and shedding light on potential differences in the healing process. We examined how cigarette smoke and nicotine affect the migration, proliferation, and osteogenic differentiation of human patient-derived MSCs in vitro, as well as the secretion of cytokines IL-6 and IL-8. We measured nicotine concentration of the cigarette smoke extract (CSE) to clarify the role of the nicotine in the effect of the cigarette smoke. MSCs exposed to nicotine-concentration-standardized CSE exhibited impaired wound healing capability, and at high concentrations, increased cell death. At lower concentrations, CSE dose-dependently impaired migration, proliferation, and osteogenic differentiation, and increased IL-8 secretion. Nicotine impaired proliferation and decreased PINP secretion. While there was a trend for elevated IL-6 levels by nicotine in undifferentiated MSCs, these changes were not statistically significant. Exposure of MSCs to equivalent concentrations of nicotine consistently elicited stronger responses by CSE and had a more pronounced effect on all studied parameters. Our results suggest that the direct effect of cigarette smoke on MSCs contributes to impaired MSC function, that adds to the nicotine effects. Cigarette smoke extract reduced the migration, proliferation, and osteogenic differentiation in MSCs in vitro, while nicotine alone reduced proliferation. Cigarette smoke impairs the osteogenic and regenerative ability of MSCs in a direct cytotoxic manner. Cytotoxic effect of nicotine alone impairs regenerative ability of MSCs, but it only partly explains cytotoxic effects of cigarette smoke. Direct effect of cigarette smoke, and partly nicotine, on MSCs could contribute to the smoking-related negative impact on long-term bone health, especially in bone healing.

The role of toll-like receptors in orchestrating osteogenic differentiation of mesenchymal stromal cells and osteoimmunology

Osteoimmunology is a concept involving molecular and cellular crosstalk between the skeletal and immune systems. Toll-like receptors (TLRs) are widely expressed both on mesenchymal stromal cells (MSCs), the hematopoietic cells, and immune cells in the osteogenic microenvironment for bone development or repair. TLRs can sense both exogenous pathogen-associated molecular patterns (PAMPs) derived from microorganisms, and damage-associated molecular patterns (DAMPs) derived from normal cells subjected to injury, inflammation, or cell apoptosis under physiological or pathological conditions. Emerging studies reported that TLR signaling plays an important role in bone remodeling by directly impacting MSC osteogenic differentiation or osteoimmunology. However, how to regulate TLR signaling is critical and remains to be elucidated to promote the osteogenic differentiation of MSCs and new bone formation for bone tissue repair. This review outlines distinct TLR variants on MSCs from various tissues, detailing the impact of TLR pathway activation or inhibition on MSC osteogenic differentiation. It also elucidates TLR pathways’ interplay with osteoclasts, immune cells, and extracellular vesicles (EVs) derived from MSCs. Furthermore, we explore biomaterial-based activation to guide MSCs’ osteogenic differentiation. Therefore, understanding TLRs’ role in this context has significant implications for advancing bone regeneration and repair strategies.

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.

Bone Marrow Stromal Cells Induce ALDH+ Stem Cell Phenotype and Chemotherapy Resistance in AML Cells through TGF-β1 Mediated Signaling: Reversal By TGFβ Inhibitor Galunisertib

