Expression Of Bone-specific Genes Research Articles

KAT2A-mediated succinylation modification of notch1 promotes the proliferation and differentiation of dental pulp stem cells by activating notch pathway

BackgroundDental pulp stem cells (DPSCs) are a kind of undifferentiated dental mesenchymal stem cells with strong self-renewal ability and multi-differentiation potential. This study aimed to investigate the regulatory functions of succinylation modification in DPSCs.MethodsDPSCs were isolated from the dental pulp collected from healthy subjects, and then stem cell surface markers were identified using flow cytometry. The osteogenic differentiation ability of DPSCs was verified by alkaline phosphatase (ALP) and alizarin red staining methods, while adipogenic differentiation was detected by oil red O staining. Meanwhile, the mRNA of two desuccinylases (SIRT5 and SIRT7) and three succinylases (KAT2A, KAT3B, and CPT1A) in DPSCs before and after mineralization induction were detected using quantitative real-time PCR. The cell cycle was measured by flow cytometry, and the expression of bone-specific genes, including COL1a1 and Runx2 were evaluated by western blotting and were combined for the proliferation and differentiation of DPSCs. Co-immunoprecipitation (co-IP) and immunofluorescence were combined to verify the binding relationship between proteins.ResultsThe specific markers of mesenchymal stem cells were highly expressed in DPSCs, while the osteogenic differentiation ability of isolated DPSCs was confirmed via ALP and alizarin red staining. Similarly, the oil red O staining also verified the adipogenic differentiation ability of DPSCs. The levels of KAT2A were found to be significantly upregulated in mineralization induction, which significantly decreased the ratio of G0/G1 phase and increased S phase cells; converse results regarding cell cycle distribution were obtained when KAT2A was inhibited. Moreover, overexpression of KAT2A promoted the differentiation of DPSCs, while its inhibition exerted the opposite effect. The elevated KAT2A was found to activate the Notch1 signaling pathway, which succinylated Notch1 at the K2177 site to increase their corresponding protein levels in DPSCs. The co-IP results showed that KAT2A and Notch1 were endogenously bound to each other, while inhibition of Notch1 reversed the effects of KAT2A overexpression on the DPSCs proliferation and differentiation.ConclusionKAT2A interacted directly with Notch1, succinylating the Notch1 at the K2177 site to increase their corresponding protein levels in DPSCs. Similarly, KAT2A-mediated succinylation modification of Notch1 promotes the DPSCs proliferation and differentiation, suggesting that targeting KAT2A and Notch1 may contribute to tooth regeneration.

Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Electrospun Propolis-coated PLGA Scaffold Enhances the Osteoinduction of Mesenchymal Stem Cells.

Major injuries that are caused by trauma and cancer can not be repaired through bone remodeling. The goal of bone regeneration by tissue engineering approaches is to fabricate bone implants in order to restore bone structure and functions. The use of stem cells and polymer scaffolds provides the conditions for tissue regeneration based on tissue engineering. This study aimed to fabricate a combined matrix of poly(lactide-co-glycolide) (PLGA) and propolis extract, which is a mixture of pollen and beeswax collected by bees from certain plants and has long been used in traditional herbal medicine, to promote the osteogenic differentiation of human adipose- derived mesenchymal stem cells (AD-MSCs). The scaffold was fabricated through electrospinning and was immersed in a propolis extract solution. Then, AD-MSCs were cultured and differentiated into the osteogenic lineage. The cell viability on the scaffold was evaluated by MTT assay. Osteogenic differentiation of the seeded stem cells was detected by evaluating calcium content, alkaline phosphatase (ALP) activity, and the expression of bonespecific genes. The viability of cells was not affected by propolis-coated and uncoated fabricated scaffolds, while higher calcium content, ALP activity, and expression of RUNX-2, type I collagen, osteocalcin, and osteonectin were observed in cells differentiated on propolis-coated PLGA scaffold on days 7, 14, and 21 of differentiation compared to PLGA scaffold. The results of this study showed that the presence of propolis in the scaffold could lead to better cell attachment and strengthen the osteoinduction process in stem cells.

Synthesis of bone biocompatible implants using human adipose-derived mesenchymal stem cells (hADMSCs) and PCL/laminin scaffold substrate.

