Promotion Of Osteogenesis Research Articles

Exosomes from bone marrow mesenchymal stem cells enhance fracture healing through the promotion of osteogenesis and angiogenesis in a rat model of nonunion

BackgroundAs important players in cell-to-cell communication, exosomes (exo) are believed to play a similar role in promoting fracture healing. This study investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exos) could improve fracture healing of nonunion.MethodsBMMSC-Exos were isolated and transplanted into the fracture site in a rat model of femoral nonunion (Exo group) every week. Moreover, equal volumes of phosphate-buffered saline (PBS) and exosome-depleted conditioned medium (CM-Exo) were injected into the femoral fracture sites of the rats in the control and CM-Exo groups. Bone healing processes were recorded and evaluated by radiographic methods on weeks 8, 14 and 20 after surgery. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative week 20. The expression levels of osteogenesis- or angiogenesis-related genes were evaluated in vitro by western blotting and immunohistochemistry. The ability to internalize exosomes was assessed using the PKH26 assay. Altered proliferation and migration of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast precursor cells (MC3TE-E1s) treated with BMMSC-Exos were determined by utilizing EdU incorporation, immunofluorescence staining, and scratch wound assay. The angiogenesis ability of HUVECs was evaluated through tube formation assays. Finally, to explore the effect of exosomes in osteogenesis via the BMP-2/Smad1/RUNX2 signalling pathway, the BMP-2 inhibitors noggin and LDN193189 were utilized, and their subsequent effects were observed.ResultsBMMSC-Exos were observed to be spherical with a diameter of approximately 122 nm. CD9, CD63 and CD81 were expressed. Transplantation of BMMSC-Exos obviously enhanced osteogenesis, angiogenesis and bone healing processes in a rat model of femoral nonunion. BMMSC-Exos were taken up by HUVECs and MC3T3-E1 in vitro, and their proliferation and migration were also improved. Finally, experiments with BMP2 inhibitors confirmed that the BMP-2/Smad1/RUNX2 signalling pathway played an important role in the pro-osteogenesis induced by BMMSC-Exos and enhanced fracture healing of nonunion.ConclusionsOur findings suggest that transplantation of BMMSC-Exos exerts a critical effect on the treatment of nonunion by promoting osteogenesis and angiogenesis. This promoting effect might be ascribed to the activation of the BMP-2/Smad1/RUNX2 and the HIF-1α/VEGF signalling pathways.

Long non-coding RNA FER1L4 promotes osteogenic differentiation of human periodontal ligament stromal cells via miR-874-3p and vascular endothelial growth factor A

BackgroundPeriodontal ligament stromal cells (PDLSCs) are ideal cell sources for periodontal tissue repair and regeneration, but little is known about what determines their osteogenic capacity. Long non-coding RNAs (lncRNAs) are important regulatory molecules at both transcriptional and post-transcriptional levels. However, their roles in the osteogenic differentiation of PDLSCs are still largely unknown.MethodsThe expression of lncRNA Fer-1-like family member 4 (FER1L4) during the osteogenic differentiation of PDLSCs was detected by quantitative reverse transcription polymerase chain reaction. Overexpression or knockdown of FER1L4 was used to confirm its regulation of osteogenesis in PDLSCs. Alkaline phosphatase and Alizarin red S staining were used to detect mineral deposition. Dual luciferase reporter assays were used to analyze the binding of miR-874-3p to FER1L4 and vascular endothelial growth factor A (VEGFA). Bone regeneration in critical-sized calvarial defects was assessed in nude mice. New bone formation was analyzed by micro-CT, hematoxylin and eosin staining, Masson’s trichrome staining, and immunohistochemical analyses.ResultsFER1L4 levels increased gradually during consecutive osteogenic induction of PDLSCs. Overexpression of FER1L4 promoted the osteogenic differentiation of PDLSCs, as revealed by alkaline phosphatase activity, Alizarin red S staining, and the expression of osteogenic markers, whereas FER1L4 knockdown inhibited these processes. Subsequently, we identified a predicted binding site for miR-874-3p on FER1L4 and confirmed a direct interaction between them. Wild-type FER1L4 reporter activity was significantly inhibited by miR-874-3p, whereas mutant FER1L4 reporter was not affected. MiR-874-3p inhibited osteogenic differentiation and reversed the promotion of osteogenesis in PDLSCs by FER1L4. Moreover, miR-874-3p targeted VEGFA, a crucial gene in osteogenic differentiation, whereas FER1L4 upregulated the expression of VEGFA. In vivo, overexpression of FER1L4 led to more bone formation compared to the control group, as demonstrated by micro-CT and the histologic analyses.ConclusionFER1L4 positively regulates the osteogenic differentiation of PDLSCs via miR-874-3p and VEGFA. Our study provides a promising target for enhancing the osteogenic potential of PDLSCs and periodontal regeneration.