Bone marrow (BM) microenvironment in acute myeloid leukemia (AML) consists of various cell types including mesenchymal stromal cells (MSCs), osteoprogenitor cells, osteoblasts, osteocytes, endothelial, immune and perivascular reticular cells that support AML cell growth and protect them from chemotherapy. We have recently reported that AML cells alter the BM microenvironment by inducing osteogenic differentiation in MSCs to gain growth advantage in the bone marrow (Battula et al, JCI Insight, 2017). However the stroma-induced functional alterations in AML cells are not well understood. Stromal cells induce chemo-resistance in AML cells and as chemo-resistance and high tumorigenic potential are the characteristics of leukemia stem cells, we hypothesize that stromal cells can induce stem cell characteristics in AML cells. To test our hypothesis we co-cultured AML cell lines including OCI-AML3 and HL60 cells with BM-derived MSCs for 3 or 5 days and determined aldehyde dehydrogenase (ALDH) enzyme activity (a well-established marker of hematopoietic and leukemia stem cells) and gene expression by RNA sequencing. We found that the percentage of ALDH+ cells in OCI-AML3 increased from 7% to 89.1% in 3 days and from 23% to 92.3% in 5 days of co-culture with MSCs. Similarly, ALDHwas increased in HL60 cells from 7% to 68% in 3 days and from 20.7% to 67.9% in 5 days of co-culture with MSCs suggesting that stromal cells can induce certain stem cell features in AML cells.Next, to better determine the mechanism, OCI-AML3 cells were co-cultured with BM-MSCs (isolated from 3 different donors) for 5 days, the OCI-AML3 cells were FACS sorted and gene expression was analyzed by RNA sequencing. Using R statistics we identified differentially expressed genes in OCI-AML3 cells and found that gene sets associated with stem cell function including ALDH2, Wnt5b, Wnt5a were up-regulated whereas inflammatory and innate immune response genes were down-regulated in OCI-AML3 cells co-cultured with MSCs compared to cells cultured alone. These genes were validated by qRT-PCR assay. Ingenuity pathway analysis® (IPA) revealed a TGFβ1 activated gene signature in OCI-AML3 cells co-cultured with MSCs compared to cells culture alone. To investigate the role of TGFβ1 in the stroma-induced stem cell phenotype in AML cells, OCI- AML3 and HL60 cells were treated with recombinant TGFβ1 (5ng/ml) for 3 or 5 days. Indeed, treatment with TGFβ1 induced ALDH positive cells from 27.7% to 89.5% in 3 days and from 3.4% to 92.5% in 5 days (OCI-AML3). Similarly, in HL60 cells treated with TGFβ1, ALDH+ cells increased from 9.8% to 25.1 in 3 days suggesting that TGFβ1 induces a stem cell phenotype in AML cells. Next, we measured levels of TGFβ1 in cell culture supernatants from MSCs by ELISA and found that MSCs secrete TGFβ1 up to 1ng/ml into the surrounding medium. In addition, to study the role of TGFβ1 in stroma-induced stem cell activity in AML cells, we treated AML cells with or without TGFβ1 type-1 receptor kinase inhibitor Galunisertib (LY2157299 monohydrate from Eli Lilly®) in the presence or absence of MSCs. Strikingly, treatment of galunisertib at 1µM completely inhibited MSCs-induced ALDH activity in OCI-AML3 and HL60 cells in 5 day co-culture setting. To determine if galunisertib sensitizes AML cells to chemotherapy in the presence of MSCs, we treated OCI-AML3 with galunisertib (1µM) or cytarabine (1µM) or in combination for 72hrs in the presence or absence of MSCs. As expected presence of MSCs inhibited cytarabine induced cell killing (positive for annexin-V and DAPI) from 47.1% to 28.3% cells. In turn, the combination of cytarabine and galunisertib negated this affect and induced cell killing in OCI-AML3 cells from 28% to 54% even in the presence of MSCs suggesting complete loss of MSC-mediated chemo-protection in OCI-AML3 cells upon inhibition of TGF-β signaling. In-vivo testing of galunisertib in combination with cytarabine and doxorubicin in AML PDX models in NSG mice is currently ongoing.Collectively, data indicate that stromal cells induce chemo-resistance in AML cell by inducing stem cells phenotype through TGF-beta mediated signaling (Fig 1). TGF-β inhibition by galunisertib, an agent in early clinical trials in solid tumors inhibits stem cell phenotype and BM microenvironment-induced chemo-resistance and facilitates cell killing of AML cells. [Display omitted] DisclosuresBattula:United Therapeutics Corporation: Research Funding.