Bone tissue engineering is considered a new method in the treatment of bone defects and can be an effective alternative to surgery and bone grafting. The use of adipose tissue mesenchymal stem cells (ADMSCs) on synthetic polymer scaffolds is one of the new approaches in bone tissue engineering. In this study, we aimed to investigate the effect of laminin coating on biocompatibility and osteogenic differentiation of ADMSCs seeded on polycaprolactone (PCL) scaffolds. The morphology of the electrospun scaffold was evaluated using a scanning electron microscope (SEM). Cell proliferation and cytotoxicity were determined by MTT assay. The adipogenic and osteogenic differentiation potential of the cells was evaluated. The osteogenic differentiation of ADMSCs cultured on the PCL scaffold coated with laminin was assessed by evaluating the level of alkaline phosphatase (ALP) activity, intracellular calcium content, and expression of bone-specific genes. The results showed that the ADMSCs cultured on PCL/laminin showed enhanced osteogenic differentiation compared to those cultured on non-coated PCL or control medium (P<0.05). It seems that laminin enhances the physicochemical properties and biocompatibility of PCL nanofiber scaffolds; and by modifying the surface of the scaffold, improves the differentiation of ADMSCs into osteogenic cells.

A forsterite-reinforced polypropylene fumarate/methoxy polyethylene glycol-hydrogel enriched with flavonoid nanoparticles enhances osteoconductivity

PPF–MPEG/Fs/Hst–GNPs caused significant enhancement in mineralization, differentiation, cell proliferation and higher expression of bone-specific genes including; collagen 1, osteocalcin, and osteopontin compared to the blank hydrogel.

Growth and Regeneration of Osteochondral Cells in Bioactive Niche: A Promising Approach for Osteochondral Tissue Repair.

Functional repair of osteochondral defects caused due to osteoarthritis still remains the greatest challenge in orthopedic therapy. A prospective clinical strategy would be exploring osteochondral tissue engineering possibilities that promote simultaneous regeneration of the articular cartilage layer as well as the underlying subchondral bone. Incorporating the appropriate cues onto the scaffolds for the regeneration of the two contrasting tissues is therefore a demanding function. In the present study, a polymer-ceramic composite scaffolding material consisting of ternary bioactive glass (67.12 SiO2/28.5 CaO/4.38 P2O5 mol %) incorporated into a semi interpenetrating polymer network of hydrophilic-hydrophobic polymer (poly(vinyl alcohol)-polycaprolactone) matrix is prepared and physicochemically characterized. In vitro bioactivity, bone-bonding ability, and biocompatibility evaluation were performed in comparison with the pristine scaffold. The degree of chondrogenic and osteogenic potential of mesenchymal stem cells in both the scaffolds was evaluated by gene expression studies. Although both the scaffolds favored the differentiation to both cell lineages in their respective medium, a higher expression of bone specific genes found with the composite scaffold suggested that this composite scaffold would serve better for osteal layer and henceforth to promote the integration of the osteochondral construct at the defect site.

The effect of decellularized cartilage matrix scaffolds combined with endometrial stem cell-derived osteocytes on osteochondral tissue engineering in rats.

Since decellularized tissues may offer the instructive niche for cell differentiation and function, their use as cell culture scaffolds is a promising approach for regenerative medicine. To repair osteochondral tissues, developing a scaffold with biomimetic structural, compositional, and functional characteristics is vital. As a result of their heterogeneous structure, decellularized articular cartilage matrix from allogeneic and xenogeneic sources are considered appropriate scaffolds for cartilage regeneration. We developed a scaffold for osteochondral tissue engineering by decellularizing sheep knee cartilage using a chemical technique. DNA content measurements and histological examinations revealed that this protocol completely removed cells from decellularized cartilage. Furthermore, SEM, MTS assay, and H&E staining revealed that human endometrial stem cells could readily adhere to the decellularized cartilage, and the scaffold was biocompatible for their proliferation. Besides, we discovered that decellularized scaffolds could promote EnSC osteogenic differentiation by increasing bone-specific gene expression. Further, it was found that decellularized scaffolds were inductive for chondrogenic differentiation of stem cells, evidenced by an up-regulation in the expression of the cartilage-specific gene. Also, in vivo study showed the high affinity of acellularized scaffolds for cell adhesion and proliferation led to an improved regeneration of articular lesions in rats after 4weeks. Finally, a perfect scaffold with high fidelity is provided by the developed decellularized cartilage scaffold for the functional reconstruction of osteochondral tissues; these types of scaffolds are helpful in studying how the tissue microenvironment supports osteocytes and chondrocytes differentiation, growth, and function to have a good osteochondral repair effect.