Mesenchymal stem cell-loaded thermosensitive hydroxypropyl chitin hydrogel combined with a three-dimensional-printed poly(ε-caprolactone) /nano-hydroxyapatite scaffold to repair bone defects via osteogenesis, angiogenesis and immunomodulation.

Chitin-derived hydrogels are commonly used in bone regeneration because of their high cell compatibility; however, their poor mechanical properties and little knowledge of the interaction between the materials and host cells have limited their practical application.Methods: To evaluate osteoinductivity and enhance the mechanical properties of a newly synthesized thermosensitive hydroxypropyl chitin hydrogel (HPCH), a mesenchymal stem cell (MSC)-encapsulated HPCH was infused into a three-dimensional-printed poly (ε-caprolactone) (PCL)/ nano-hydroxyapatite (nHA) scaffold to form a hybrid scaffold. The mechanical properties and cell compatibility of the scaffold were tested. The interaction between macrophages and scaffold for angiogenesis and osteogenesis were explored in vitro and in vivo.Results: The hybrid scaffold showed improved mechanical properties and high cell viability. When MSCs were encapsulated in HPCH, osteo-differentiation was promoted properly via endochondral ossification. The co-culture experiments showed that the hybrid scaffold facilitated growth factor secretion from macrophages, thus promoting vascularization and osteoinduction. The Transwell culture proved that MSCs modulated the inflammatory response of HPCH. Additionally, subcutaneous implantation of MSC-encapsulated HPCH confirmed M2 activation. In situ evaluation of calvarial defects confirmed that the repair was optimal in the MSC-loaded HPCH + PCL/nHA group.Conclusions: PCL/nHA + HPCH hybrid scaffolds effectively promoted vascularization and osteoinduction via osteogenesis promotion and immunomodulation, which suggests promising applications for bone regeneration.

Effects of testosterone and 17β-estradiol on osteogenic and adipogenic differentiation capacity of human bone-derived mesenchymal stromal cells of postmenopausal women

Progressive bone loss is a predominant symptom of aging and osteoporosis. Therefore, the effects of sex steroids (i.e. testosterone and 17β-estradiol) on the differentiation capacity of human bone-derived mesenchymal stromal cells (hMSCs), as progenitors of osteoblasts and adipocytes, are of particular interest. The objectives of the present study were, thus, to elucidate whether bone-derived hMSCs of postmenopausal women produce aromatase (CYP19A1) and, whether they modulate their differentiation behaviour in response to testosterone and 17β-estradiol (E2), in relation to their steroid receptor expression. Supplementation of testosterone resulted in a considerable formation of E2 under osteogenic and adipogenic culture conditions, whereas E2 synthesis remained minimal in the cells cultured in basal medium. Concomitant with high aromatase expression and 17β-estradiol formation of the cells cultured in osteogenic medium supplemented with testosterone, a distinct promotion of late-stage osteogenesis was found, as shown by significant matrix mineralization and a notable increase in osteogenic markers. These effects were abrogated by the aromatase inhibitor anastrozole. Under adipogenic conditions, testosterone reduced the occurrence of lipid droplets and led to a decrease in PPARγ and AR expression, independent of anastrozole. Regardless of the culture conditions, ERα was detectable whilst ERβ was not. In conclusion, aromatase activity is limited to differentiated hMSCs and the resulting 17β-estradiol enhances late osteogenic differentiation stages via ERα. Adipogenic differentiation, on the other hand, is reduced by both sex steroids: testosterone via AR and 17β-estradiol.