Hydrogel mechanics are a key driver of bone formation by mesenchymal stromal cell spheroids

Mesenchymal stromal cells (MSCs) can promote tissue repair in regenerative medicine, and their therapeutic potential is further enhanced via spheroid formation. Stress relaxation of hydrogels has emerged as a potent stimulus to enhance MSC spreading and osteogenic differentiation, but the effect of hydrogel viscoelasticity on MSC spheroids has not been reported. Herein, we describe a materials-based approach to augment the osteogenic potential of entrapped MSC spheroids by leveraging the mechanical properties of alginate hydrogels. Compared to spheroids entrapped in covalently crosslinked elastic alginate, calcium deposition of MSC spheroids was consistently increased in ionically crosslinked, viscoelastic hydrogels. We previously demonstrated that intraspheroidal presentation of Bone Morphogenetic Protein-2 (BMP-2) on hydroxyapatite (HA) nanoparticles resulted in more spatially uniform MSC osteodifferentiation, providing a method to internally influence spheroid phenotype. In these studies, we observed significant increases in calcium deposition by MSC spheroids loaded with BMP-2-HA in viscoelastic gels compared to soluble BMP-2, which was greater than spheroids entrapped in all elastic alginate gels. Upon implantation in critically sized calvarial bone defects, bone formation was greater in all animals treated with viscoelastic hydrogels. Increases in bone formation were evident in viscoelastic gels, regardless of the mode of presentation of BMP-2 (i.e., soluble delivery or HA nanoparticles). These studies demonstrate that the dynamic mechanical properties of viscoelastic alginate are an effective strategy to enhance the therapeutic potential of MSC spheroids for bone formation and repair.

3D additive manufactured composite scaffolds with antibiotic-loaded lamellar fillers for bone infection prevention and tissue regeneration

Bone infections following open bone fracture or implant surgery remain a challenge in the orthopedics field. In order to avoid high doses of systemic drug administration, optimized local antibiotic release from scaffolds is required. 3D additive manufactured (AM) scaffolds made with biodegradable polymers are ideal to support bone healing in non-union scenarios and can be given antimicrobial properties by the incorporation of antibiotics. In this study, ciprofloxacin and gentamicin intercalated in the interlamellar spaces of magnesium aluminum layered double hydroxides (MgAl) and α-zirconium phosphates (ZrP), respectively, are dispersed within a thermoplastic polymer by melt compounding and subsequently processed via high temperature melt extrusion AM (~190 °C) into 3D scaffolds. The inorganic fillers enable a sustained antibiotics release through the polymer matrix, controlled by antibiotics counterions exchange or pH conditions. Importantly, both antibiotics retain their functionality after the manufacturing process at high temperatures, as verified by their activity against both Gram + and Gram - bacterial strains. Moreover, scaffolds loaded with filler-antibiotic do not impair human mesenchymal stromal cells osteogenic differentiation, allowing matrix mineralization and the expression of relevant osteogenic markers. Overall, these results suggest the possibility of fabricating dual functionality 3D scaffolds via high temperature melt extrusion for bone regeneration and infection prevention.

Influence of platelet storage time on human platelet lysates and platelet lysate-expanded mesenchymal stromal cells for bone tissue engineering