Promoting in-vivo bone regeneration using facile engineered load-bearing 3D bioactive scaffold

The worldwide incidence of bone disorders has trended steeply upward and is expected to get doubled by 2030. The biological mechanism of bone repair involves both osteoconductivity and osteoinductivity. Despite the self-healing functionality after injury, bone tissue faces a multitude of pathological challenges. Several innovative approaches have been developed to prepare biomaterial-based bone grafts. To design a suitable bone material, the freeze-drying technique has achieved significant importance among the other conventional methods. However, the functionality of the polymeric freeze-dried scaffold in in-vivo osteogenesis is in a nascent stage. In this study facile, freeze-dried, biomaterial-based load-bearing three-dimensional porous composite scaffolds have been prepared. The biocompatible scaffolds have been made by using chitosan (C), polycaprolactone (P), hydroxyapatite (H), glass ionomer (G), and graphene (gr). Scaffolds of eight different groups (C, P, CP, CPH, CPHG, CPHGgr1, CPHGgr2, CPHGgr3) have been designed and characterized to evaluate their applicability in orthopedics. To evaluate the efficacy of the scaffolds a series of physio-chemical, morphological, and in-vitro and in-vivo biological experiments have been performed. From the obtained results it was observed that the CPHGgr1 is the ideal compatible material for Wharton’s jelly-derived mesenchymal stem cells (MSCs) and the blood cells. The in-vitro bone-specific gene expression study revealed that the scaffold assists MSCs osteogenic differentiation. Additionally, the in-vivo study on the mice model was also performed for a period of four and eight weeks. The subcutaneous implantation of the designed scaffolds did not show any altered physiological condition in the animals, which indicated the in-vivo biocompatibility of the designed material. The histopathological study revealed that after eight weeks of implantation, the CPHGgr1 scaffold supported significantly better collagen deposition and calcification. The facile designing of the CPHGgr1 multicomponent nanocomposite provided an osteo-regenerative biomaterial with desired mechanical strength as an ideal regenerative material for cancellous bone tissue regeneration.

Effect of bassorin (derived from gum tragacanth) and halloysite nanotubes on physicochemical properties and the osteoconductivity of methylcellulose-based injectable hydrogels

Injectable hydrogels have been known as promising materials for the regeneration of irregular shape tissue defects. In this study, novel thermosensitive methylcellulose (MC) hydrogels containing bassorin (Ba) and halloysite nanotubes (HNTs) have been developed for application in bone tissue engineering. Bassorin isolated from gum tragacanth (GT) with the concentration of 0.25-1.5 w/v% was blended with MC. The best MC/Ba gel (containing 0.5% bassorin) was chosen based on the results of injectability and rheological tests. HNTs (1-7%) were added to this formulation and tested for the physicochemical, mechanical, rheological, degradation, swelling, and biological properties. In vitro biological evaluations including cell proliferation (by MTT assay), cell attachment (by SEM), osteogenic activity (by Alizarin Red staining and alkaline phosphatase assay), and osteogenic gene expression (by quantitative real-time polymerase chain reaction) were done using MG-63 cells. Results showed that bassorin led to the increased gel-forming ability (at a lower temperature) and mechanical properties of MC hydrogel. The presence of HNTs and bassorin affected the degradation rate and swelling degree of MC-based hydrogel. Results showed significant enhancement in cell proliferation, differentiation, and mineralization, as well as higher bone-specific gene expression of the cell on bassorin and HNTs incorporated MC compared to pure MC hydrogel.

Role of calcium phosphate and bioactive glass coating on in vivo bone healing of new Mg\u2013Zn\u2013Ca implant