TAZ promotes the proliferation and osteogenic differentiation of human periodontal ligament stem cells via the p‐SMAD3

This study aims to offer insights about the biological influence of TAZ, which is a transcriptional coactivator containing a PDZ-binding motif, upon the apoptosis, proliferation, and osteogenic differentiation of human periodontal ligament stem cells (h-PDLSCs). We used the green fluorescence protein lentivirus infection system to knockdown or overexpress TAZ in h-PDLSCs. 5-ethynyl-2'-deoxyuridine (EdU) staining detected the proliferative activity, and h-PDLSC apoptosis was analyzed by Annexin V-APC staining. TAZ knockdown or overexpression was performed to determine the osteogenic differentiation function of TAZ during the osteogenic induction of h-PDLSCs. The molecular mechanism of TAZ in the promotion of h-PDLSC osteogenesis was also explored. The chemical inhibitor of SMAD2/3 SIS3 HCL was used to identify the effects in vitro osteogenic differentiation and bone formation in h-PDLSCs overexpressing TAZ. TAZ overexpression resulted in enhanced cell rapid multiplication, which increased the expression of messenger RNA in stemness-related genes. By comparison, TAZ knockdown reduced proliferative activity and increased the apoptosis of h-PDLSCs. After the 7-day osteogenic induction period, alkaline phosphatase activity in the TAZ-overexpression group was significantly increased, and mineralized nodules increased significantly after osteogenic induction for 21 days. Similarly, osteoblast differentiation of h-PDLSCs was impaired after TAZ knockdown. However, the osteogenic potential of the group exposed to the p-SMAD3 inhibitor was restored to its original level. Hippo/TAZ plays a positive role inside the proliferation, stemness maintenance, and osteogenic specialization of h-PDLSCs, and the specific downstream factor of osteogenic differentiation is SMAD3.

Nanotopographical Cues Mediate Osteogenesis of Stem Cells on Virus Substrates through BMP-2 Intermediate.

Recent studies have demonstrated rapid osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) on substrates with plant virus modified nanotopographical cues as a promising strategy for bone repair; however, the mechanisms remain unclear. We hypothesized that the highly structurally ordered virus coat proteins, responsible for targeting specific cellular components, are critical for the osteogenesis promotion. In this study, hybrid viral gold nanorods were prepared to explore the effects of highly ordered arranged virus coat proteins on osteogenic differentiation of BMSCs. The results herein indicate that it is the nanotopographical cues modified by structurally ordered virus nanoparticles, not the chemical properties of virus surface, that mediate osteogenesis. Bone morphogenetic protein 2 (BMP-2) expression is significantly increased and serves as a modulator that mediates the osteogenic differentiation in response to the viral particle coatings. After BMP-2 is inhibited by Noggin, the osteogenesis promoting effects are significantly compromised, demonstrated by lower alkaline phosphatase activity and calcium sequestration. This study reveals that plant virus modified nanotopographical substrates promote osteogenic differentiation of BMSCs through increasing BMP-2 autocrine. It provides key insights to engineering functional materials for rapid bone repair.

Synergistic Effects of Novel Superparamagnetic/Upconversion HA Material and Ti/Magnet Implant on Biological Performance and Long-Term In Vivo Tracking.

To solve the clinical challenges presented by the long-term tracking of implanted hydroxyapatite (HA) bone repair material and to investigate the synergistic effects of superparamagnetic HA and a static magnetic field (SMF) on the promotion of osteogenesis, herein a new type of superparamagnetic/upconversion-generating HA material (HYH-Fe) is developed via a two-step doping method, as well as a specially-designed titanium implant with a built-in magnet to provide a local static magnetic field in vivo. The results show that the prepared HYH-Fe material maintains the crystal structure of HA and exhibits good cytocompatibility. The combined use of the superparamagnetic HYH-Fe material and SMF can effectively and synergistically promote osteogenesis/osteointegration surrounding the Ti implants. In addition, the HYH-Fe material exhibits distinct advantages in terms of both long-term fluorescence tracking and microcomputed tomography (micro-CT) tracking. The new material and tracking strategy in this study provide scientific feasibility and will have important clinical value for long-term tracking and evaluation of implanted materials and the bone repair effect.