BackgroundHuman platelet lysate (HPL) is emerging as the preferred xeno-free supplement for the expansion of mesenchymal stromal cells (MSCs) for bone tissue engineering (BTE) applications. Due to a growing demand, the need for standardization and scaling-up of HPL has been highlighted. However, the optimal storage time of the source material, i.e., outdated platelet concentrates (PCs), remains to be determined. The present study aimed to determine the optimal storage time of PCs in terms of the cytokine content and biological efficacy of HPL.MethodsDonor-matched bone marrow (BMSCs) and adipose-derived MSCs (ASCs) expanded in HPL or fetal bovine serum (FBS) were characterized based on in vitro proliferation, immunophenotype, and multi-lineage differentiation. Osteogenic differentiation was assessed at early (gene expression), intermediate [alkaline phosphatase (ALP) activity], and terminal stages (mineralization). Using a multiplex immunoassay, the cytokine contents of HPLs produced from PCs stored for 1–9 months were screened and a preliminary threshold of 4 months was identified. Next, HPLs were produced from PCs stored for controlled durations of 0, 1, 2, 3, and 4 months, and their efficacy was compared in terms of cytokine content and BMSCs’ proliferation and osteogenic differentiation.ResultsBMSCs and ASCs in both HPL and FBS demonstrated a characteristic immunophenotype and multi-lineage differentiation; osteogenic differentiation of BMSCs and ASCs was significantly enhanced in HPL vs. FBS. Multiplex network analysis of HPL revealed several interacting growth factors, chemokines, and inflammatory cytokines. Notably, stem cell growth factor (SCGF) was detected in high concentrations. A majority of cytokines were elevated in HPLs produced from PCs stored for ≤ 4 months vs. > 4 months. However, no further differences in PC storage times between 0 and 4 months were identified in terms of HPLs’ cytokine content or their effects on the proliferation, ALP activity, and mineralization of BMSCs from multiple donors.ConclusionsMSCs expanded in HPL demonstrate enhanced osteogenic differentiation, albeit with considerable donor variation. HPLs produced from outdated PCs stored for up to 4 months efficiently supported the proliferation and osteogenic differentiation of MSCs. These findings may facilitate the standardization and scaling-up of HPL from outdated PCs for BTE applications.

MiR-1323 suppresses bone mesenchymal stromal cell osteogenesis and fracture healing via inhibiting BMP4/SMAD4 signaling

BackgroundAtrophic non-union fractures show no radiological evidence of callus formation within 3 months of fracture. microRNA dysregulation may underlie the dysfunctional osteogenesis in atrophic non-union fractures. Here, we aimed to analyze miR-1323 expression in human atrophic non-union fractures and examine miR-1323’s underlying mechanism of action in human mesenchymal stromal cells.MethodsHuman atrophic non-union and standard healing fracture specimens were examined using H&E and Alcian Blue staining, immunohistochemistry, qRT-PCR, immunoblotting, and ALP activity assays. The effects of miR-1323 mimics or inhibition on BMP4, SMAD4, osteogenesis-related proteins, ALP activity, and bone mineralization were analyzed in human mesenchymal stromal cells. Luciferase reporter assays were utilized to assay miR-1323’s binding to the 3'UTRs of BMP4 and SMAD4. The effects of miR-1323, BMP4, and SMAD4 were analyzed by siRNA and overexpression vectors. A rat femur fracture model was established to analyze the in vivo effects of antagomiR-1323 treatment.ResultsmiR-1323 was upregulated in human atrophic non-union fractures. Atrophic non-union was associated with downregulation of BMP4 and SMAD4 as well as the osteogenic markers ALP, collagen I, and RUNX2. In vitro, miR-1323 suppressed BMP4 and SMAD4 expression by binding to the 3'UTRs of BMP4 and SMAD4. Moreover, miR-1323’s inhibition of BMP4 and SMAD4 inhibited mesenchymal stromal cell osteogenic differentiation via modulating the nuclear translocation of the transcriptional co-activator TAZ. In vivo, antagomiR-1323 therapy facilitated the healing of fractures in a rat model of femoral fracture.ConclusionsThis evidence supports the miR-1323/BMP4 and miR-1323/SMAD4 axes as novel therapeutic targets for atrophic non-union fractures.

Matrine attenuates heterotopic ossification by suppressing TGF-β induced mesenchymal stromal cell migration and osteogenic differentiation