Present investigation focuses on development and detailed characterization of a new Mg alloy sample (BM) with and without coating of hydroxyapatite (BMH) and bioactive glass (BMG) by air plasma spray method. After detailed mechano-physico-chemical characterization of powders and coated samples, electrochemical corrosion and SBF immersion tests were carried out. Detailed in vitro characterizations for cell viability were undertaken using MG-63 cell line followed by in vivo tests in rabbit model for studying bone healing up to 60 days. Starting current density increases from BM to BMH to BMG indicating highest resistance towards corrosion in case of BMG samples, however BMH also showed highest icorr value suggesting slowest rate of corrosion than BM and BMG samples. Dissolution of calcium ion in case of BMH and BMG control formation of apatite phases on surface. Ca2+ ions of coatings and from SBF solution underwent reduction reaction simultaneously with conversion of Mg to MgCl2 releasing OH− in the solution, which increases pH. Viability and propagation of human osteoblast-like cells was verified using confocal microscopy observations and from expression of bone specific genes. Alkaline phosphatase assay and ARS staining indicate cell proliferation and production of neo-osseous tissue matrix. In vivo, based on histology of heart, kidney and liver, and immune response of IL-2, IL-6 and TNFα, all the materials show no adverse effects in body system. The bone creation was observed to be more for BMH. Although both BMH and BMG show rays of possibilities in early new bone formation and tough bone–implant bonding at interface as compared to bare Mg alloy, however, BMG showed better well-sprayed coating covering on substrate and resistance against corrosion prior implanting in vivo. Also, better apatite formation on this sample makes it more favourable implant.

Electrospun PCL scaffold modified with chitosan nanoparticles for enhanced bone regeneration

The encapsulation of ascorbic acid within chitosan nanoparticles (CHNs), embedded in a fibrous structure of a dexamethasone (Dex)-loaded PCL scaffold, provides a new plan for osteogenic differentiation of mesenchymal stem cells. This electrospun PCL fibrous scaffold can release Dex, as bone differentiation initiator, and ascorbic acid, as bone differentiation enhancer, in an approximately sustained release pattern for about 2weeks. Ascorbic acid-loaded CHNs were prepared by electrospraying a mixture of chitosan and ascorbic acid, and Dex-containing PCL fibers were prepared by electrospinning a mixture of PCL and Dex. The final PCL/chitosan bilayer scaffolds were obtained by the sequential employment of electrospinning and electrospraying methods. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) confirmed that the CHNs were successfully incorporated into the fibrous PCL matrix. The improved proliferation of hMSCs cultured on the PCL/chitosan scaffolds was also verified. Osteogenic assays showed an increase in alkaline phosphatase activity and mineral deposits. The expression of bone-specific genes also confirmed the osteogenic differentiation of cells cultured on these PCL/chitosan bilayer scaffolds. Dual-drug-loaded PCL/chitosan scaffold enhanced the osteoblast differentiation of hMSC cells and can be served as a potential scaffold for bone tissue engineering.

A Biomimetic Biphasic Scaffold Consisting of Decellularized Cartilage and Decalcified Bone Matrixes for Osteochondral Defect Repair.

It is challenging to develop a biphasic scaffold with biomimetic compositional, structural, and functional properties to achieve concomitant repair of both superficial cartilage and subchondral bone in osteochondral defects (OCDs). This study developed a biomimsubchondraletic biphasic scaffold for OCD repair via an iterative layered lyophilization technique that controlled the composition, substrate stiffness, and pore size in each phase of the scaffold. The biphasic scaffold consisted of a superficial decellularized cartilage matrix (DCM) and underlying decalcified bone matrix (DBM) with distinct but seamlessly integrated phases that mimicked the composition and structure of osteochondral tissue, in which the DCM phase had relative low stiffness and small pores (approximately 134 μm) and the DBM phase had relative higher stiffness and larger pores (approximately 336 μm). In vitro results indicated that the biphasic scaffold was biocompatible for bone morrow stem cells (BMSCs) adhesion and proliferation, and the superficial DCM phase promoted chondrogenic differentiation of BMSCs, as indicated by the up-regulation of cartilage-specific gene expression (ACAN, Collagen II, and SOX9) and sGAG secretion; whereas the DBM phase was inducive for osteogenic differentiation of BMSCs, as indicated by the up-regulation of bone-specific gene expression (Collagen I, OCN, and RUNX2) and ALP deposition. Furthermore, compared with the untreated control group, the biphasic scaffold significantly enhanced concomitant repair of superficial cartilage and underlying subchondral bone in a rabbit OCD model, as evidenced by the ICRS macroscopic and O’Driscoll histological assessments. Our results demonstrate that the biomimetic biphasic scaffold has a good osteochondral repair effect.