Osteogenic potential of zingerone, a phenolic compound in mouse mesenchymal stem cells.

Zingerone, 4-(4-hydroxy-3-methoxyphenyl)-2-butanone (Zg), a phenolic compound isolated from ginger is reported to have anti-inflammatory and antidiabetic properties. However, its role in the promotion of osteogenesis is not known. In this study, we investigated the therapeutic effect of Zg on osteogenesis at the cellular and molecular levels. Zg treatment was nontoxic to mouse mesenchymal stem cells (mMSCs). At the cellular level, it enhanced osteoblast differentiation as evidenced by more calcium deposits. At the molecular level, Zg stimulated the expression of Runx2 (a bone transcription factor) and other marker genes of osteoblast differentiation in mMSCs. Recent studies indicated that microRNAs (miRNAs) regulate bone metabolism, and we identified that Zg treatment in mMSCs upregulated mir-590, a positive regulator of Runx2 by targeting Smad7, an antagonist of TGF-β1 signaling. Thus, the osteogenic potential of Zg would be beneficial for treating bone and bone-related diseases.

Tripeptide IRW initiates differentiation in osteoblasts via the RUNX2 pathway

BackgroundOsteoblasts maintain the structural integrity of bone via differentiation and mineralization; therefore, their malfunction or reduced activity can cause serious bone disorders. Although studies have demonstrated the association between nutrients and bone, research on food-derived bioactive peptides and bone health are scanty. MethodsOsteoblasts MC3T3-E1 were treated with IRW (50 and 25 μM). Cell proliferation, cell cycle, osteoblastic differentiation, and mineralization were tested to evaluate the effects of IRW on osteogenesis promotion. The activation of PI3K-Akt-RUNX2 pathway and collagen synthesis were investigated to better understand the functions of IRW. ResultsIRW treatment (50 and 25 μM) in MC3T3-E1 cells caused a significant increase in cell proliferation by increasing the percentage of S and G2/M phase. Furthermore, IRW promoted mineralization in MC3T3-E1 cells. Mechanistically, we found that IRW treatment resulted in a 4-fold increase of Akt serine phosphorylation and a 2-fold increase of its downstream target RUNX2. Expression levels of RUNX2 associated proteins were concomitantly altered: ALP (2-fold increase), Col1A2 (2-fold increase), RANKL (2-fold decrease), and OPG (2-fold increase). Meanwhile, a parallel collagen synthesis pathway was found to contribute to IRW-stimulated osteogenesis. ConclusionsIRW, an egg-derived small bioactive peptide enhances osteoblastic activity and stimulates osteogenesis. The stimulation is primarily due to the activation of PI3K-Akt-RUNX2 pathway and its downstream effectors, accompanied by a secondary collagen synthesis pathway. General significanceOur results revealed the positive effects of tripeptide IRW on regulating osteogenesis and collagen synthesis, indicating its potential for the prevention or treatment of osteoporosis.

ERα and Wnt/β‑catenin signaling pathways are involved in angelicin‑dependent promotion of osteogenesis.

Reports of the ameliorative effect of angelicin on sex hormone deficiency-induced osteoporosis have highlighted this compound as a candidate for the treatment of osteoporosis. However, the molecular mechanisms of action of angelicin on osteoblast differentiation have not been thoroughly researched. The aim of the present study was to evaluate the effect of angelicin on the proliferation, differentiation and mineralization of rat calvarial osteoblasts using a Cell Counting Kit-8, alkaline phosphatase activity and the expression of osteogenic genes and proteins. Treatment with angelicin promoted the proliferation, matrix mineralization and upregulation of osteogenic marker genes including collagen type I α 1 and bone γ-carboxyglutamate in fetal rat calvarial osteoblasts. Furthermore, angelicin promoted the expression of β-catenin and runt related transcription factor 2, which serve a vital role in the Wnt/β-catenin signaling pathway. Consistently, the osteogenic effect of angelicin was attenuated by the use of a Wnt inhibitor. Moreover, angelicin increased the expression of estrogen receptor α (ERα), which also serves a key role in osteoblast differentiation. Taken together, these results demonstrated that angelicin may promote osteoblast differentiation through activation of ERα and the Wnt/β-catenin signaling pathway.