Purpose of the studyHeterotopic ossification (HO) is a debilitating disease characterized by extraskeletal bone formation. Active TGF-β recruits mesenchymal stromal cells (MSCs), which contribute to trauma-induced HO. Inhibiting TGF-β induced MSC migration and osteogenic differentiation could be a promising treatment for HO. Matrine is an alkaloid from the genus Sophora that can suppress pancreatic and hepatic fibrosis by regulating TGF-β/Smad signaling. We conducted this study to evaluate the effects of matrine on HO and explore the mechanisms, we carried out this study. Materials and methodsAchilles tendon puncture was performed in C57BL/6J male mice to establish the HO model. Following treatment with matrine for 3, 6, 9, and 15 weeks, mice were sacrificed and tendons were collected. In vivo, micro-CT, hematoxylin and eosin staining, CD73 and CD90 immunofluorescence, and osteocalcin staining were used to evaluate the development of HO. In vitro, a transwell migration assay was used to evaluate MSC migration. Immunohistochemistry, immunofluorescence and western blotting were used to evaluate the TGF-β/Smad2/3 pathway. Real-time PCR was conducted to analyze the transcription of alkaline phosphatase (Alp), runt-related transcription factor-2 (Runx2), osteocalcin (Ocn), osteopontin (Opn), and type I collagen (Col1). ALP activity and alizarin red staining were used to assess MSC osteogenic differentiation. ResultsIn vivo, matrine significantly reduced ossification and inhibited HO progression. In vitro, matrine significantly suppressed MSC migration, ALP activity, and mineralization of MSCs. Mechanistically, matrine inhibited TGF-β induced Smad2/3 phosphorylation and transcription of Runx2, Alp, and Ocn after osteoinduction. ConclusionsMatrine inhibited HO by suppressing the migration and osteogenic differentiation of TGF-β-induced-MSCsin mice.

Experimental Study of the Possibility of Culturing of Mesenchymal Stromal Cell and Induction of Osteogenic Differentiation on Collagen-Based Scaffolds of Various Modifications.

We analyzed the suitability of various collagen-based scaffolds for culturing and osteogenic differentiation of mesenchymal stromal cells (MSC). Decellularized and lyophilized swine intestinal submucosa (SIS) and porous collagen sponge made from reconstructed bovine derma (PCS) were the most effective in promoting MSC adhesion, survival, and growth. MSC from rat and mouse bone marrow and rat adipose tissue successfully adhered to the scaffold surface and penetrated into its deep layers. These scaffolds were also the most effective in inducing osteogenesis. These results indicate that microarchitectonics of PCS and SIS is optimal for support of MSC growth and osteogenic differentiation.

Neuroblastoma-secreted exosomes carrying miR‐375 promote osteogenic differentiation of bone-marrow mesenchymal stromal cells

ABSTRACT Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.

Load-induced osteogenic differentiation of mesenchymal stromal cells is caused by mechano-regulated autocrine signaling.

Mechanical boundary conditions critically influence the bone healing process. In this context, previous in vitro studies have demonstrated that cyclic mechanical compression alters migration and triggers osteogenesis of mesenchymal stromal cells (MSC), both processes being relevant to healing. However, it remains unclear whether this mechanosensitivity is a direct consequence of cyclic compression, an indirect effect of altered supply or a specific modulation of autocrine bone morphogenetic protein (BMP) signaling. Here, we investigate the influence of cyclic mechanical compression (ε=5% and 10%, f=1Hz) on human bone marrow MSC (hBMSC) migration and osteogenic differentiation in a 3D biomaterial scaffold, an in vitro system mimicking the mechanical environment of the early bone healing phase. The open-porous architecture of the scaffold ensured sufficient supply even without cyclic compression, minimizing load-associated supply alterations. Furthermore, a large culture medium volume in relation to the cell number diminished autocrine signaling. Migration of hBMSCs was significantly downregulated under cyclic compression. Surprisingly, a decrease in migration was not associated with increased osteogenic differentiation of hBMSCs, as the expression of RUNX2 and osteocalcin decreased. In contrast, BMP2 expression was significantly upregulated. Enabling autocrine stimulation by increasing the cell-to-medium ratio in the bioreactor finally resulted in a significant upregulation of RUNX2 in response to cyclic compression, which could be reversed by rhNoggin treatment. The results indicate that osteogenesis is promoted by cyclic compression when cells condition their environment with BMP. Our findings highlight the importance of mutual interactions between mechanical forces and BMP signaling in controlling osteogenic differentiation.

Pooling of Patient-Derived Mesenchymal Stromal Cells Reduces Inter-Individual Confounder-Associated Variation without Negative Impact on Cell Viability, Proliferation and Osteogenic Differentiation.