Runt-related transcription factor 1 is required for murine osteoblast differentiation and bone formation

Despite years of research investigating osteoblast differentiation, the mechanisms by which transcription factors regulate osteoblast maturation, bone formation, and bone homeostasis is still unclear. It has been reported that runt-related transcription factor 1 (Runx1) is expressed in osteoblast progenitors, pre-osteoblasts, and mature osteoblasts; yet, surprisingly, the exact function of RUNX1 in osteoblast maturation and bone formation remains unknown. Here, we generated and characterized a pre-osteoblast and differentiating chondrocyte-specific Runx1 conditional knockout mouse model to study RUNX1's function in bone formation. Runx1 ablation in osteoblast precursors and differentiating chondrocytes via osterix-Cre (Osx-Cre) resulted in an osteoporotic phenotype and decreased bone density in the long bones and skulls of Runx1f/fOsx-Cre mice compared with Runx1f/f and Osx-Cre mice. RUNX1 deficiency reduced the expression of SRY-box transcription factor 9 (SOX9), Indian hedgehog signaling molecule (IHH), Patched (PTC), and cyclin D1 in the growth plate, and also reduced the expression of osteocalcin (OCN), OSX, activating transcription factor 4 (ATF4), and RUNX2 in osteoblasts. ChIP assays and promoter activity mapping revealed that RUNX1 directly associates with the Runx2 gene promoter and up-regulates Runx2 expression. Furthermore, the ChIP data also showed that RUNX1 associates with the Ocn promoter. In conclusion, RUNX1 up-regulates the expression of Runx2 and multiple bone-specific genes, and plays an indispensable role in bone formation and homeostasis in both trabecular and cortical bone. We propose that stimulating Runx1 activity may be useful in therapeutic approaches for managing some bone diseases such as osteoporosis.

Acellular pericardium: A naturally hierarchical, osteoconductive, and osteoinductive biomaterial for guided bone regeneration.

There is great demand for an improved barrier membrane with osteogenic potential for guided bone regeneration (GBR). Natural acellular porcine pericardium (APP) is increasingly used in regenerative medicine as a kind of common extracellular matrix materials. This study aimed to investigate its potential application in GBR, especially its osteoconductive and osteoinductive properties. Bio-Gide (BG), a commercial collagen membrane, was set as the control group. APP samples were characterized by physicochemical analyses and their biological effects on human bone mesenchymal stem cells (hBMSCs) and human gingival fibroblasts (hGFs) were also examined. Additionally, the osteogenic potential of APP was tested on a bilateral critical-sized calvarial defect model. We discovered that the smooth surface of APP tended to recruit more hBMSCs. Moreover, promoted proliferation and osteogenic differentiation of hBMSCs was detected on this side of APP, with increased alkaline phosphatase activity and upregulated expression of bone-specific genes. Besides, the rough side of APP showed good biocompatibility and barrier function with hGFs. Histologic observation and analysis of calvarial defect healing over 4 weeks revealed enhanced bone regeneration under APP compared with BG and the control group. The results of this study indicate that APP is a potential osteoconductive and osteoinductive biomaterial for GBR.

Directed differentiation of BMSCs on structural/compositional gradient nanofibrous scaffolds for ligament-bone osteointegration.

Nanofibrous scaffolds with structural and compositional gradients exhibit great potential to modulate zonal differentiation of stem cells for the regeneration of soft-to-hard tissue interface. Here, the response of bone marrow stem cells (BMSCs) to electrospun gradient nanofibrous scaffolds was investigated to demonstrate their potential capabilities for interfacial tissue regeneration. The electrospun scaffolds showed gradient distribution of BMP-2/nanoHA contents and the fiber orientations gradually changed from random to align. Biomimetic mineralization demonstrated that calcium and phosphorus elements can deposit onto the surface of the nanofibers in a gradient manner similar to nanoHA content. BMSCs cultured on the gradient nanofibrous scaffolds exhibited high cell viability and cell morphology gradually changed from disorder to highly align similar to the underlying fiber orientation. BMP-2/nanoHA content gradients in the nanofibrous scaffolds were found to effectively promote the zonal expression of bone-specific genes like osteocalcin (OCN), Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). Immunofluorescent staining of osteopontin (OPN) and OCN further confirmed osteoblastic phenotypic maturation on the regions of the scaffolds with a higher level of nanoHA and BMP-2 contents after cultured 28days. These results indicated that the gradient nanofibrous scaffolds enable to guide zonal differentiation of BMSCs in vitro, which might be useful to realize multitissue regeneration in one construct for the regeneration of soft-to-hard tissue interface.