Hypermethylation of Frizzled1 is associated with Wnt/\u03b2-catenin signaling inactivation in mesenchymal stem cells of patients with steroid-associated osteonecrosis

The Wnt/β-catenin signaling pathway is associated with the pathogenesis of steroid-induced osteonecrosis. Our investigation studied whether aberrant CpG island hypermethylation of the FZD1 gene was present in patients with osteonecrosis of the femoral head (ONFH), which results in Wnt/β-catenin signaling inactivation and subsequent cell dysfunction. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 21) and patients with new femoral neck fractures (n = 22), and then mesenchymal stem cells (MSCs) were isolated. We investigated cell viability, the transcription and translation levels of Wnt/β-catenin signaling-related genes, the extent of methylation at CpG islands of the FZD1 promoter, and the osteogenic and adipogenic differentiation abilities of MSCs from the control group and from the ONFH group treated with or without 5′-Aza-dC. According to the results, MSCs from the ONFH group showed a reduced proliferation ability, low transcription and translation levels of FZD1, inhibition of the Wnt/β-catenin signaling pathway, weakened osteogenesis and enhanced adipogenesis ability. Aberrant CpG island hypermethylation of FZD1 was observed in the ONFH group. Treatment with 5’-Aza-dC resulted in de novo FZD1 expression, reactivation of the Wnt/β-catenin signaling pathway and promotion of osteogenesis. Taken together, our study not only provides novel insights into the regulation of the Wnt/β-catenin signaling pathway in this disease but also reveals potential for the use of demethylating agents for the treatment of GC-associated ONFH.

Delivery vehicle of muscle-derived irisin based on silk/calcium silicate/sodium alginate composite scaffold for bone regeneration.

Irisin is a cytokine produced by skeletal muscle and usually plays a pivotal role in inducing fat browning and regulating energy expenditure. In recent years, it was found that irisin might be the molecular entity responsible for muscle-bone connectivity and is useful in osteogenesis induction. To study its effect on bone regeneration, we developed silk/calcium silicate/sodium alginate (SCS) composite scaffold based on an interpenetrating network hydrogel containing silk fibroin, calcium silicate, sodium alginate. Then we loaded irisin on the SCS before coating it with polyvinyl alcohol (PVA). The SCS/P scaffold was physically characterized and some in vitro and in vivo experiments were carried out to evaluate the scaffold effect on bone regeneration. The SCS/P scaffold was showed a porous sponge structure pursuant to scanning electron microscopy analysis. The release kinetics assay demonstrated that irisin was stably released from the irisin-loaded hybrid system (i/SCS/P system) to 50% within 7 days. Moreover, osteoinductive studies using bone marrow stem cells (BMSCs) in vitro exhibited the i/SCS/P system improved the activity of alkaline phosphatase (ALP) and enhanced the expression levels of a series of osteogenic markers containing Runx-2, ALP, BMP2, Osterix, OCN, and OPN. Alizarin red staining also demonstrated the promotion of osteogenesis induced by i/SCS/P scaffolds. In addition, in vivo studies showed that increased bone regeneration with better mineralization and higher quality was found during the repair of rat calvarial defects through utilizing the i/SCS/P system. These data provided strong evidence that the composite i/SCS/P would be a promising substitute for bone tissue engineering.