Patient-derived mesenchymal stromal cells (MSCs) play a key role in bone tissue engineering. Various donor-specific factors were identified causing significant variability in the biological properties of MSCs impairing quality of data and inter-study comparability. These limitations might be overcome by pooling cells of different donors. However, the effects of pooling on osteogenic differentiation, proliferation and vitality remain unknown and have, therefore, been evaluated in this study. MSCs of 10 donors were cultivated and differentiated into osteogenic lineage individually and in a pooled setting, containing MSCs of each donor in equal parts. Proliferation was evaluated in expansion (assessment of generation time) and differentiation (quantification of dsDNA content) conditions. Vitality was visualized by a fluorescence-microscopy-based live/dead assay. Osteogenic differentiation was assessed by quantification of alkaline phosphatase (ALP) activity and extracellular calcium deposition. Compared to the individual setting, generation time of pooled MSCs was shorter and proliferation was increased during differentiation with significantly lower variances. Calcium deposition was comparable, while variances were significantly higher in the individual setting. ALP activity showed high variance in both groups, but increased comparably during the incubation period. In conclusion, MSC pooling helps to compensate donor-dependent variability and does not negatively influence MSC vitality, proliferation and osteogenic differentiation.

Oxidized alginate beads for tunable release of osteogenically potent mesenchymal stromal cells.

Bone defect repair can benefit from local delivery of mesenchymal stromal cells (MSCs). However, local harsh environmental conditions after injury may necessitate a cell therapy strategy that shields MSCs initially and releases them locally over time. This may be possible by using biomaterials that exhibit stimuli-responsive degradability, such as oxidized alginate hydrogels that undergo hydrolytic degradation. However, it remains unknown whether varying encapsulation periods compromise MSC osteogenic differentiation capacity after release. To address this, we cultured MSCs in 3D alginate beads with tunable degradability before characterizing the function of released cells. Alginates were oxidized to different degrees (2%, 3%, and 4%) to achieve distinct rates of degradation (days to weeks), then functionalized with RGD peptides to enable cell adhesion, and modified additionally with 6-aminofluorescin to enable fluorescence-based detection. Bead morphology, degradation kinetics, cell morphology, and cell release kinetics were monitored over time. Cells that were released from the beads were stimulated to differentiate into the osteogenic lineage. Our results indicate that MSCs released from all bead groups retained a strong ability to deposit mineralized matrix under osteogenic differentiation conditions. These findings provide the basis for designing and implementing biomaterial-based strategies for the in-situ temporal delivery of potent MSCs at bone defect sites.

Generating biomimetic mineralized collagen scaffolds for bone regeneration

Background & Aim In bone healing, osteoblasts derived from periosteal mesenchymal stromal cells (MSCs) form new bone. Osteoblastic differentiation from MSCs (osteoinduction), is partly driven by the components and mechanics of the extracellular matrix (ECM). Bone ECM comprises fibrillar collagen with apatitic mineral in gap zones between collagen molecules (intrafibrillar) and surrounding fibrils (extrafibrillar). This project aims to optimize mineralization conditions for fabricating bone biomimetic hydroxyapatite (HA)-collagen scaffolds. These biomimetic scaffolds will later be tested for their osteoinductive potential in mesenchymal stromal cells (MSCs). Methods, Results & Conclusion Methods EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-fixed fibrillar collagen scaffolds were mineralized with a metastable, supersaturated Ca and P solution for 2, 4, 6, and 8 days. This process permits biomimetic, intrafibrillar mineralization seen in native bone. Non-biomimetic scaffolds were mineralized in an unstable, supersaturated Ca and P solution. Scanning electron microscopy (SEM) assessed mineral morphology and location, and backscattered electrons detected heavier elements such as Ca and P. Transmission electron microscopy (TEM) identified HA and its alignment with selected area electron diffraction (SAED). To compare osteoinduction by biomimetic scaffolds vs. non-biomimetic scaffolds, qRT-PCR and immunofluorescent staining will be used to quantify gene and protein expression of early (ie. type I collagen and fibronectin) vs. late osteoblastic markers (ie. alkaline phosphatase and osteocalcin). Biomimetic scaffolds are expected to induce greater expression of later osteoblast markers in MSCs compared to non-biomimetic scaffolds. Results Biomimetic scaffolds mineralized for 6 and 8 days exhibited unaligned, extrafibrillar mineral compared to those mineralized for 4 days (Fig.1). SAED confirmed aligned, intrafibrillar mineral. Scaffolds mineralized for 2 days exhibited no detectable mineralization with SEM and backscatter SEM. Non-biomimetic scaffolds demonstrated extrafibrillar amorphous and crystalline mineral phases. Conclusion Mineralization for 4 days with pAsp is the optimal time point for biomimetic mineralized collagen scaffolds for future studies regarding osteogenic differentiation in MSCs. Ultimately, this biomimetic approach is expected to enhance the osteoinductivity of scaffolds as alternatives to autografts in the treatment of non-union fractures.