Extracellular matrix decorated polycaprolactone scaffolds for improved mesenchymal stem/stromal cell osteogenesis towards a patient-tailored bone tissue engineering approach.

The clinical demand for tissue-engineered bone is growing due to the increase of non-union fractures and delayed healing in an aging population. Herein, we present a method combining additive manufacturing (AM) techniques with cell-derived extracellular matrix (ECM) to generate structurally well-defined bioactive scaffolds for bone tissue engineering (BTE). In this work, highly porous three-dimensional polycaprolactone (PCL) scaffolds with desired size and architecture were fabricated by fused deposition modeling and subsequently decorated with human mesenchymal stem/stromal cell (MSC)-derived ECM produced in situ. The successful deposition of MSC-derived ECM onto PCL scaffolds (PCL-MSC ECM) was confirmed after decellularization using scanning electron microscopy, elemental analysis, and immunofluorescence. The presence of cell-derived ECM within the PCL scaffolds significantly enhanced MSC attachment and proliferation, with and without osteogenic supplementation. Additionally, under osteogenic induction, PCL-MSC ECM scaffolds promoted significantly higher calcium deposition and elevated relative expression of bone-specific genes, particularly the gene encoding osteopontin, when compared to pristine scaffolds. Overall, our results demonstrated the favorable effects of combining MSC-derived ECM and AM-based scaffolds on the osteogenic differentiation of MSC, resulting from a closer mimicry of the native bone niche. This strategy is highly promising for the development of novel personalized BTE approaches enabling the fabrication of patient defect-tailored scaffolds with enhanced biological performance and osteoinductive properties.

Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells

Osteoarthritis (OA) involves the degeneration of articular cartilage and subchondral bone. The capacity of articular cartilage to repair and regenerate is limited. A biodegradable, fibrous scaffold containing zinc oxide (ZnO) was fabricated and evaluated for osteochondral tissue engineering applications. ZnO has shown promise for a variety of biomedical applications but has had limited use in tissue engineering. Composite scaffolds consisted of ZnO nanoparticles embedded in slow degrading, polycaprolactone to allow for dissolution of zinc ions over time. Zinc has well-known insulin-mimetic properties and can be beneficial for cartilage and bone regeneration. Fibrous ZnO composite scaffolds, having varying concentrations of 1-10 wt.% ZnO, were fabricated using the electrospinning technique and evaluated for human mesenchymal stem cell (MSC) differentiation along chondrocyte and osteoblast lineages. Slow release of the zinc was observed for all ZnO composite scaffolds. MSC chondrogenic differentiation was promoted on low percentage ZnO composite scaffolds as indicated by the highest collagen type II production and expression of cartilage-specific genes, while osteogenic differentiation was promoted on high percentage ZnO composite scaffolds as indicated by the highest alkaline phosphatase activity, collagen production, and expression of bone-specific genes. This study demonstrates the feasibility of ZnO-containing composites as a potential scaffold for osteochondral tissue engineering.

Abstract 1796: p53 regulation of osteoblast differentiation is mediated through specific microRNAs

Abstract We have previously shown a role for tumor suppressor gene p53 in the transcriptional activation of several bone-specific genes in osteoblast differentiation. In this study, we used a screen for microRNAs to look for ones that were p53 dependent and differentiation dependent. In a model of invitro osteoblast differentiation, our previous work has established that p53 levels generally increase around day 4 of differentiation and represent the timeline when p53 transcriptionally regulates bone-specific gene expression. In these experiments, we used MC3T3-E1 osteoblasts to stably reduced p53 levels using specific shRNAs. These cells were initially characterized for the reduction in p53 levels and then compared with MC3T3E1 cells stably transfected with a scrambled control (Control MC3T3E1 cells). MicroRNAs were isolated from day 0 and day 4 after treatment with differentiation promoting (DP) media. A microRNA profiling service utilizing a microarray detection system analyzed over 1000 different microRNAs in control and DP treated cells to determine differentiation specific microRNAs expression. This array provided us with information about a number of genes that underwent alterations during differentiation in a p53 dependent manner. We validated several of the changes using realtime PCR and selected two microRNAs to study in detail. These two microRNAs (mIR 34b and 140) were increased two fold during normal differentiation but showed a dramatic reduction in expression when p53 levels were reduced. We ectopically expressed these microRNAs in MC3T3-E1 cells and created stable lines. Cells carrying mIR34b showed a decrease in cell proliferation rates when compared to mIR140 expressing and control cells. P53 levels directly correlated with microRNA 34b expression but not mIR140. MiR 140 ectopic expression affected Bone morphogenetic protein expression (BMP2), an important bone anabolic agent that is also p53 regulated. Putative target binding sites for bone-specific transcription factors Runx2, SP7, Vitamin D receptor (VDR) were found for mIR34b and 140 and were utilized in luciferase reporter assays to confirm microRNA specific interactions. These studies provided evidence and confirmed for us that the p53 regulation of osteoblast differentiation is also mediated through specific microRNAs that directly target important bone-specific genes. Citation Format: Elisha Pendleton, Shivang Shah, Oliver Couture, Teresa Kusper, Mustafa Broachwalla, Lauren Alt, Michael Fay, Nalini Chandar. p53 regulation of osteoblast differentiation is mediated through specific microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1796.