Comparing hydroxyapatite with osteogenic medium for the osteogenic differentiation of mesenchymal stem cells on PHBV nanofibrous scaffolds

Having advantageous biocompatibility and osteoconductive properties known to enhance the osteogenic differentiation of mesenchymal stem cells (MSCs), hydroxyapatite (HA) is a commonly used material for bone tissue engineering. What remains unclear, however, is whether HA holds a similar potential for stimulating the osteogenic differentiation of MSCs to that of a more frequently used osteogenic-inducing medium (OIM). To that end, we used PHBV electrospun nanofibrous scaffolds to directly compare the osteogenic capacities of HA with OIM over MSCs. Through the observation of cellular morphology, the staining of osteogenic markers, and the quantitative measuring of osteogenic-related genes, as well as microRNA analyses, we not only found that HA was as capable as OIM for differentiating MSCs down an osteogenic lineage; albeit, at a significantly slower rate, but also that numerous microRNAs are involved in the osteogenic differentiation of MSCs through multiple pathways involving the inhibition of cellular proliferation and stemness, chondrogenesis and adipogenesis, and the active promotion of osteogenesis. Taken together, we have shown for the first time that PHBV electrospun nanofibrous scaffolds combined with HA have a similar osteogenic-inducing potential as OIM and may therefore be used as a viable replacement for OIM for alternative in vivo-mimicking bone tissue engineering applications.

Promotion of osteogenesis by bioactive glass–ceramic coating: Possible involvement of the Hedgehog signaling pathway

PurposeBioactive glass–ceramic (BGC) coatings have been extensively studied and clinically used as bone substitute materials because of their osteogenesis, osteoinduction, and osteoconduction characteristics. Although the Hedgehog (Hh) signaling pathway plays an important role in skeletal development, the relationship between BGC coatings and the Hh signaling pathway is unknown. MethodsIn this study, a BGC coating is fabricated by furnace sintering, and its surface is investigated by a scanning electron microscope (SEM) and a transmission electron microscope (TEM). Furthermore, the expression of Ki67 is evaluated using immunofluorescence, and osteogenesis-related factors and Hh signaling pathway molecules on the BGC coating are examined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blotting in bone marrow mesenchymal stem cells (BMSCs). ResultsThe SEM and the TEM show that the BGC coating surface is smooth, without cracks, and composed of particles with mesoporous structure. The expression of Ki67 positive BMSCs of the BGC group is higher than that of the control group. Real-time RT-PCR and Western blotting assay reveal that the expression levels of osteoblast-related genes (BMP2, Osteocalcin, ALP, Runx2) and Hh signaling pathway molecules (Gli1, Smo) are much higher for the BGC coating group than those for the control group. Furthermore, after treating with Smo inhibitor cyclopamine, the Smo and Gli1 expressions in BMSCs are dramatically down-regulation for the BGC coating compared to those for the control group. Both mRNA and protein expression levels of osteogenesis-related factors was downregulated after treating Smo inhibitor cyclopamine in BMSCs with the BGC coating. ConclusionsThe BGC coatings promote osteogenesis probably via the Hh signaling pathway, which provides a theory reference for future clinical application of bone formation.

The effects of substrate-mediated electrical stimulation on the promotion of osteogenic differentiation and its optimization.

To explore the effect of electrical stimulation (ES) on osteogenesis, a polypyrrole (PPy)-made electrical culture system was developed to provide a direct-current electric field (DCEF). This DCEF device was applied to treat differentiated rat bone marrow stromal cells (rBMSCs) once in different stages of osteo-differentation to investigate its temporal effects. The mineralization results showed that the DCEF treatment not only accelerated cell differentiation but also promoted the saturation levels, and the ES on day 8 was the group demonstrated the optimal result. The gene regulation analysis indicated that the DCEF treatment immediately increased the levels of genes related to osteo-differentiation, especially Runx2. Because Runx2 is a crucial transcriptional factor of osteogenesis, the ES-caused improvement of mineralization was likely contributed by the extension of its expression. Further, different ES modes were investigated of their efficacy on bone matrix deposition. Square waves with different parameters including frequency, offset, amplitude, and duty cycle were systematically examined. In contrast to constant voltage, square waves demonstrated periodical changes of current through substrate to significantly improve mineralization, and the efficiencies highly depended on both frequency and intensity. Through this comprehensive study, DCEF treating condition was optimized, which should be beneficial to its application on osteogenesis promotion. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1607-1619, 2019.