Coculture with monocytes/macrophages modulates osteogenic differentiation of adipose-derived mesenchymal stromal cells on poly(lactic-co-glycolic) acid/polycaprolactone scaffolds.

The effects of immune cells, in particular macrophages, on the behaviour of mesenchymal stromal cells (MSCs) have recently gained much attention for MSCs‐based tissue‐engineered constructs. This study aimed to evaluate the effect of monocytes/macrophages on the osteogenic differentiation of adipose‐derived mesenchymal stromal cells (ADMSCs) in three‐dimensional (3D) cocultures. For this, we cocultured THP‐1 monocytes, M1 macrophages, or M2 macrophages with ADMSCs on 3D poly(lactic‐co‐glycolic) acid (PLGA)/polycaprolactone (PCL) scaffolds using osteogenic medium for up to 42 days. We found that osteogenic differentiation of ADMSCs was inhibited by monocytes and both macrophage subtypes in 3D scaffolds. Furthermore, coculture of monocytes/macrophages with ADMSCs resulted in downregulated secretion of oncostatin M (OSM) and bone morphogenetic protein 2 (BMP‐2) and inhibited expression of osteogenic markers alkaline phosphatase (ALP), bone sialoprotein (BSP), and runt‐related transcription factor 2 (RUNX2). Compared with both macrophage subtypes, monocytes inhibited osteogenic differentiation of ADMSCs more significantly. These data suggest that the mutual interactions between monocytes/macrophages and ADMSCs negatively affect MSC osteogenic differentiation and thus possibly bone healing capacity, which highlights the importance of the micro‐environment in influencing cell‐based constructs to treat bone defects and the potential to improve their performance by resolving the inflammation ahead of treatment.

α2-HS Glycoprotein in Plasma Extracellular Vesicles Inhibits the Osteogenic Differentiation of Human Mesenchymal Stromal Cells In Vitro.

Extracellular vesicles (Evs) contain diverse functional proteins, mRNAs, miRNAs, and DNA fragments, are secreted by various types of cells, and play important roles in cellular communication. Here, we show for the first time that plasma Evs inhibited the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro and the level of inhibition was positively correlated with the plasma Evs concentration. Plasma Evs downregulated the expression of markers such as osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), and Osterix at mRNA levels required for osteogenic differentiation and reduced pSmad1/5/8 levels in MSCs. Furthermore, pSmad1/5/8 levels increased and MSCs underwent normal osteogenic differentiation after Evs-derived α2-HS glycoprotein (AHSG) function was inhibited with an anti-AHSG neutralizing antibody. However, the levels of pERK1/2, active β-catenin, and HES1 were not significantly altered. Therefore, we propose that as essential components of the extracellular microenvironment of MSCs, plasma Evs are taken up by MSCs and subsequently repress osteogenic differentiation through an AHSG-mediated decrease in pSmad1/5/8 levels. Our work identifies plasma Evs as novel regulators of MSC osteogenic differentiation.

Dosage and composition of bioactive glasses differentially regulate angiogenic and osteogenic response of human MSCs.