Mummy Material Can Promote Differentiation of Adipose Derived Stem Cells into Osteoblast through Enhancement of Bone Specific Transcription Factors Expression

Purpose: Application of Mummy material for treatment of different diseases such as bone fracture, cutaneous wounds and joint inflammation has been advised since hundred years ago in Persian traditional medicine. Due to the claims of indigenous people and advice of traditional medicine for application of this material in healing of bone fractures, this study has been designed to evaluate whether Mummy material can promote the differentiation of mesenchymal stem cells into osteoblasts and enhance the expression of bone specific genes and proteins.Methods: Adipose derived stem cells (ASCs) at fourth cell passage were divided into control, osteogenesis group (received osteogenic medium), Mummy group (received Mummy at concentration of 500 µg/ml). ASCs in the fourth group were treated with both osteogenic medium and Mummy (500µg/ml). Cells in all groups were harvested on days 7, 14 and 21 days for further evaluation through Real time RT-PCR, Von kossa staining, Immunocytochemistry and flowcytometery.Results: Treatment of ASCs with Mummy at concentration of 500µg/ml promotes the expression level of Osteocalcin, RUNX-2 and β1-integrin genes in different time points but that of the Osterix did not changed. Furthermore the expression of Osteocalcin protein enhanced significantly in ASCs treated with Mummy detected by Immunocytochemistry and flowcytometery technique compared to the control groups. The results of this study also showed that treatment of ASCs with Mummy resulted in formation of mineral deposits which was evaluated by Von Kossa staining method.Conclusion: Obtained data from this study reveals that Mummy is a potent enhancer for differentiation of ASCs into osteoblasts in in vitro system, probably through increasing the level of bone specific genes and proteins.

Bone morphogenetic protein-2 is a stronger inducer of osteogenesis within muscle tissue than heterodimeric bone morphogenetic protein-2/6 and -2/7: Implications for expedited gene-enhanced bone repair.

Bone morphogenetic protein (BMP)-2 gene-activated muscle tissue fragments can regenerate large bone defects in preclinical animal models. The use of tissue fragments instead of isolated cells expedites gene-enhanced tissue engineering and may increase the possibility of clinical translation. The present in vitro study investigated whether the osteoinductive effect of BMP-2 on muscle tissue fragments can be enhanced using the heterodimers BMP-2/6 or BMP-2/7. Skeletal muscle tissue fragments from rats were cultured in vitro for up to 20days in normal medium, osteogenic medium or osteogenic medium supplemented with either a low (50ng/ml) or high (200ng/ml) concentration of recombinant human BMP-2, BMP-2/6 or BMP-2/7. Osteoinduction was evaluated by a quantitative reverse transcriptase-polymerase chain reaction, Alizarin red S staining, immunohistology and histomorphometry. Interestingly, BMP-2 was a significantly stronger inducer of osteogenic differentiation within muscle tissue than both heterodimers. Even the low concentration of BMP-2 elicited significantly higher levels of calcium deposition, bone-specific gene expression and protein production than the high concentration of both heterodimers. At the high concentration, BMP-2/7 had a significantly stronger osteogenic effect on muscle than BMP-2/6. The homodimer BMP-2 induced osteoblastogenesis in muscle faster, at a lower concentration and with a higher potency than the heterodimers BMP-2/6 or BMP-2/7. The findings of this in vitro study encourage bone repair by muscle implants in combination with BMP-2 single growth factor delivery, which might be beneficial with respect to clinical translation.