Epigenetic Control of Mesenchymal Stem Cell Fate Decision via Histone Methyltransferase Ash1l.

Previous research indicates that knocking out absent, small, or homeotic-like (Ash1l) in mice, a histone 3 lysine 4 (H3K4) trimethyltransferase, can result in arthritis with more severe cartilage and bone destruction. Research has documented the essential role of Ash1l in stem cell fate decision such as hematopoietic stem cells and the progenitors of keratinocytes. Following up on those insights, our research seeks to document the function of Ash1l in skeletal formation, specifically whether it controls the fate decision of mesenchymal progenitor cells. Our findings indicate that in osteoporotic bones, Ash1l was significantly decreased, indicating a positive correlation between bone mass and the expression of Ash1l. Silencing of Ash1l that had been markedly upregulated in differentiated C3H10T1/2 (C3) cells hampered osteogenesis and chondrogenesis but promoted adipogenesis. Consistently, overexpression of an Ash1l SET domain-containing fragment 3 rather than Ash1lΔN promoted osteogenic and chondrogenic differentiation of C3 cells and simultaneously inhibited adipogenic differentiation. This indicates that the role of Ash1l in regulating the differentiation of C3 cells is linked to its histone methyltransferase activity. Subcutaneous ex vivo transplantation experiments confirmed the role of Ash1l in the promotion of osteogenesis. Further experiments proved that Ash1l can epigenetically affect the expression of essential osteogenic and chondrogenic transcription factors. It exerts this impact via modifications in the enrichment of H3K4me3 on their promoter regions. Considering the promotional action of Ash1l on bone, it could potentially prompt new therapeutic strategy to promote osteogenesis. Stem Cells 2019;37:115-127.

Promoted Angiogenesis and Osteogenesis by Dexamethasone-loaded Calcium Phosphate Nanoparticles/Collagen Composite Scaffolds with Microgroove Networks

Reconstruction of large bone defects remains a clinical challenge because current approaches involving surgery and bone grafting often do not yield satisfactory outcomes. For artificial bone substitutes, angiogenesis plays a pivotal role to achieve the final success of newly regenerated bone. In this study, dexamethasone-loaded biphasic calcium phosphate nanoparticles/collagen composite scaffolds with several types of concave microgrooves were prepared for simultaneous promotion of angiogenesis and osteogenesis. Microgrooves in the scaffolds were supposed to guide the assembly of human umbilical vascular endothelial cells (HUVECs) into well aligned tubular structures, thus promoting rapid angiogenesis. The scaffolds were used for co-culture of HUVECs and human bone marrow-derived mesenchymal stem cells. Subcutaneous implantation in mice showed that more blood vessels and newly formed bone were observed in the microgrooved composite scaffolds than in the control scaffold. Scaffold bearing parallel microgrooves with a concave width of 290 µm and a convex ridge width of 352 µm showed the highest promotion effect on angiogenesis and osteogenesis among the parallelly microgrooved composite scaffolds. The scaffolds bearing a grid network had further superior promotion effect to the scaffolds bearing parallel microgrooves. The results indicated that microgrooves in the composite scaffolds facilitated angiogenesis and stimulated new bone formation. The microgrooved composite scaffolds should be useful for repairing of large bone defects.

Progranulin Promotes Regeneration of Inflammatory Periodontal Bone Defect in Rats via Anti-inflammation, Osteoclastogenic Inhibition, and Osteogenic Promotion