Vascularization of the fracture site and cell-mediated deposition of the mineralized matrix are crucial determinants for successful bone regeneration after injury. Ceramic biomaterials such as bioactive glasses (BAGs) that release bioactive ions have shown promising results in bone defect regeneration. However, it remains unclear how the dosage and composition of bioactive ions influence the angiogenic and osteogenic behavior of primary human mesenchymal stromal cells (MSCs). Here, we show that exposure to ionic dissolution products from 1393 and 45S5 BAGs can evoke distinct angiogenic and osteogenic responses from primary MSCs in a dose- and composition-dependent manner. Significantly higher concentrations of the pro-angiogenic factors VEGF, HGF, PIGF, angiopoietin, and angiogenin were detected in conditioned media (CM) from MSCs exposed to 45S5, but not 1393, BAGs. Application of this CM to human umbilical vein endothelial cells (HUVECs) resulted in robust 2D tube formation in vitro. Osteogenic differentiation of MSCs was assessed by gene expression analysis and mineralization assays. Low concentrations (0.1% w/v) of 1393 BAGs significantly enhanced the gene expression of RUNX2 and ALP and induced an earlier onset of matrix mineralization compared to all other groups. We further tested whether simultaneous exposure to both BAGs would improve both angiogenic secretion and osteogenic differentiation of MSCs, and did not find evidence to support this hypothesis. Our results provide evidence of BAG composition-dependent enhancement of primary human MSCs' regenerative function, besides also underlining the importance of an in vitro evaluation of the dose-response relationship to translate BAG based approaches into safe and effective clinical therapies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2827-2837, 2018., 2018.

Human Osteoblast-Derived Extracellular Matrix with High Homology to Bone Proteome Is Osteopromotive.

Efficient osteogenic differentiation of mesenchymal stromal cells (MSCs) is crucial to accelerate bone formation. In this context, the use of extracellular matrix (ECM) as natural 3D framework mimicking in vivo tissue architecture is of interest. The aim of this study was to generate a devitalized human osteogenic MSC-derived ECM and to investigate its impact on MSC osteogenic differentiation to improve MSC properties in bone regeneration. The devitalized ECM significantly enhanced MSC adhesion and proliferation. Osteogenic differentiation and mineralization of MSCs on the ECM were quicker than in standard conditions. The presence of ECM promoted in vivo bone formation by MSCs in a mouse model of ectopic calcification. We analyzed the ECM composition by mass spectrometry, detecting 846 proteins. Of these, 473 proteins were shared with the human bone proteome we previously described, demonstrating high homology to an in vivo microenvironment. Bioinformatic analysis of the 846 proteins showed involvement in adhesion and osteogenic differentiation, confirming the ECM composition as key modulator of MSC behavior. In addition to known ECM components, proteomic analysis revealed novel ECM functions, which could improve culture conditions. In summary, this study provides a simplified method to obtain an in vitro MSC-derived ECM that enhances osteogenic differentiation and could be applied as natural biomaterial to accelerate bone regeneration.

Coacervation-Mediated Combinatorial Synthesis of Biomatrices for Stem Cell Culture and Directed Differentiation.

Combinatorial screening represents a promising strategy to discover biomaterials for tailored cell culture applications. Although libraries incorporating different biochemical cues have been investigated, few simultaneously recapitulate relevant biochemical, physical, and dynamic features of the extracellular matrix (ECM). Here, a noncovalent system based on liquid-liquid phase separation (coacervation) and gelation mediated by glycosaminoglycan (GAG)-peptide interactions is reported. Multiple biomaterial libraries are generated using combinations of sulfated glycosaminoglycans and poly(ethylene glycol)-conjugated peptides. Screening these biomaterials reveals preferred biomatrices for the attachment of six cell types, including primary mesenchymal stromal cells (MSCs) and primary neural precursor cells (NPCs). Incorporation of GAGs sustains the expansion of all tested cell types comparable to standard cell culture surfaces, while osteogenic differentiation of MSC and neuronal differentiation of NPC are promoted on chondroitin and heparan biomatrices, respectively. The presented noncovalent system provides a powerful tool for developing tissue-specific ECM mimics.