Progranulin (PGRN) has been proved to play a crucial role in anti-inflammation and osteogenesis promotion; thus, it was hypothesized that PGRN could promote bone regeneration in periodontal disease. In this experiment, the periodontal bone defects were established in periodontitis rats; recombinant human progranulin (rhPGRN), tumor necrosis factor alpha inhibitor (anti-TNF-α), or phosphate buffer saline (PBS)-loaded collagen membrane scaffolds were implanted within defects and the rats were sacrificed at scheduled time points. Volume of new bone was assessed by radiological and histomorphometric analyses. Expression of osteogenesis-related markers and tumor necrosis factor-α (TNF-α) was evaluated using immunohistochemistry. Tartrate-resistant acid phosphatase (TRAP) staining was also performed to determine the number of osteoclasts. Immunofluorescence (IF) staining was performed to explore the interaction between rhPGRN and tumor necrosis factor receptors (TNFRs). The results showed that the rhPGRN group had significantly superior quantity and quality of newly formed bone, higher expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and TNFR2 compared with the PBS group and the anti-TNF-α group. Similarly to the anti-TNF-α group, the rhPGRN group also exhibited the significant inhibitory effect on the expression of TNF-α and the number of TRAP-positive cells compared with the PBS group. Hence, our experiment suggests that PGRN promotes regeneration of inflammatory periodontal bone defect in rats via anti-inflammation, osteoclastogenic inhibition, and osteogenic promotion. Local administration of PGRN may provide a new therapeutic strategy for periodontal bone regeneration.

Square prism micropillars improve osteogenicity of poly(methyl methacrylate) surfaces.

Osteogenicity and osteointegration of materials is one of the key elements of the success of bone implants. Poly(methyl methacrylate) (PMMA) is the basic compound of bone cement and has been widely investigated for other orthopedic applications, but its poor osteointegration and the subsequent loosening of implant material limits its widespread use as bone implants. Micropillar features on substrate surfaces were recently reported to modulate cell behavior through alteration of cell morphology and promotion of osteogenesis. Utilization of this pillar-decorated topography may be an effective approach to enhance osteogenicity of polymeric surfaces. The aim of this study was to investigate the effect of cell morphology on the micropillar features on attachment, proliferation, and osteogenic activity of human osteoblast-like cells. A series of solvent cast PMMA films decorated with 8 µm high square prism micropillars with pillar width and interpillar distances of 4, 8 and 16 µm were prepared from photolithographic templates, and primary human osteoblast-like cells (hOB) isolated from bone fragments were cultured on them. Micropillars increased cell attachment and early proliferation rate compared to unpatterned surfaces, and triggered distinct morphological changes in cell body and nucleus. Surfaces with pillar dimensions and gap width of 4 µm presented the best osteogenic activity. Expression of osteogenic marker genes was upregulated by micropillars, and cells formed bone nodule-like aggregates rich in bone matrix proteins and calcium phosphate. These results indicated that micropillar features enhance osteogenic activity on PMMA films, possibly by triggering morphological changes that promote the osteogenic phenotype of the cells.

Effect of Concentrated Growth Factor (CGF) on the Promotion of Osteogenesis in Bone Marrow Stromal Cells (BMSC) in vivo

The therapeutic method traditionally used in bone defect reconstruction is autologous bone grafting. The most common problems affecting this type of repair approach are bone absorption and donor trauma. The approach taken in this study overcomes these problems. Bone marrow stromal cells (BMSCs) provided the crucial seed cells. Fibrin biological scaffolds were formed by combining the BMSCs with concentrated growth factor (CGF). BMSCs were isolated from Wistar rat femurs; CGF was prepared from rat heart blood. Five repair groups were created for comparative purposes: (A) CGF + BMSCs; (B) CGF; (C) collagen + BMSCs; (D) collagen; (E) blank. After three months, the rats were sacrificed, and histopathology and three-dimensional CT images produced. Bone regeneration was significantly higher in the (A) CGF + BMSC group; osteogenesis was lower in the (B) CGF and (C) collagen + BMSC groups, at very similar levels; the (D) collagen and (E) blank groups scored the lowest results. Our research suggests that combining CGF with BMSCs leads to the formation of fibrin scaffolds that have a powerful effect on osteogenesis as well as a subsidiary angiogenic effect. SEM images of the CGF scaffolds cultured with BMSCs confirmed good CGF biocompatibility. The superior osteoinductive activity of the CGF + BMSC combination makes it an excellent biomaterial for bone regeneration.