Osteoinductive Effect Research Articles

Hyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic rats.

ABSTRACTTobacco mosaic virus (TMV) has been studied as a multi-functional agent for bone tissue engineering. An osteo-inductive effect of wild-type TMV has been reported, as it can significantly enhance the bone differentiation potential of bone marrow stromal cells both on a two-dimensional substrate and in a three-dimensional (3D) hydrogel system. A TMV mutant (TMV-RGD1) was created which featured the adhesion peptide arginyl-glycyl-aspartic acid (RGD), the most common peptide motif responsible for cell adhesion to the extracellular matrix, on the surface of the virus particle to enhance the bio-functionality of the scaffold material. We hypothesised that the incorporation of either wild-type TMV or TMV-RGD1 in the 3D hydrogel scaffold would induce bone healing in critical size defects of the cranial segmental bone. We have previously tested the virus-functionalised scaffolds, in vitro, with a hyaluronic acid-based system as an in-situ hydrogel platform for 3D cell encapsulation, culture, and differentiation. The results of these experiments suggested the potential of the virus-functionalised hydrogel to promote in vitro stem cell differentiation. The hydrogel-forming system we employed was shown to be safe and biocompatible in vivo. Here, we further explored the physiological responses regarding bone regeneration of a calvarial defect in both normal and osteoporotic ovariectomized rat models. Our results, based on histological analysis in both animal models, suggested that both wild-type TMV and TMV-RGD1 functionalised hydrogels could accelerate bone regeneration, without systemic toxicity, evaluated by blood counts. New bone formation was intensified by the incorporation of the RGD-mutant viral particles. This finding increased the potential for use of the rod-shaped plant virus as a platform for the addition of powerful biofunctionality for tissue engineering applications. This study was approved by the Ethics Committee on Animal Use of the Zhenjiang Affiliated First People’s Hospital affiliated to Jiangsu University

The Calcium Phosphate Modified Titanium Implant Combined With Platelet-Rich Plasma Treatment Promotes Implant Stabilization in an Osteoporotic Model.

Osteoporosis as a kind of systemic bone metabolic disease has become one of the most prevalent diseases among the middle- and old-age, characterized with low bone mass and disruptive osseous microenvironment. The poor bone condition both in quantity and quality makes it daunting for osteoporotic patients who are submitted to dental implantation, joint replacement therapy, or orthopedic surgery. Since calcium phosphate (CaP) and platelet-rich plasma (PRP) treatment, all have improving the effect on bone regeneration. Inspired by this fact, the authors introduced a kind of novel implant with CaP modified surface by HPT (hydrothermal & pressure) treatment in this study. After producing, the authors tested its physicochemical properties through scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscope (XPS) and contact-angle measurement. Then the authors desired to investigate the effect of this CaP-modified implant on bone regeneration and stabilization maintenance combined with PRP treatment by establishing an osteoporotic rat model. After 3 months of surgery, the authors collected all the specimens and evaluated new bone formation by micro-computed tomography (micro-CT) analysis, biomechanical test, and histologic assessment. All the results in vivo experiment showed the CaP modified implant combined with PRP treatment could improve the osteoinductive effect under osteoporotic condition, leading to better maintenance for stabilization between bone and implant interface, which might be rendered as a promising clinical method for osteoporotic patients when they receive orthopedic surgeries.

Small Molecule–Mediated Enhanced Osteogenesis of Human Mesenchymal Stem Cells: a Probable Alternate for BMP-2

The use of bone morphogenic protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. An equipotential and a cost-effective replacement for BMP-2 is therefore required. In this study, different combinations of small molecules like FK506 (F), an immunosuppressant; purmorphamine (P), a hedgehog agonist; and Ku-0063794 (K), an mTOR inhibitor, were evaluated on human mesenchymal stem cells (hMSCs) to assess the osteoinduction behavior. Dose optimization and various combinations of small molecules were evaluated using MTT and ALP assay. The gene and protein expression of osteogenic markers RunX2, Osteopontin (OPN), and collagen type 1 on treatment with small molecules was evaluated and compared with BMP-2 expression. The confirmation of the osteogenic potential of small molecules was calcium deposits formed in hMSCs after staining with Alizarin Red S. Optimum concentration of individual small molecule (F 5 nM, K 10 nM, and P 2 μM) was determined based on the MTT and ALP activity. Small molecules (F + K + P) with osteogenic media (OS) showed 1.5-fold higher ALP activity with respect to OS. The synergistic effect of F + K + P and BMP-2/OS demonstrated an upregulation of RUNX2, OPN, and collagen 1 proteins and genes. Small molecules alone (F- 5 nM, K- 10 nM, and P- 2 μM) expressed the formation of calcium ions stained by Alizarin Red S which confirmed their osteoinductive effect. Thus, small molecules may have the potential to substitute or enable the use of reduced dose of BMP-2 for enhanced osteogenesis of progenitor cells. The use of Bone Morphogenic Protein-2 (BMP-2) to accelerate the healing of bone defects has resulted in side effects such as ectopic bone formation, soft tissue swelling, and cyst-like bone void formation. We show that cost-effective small molecules, in combination with osteogenic differentiation cocktail, have the potential to substitute BMP-2 for enhanced osteogenesis of progenitor cells. Graphical abstract

Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes

BackgroundExcessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process.MethodsBMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4.ResultsIn SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes.ConclusionsOur results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes

Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

Osteogenic and Anti-Inflammatory Behavior of Injectable Calcium Phosphate Loaded with Therapeutic Drugs.

Bone fractures related to musculoskeletal disorders determine long-term disability in older people with a consequent significant economic burden. The recovery of pathologically impaired tissue architecture allows avoiding bone loss-derived consequences such as bone height reduction, deterioration of bone structure, inflamed bone pain, and high mortality for thighbone fractures. Actually, standard therapy for osteoporosis treatment is based on the systemic administration of biphosphonates and anti-inflammatory drugs, which entail several side effects including gastrointestinal (GI) diseases, fever, and articular pain. Hence, the demand of innovative therapeutic approaches for locally treating bone lesions has been increasing in the last few years. In this scenario, the development of injectable materials loaded with therapeutically active agents (i.e., anti-inflammatory drugs, antibiotics, and peptides mimicking growth factors) could be an effective tool to treat bone loss and inflammation related to musculoskeletal diseases, including osteoporosis and osteoarthritis. According to this challenge, here, we propose three different compositions of injectable calcium phosphates (CaP) as new carrier materials of therapeutic compounds such as bisphosphonates (i.e., alendronate), anti-inflammatory drugs (i.e., diclofenac sodium), and natural molecules (i.e., harpagoside) for the local bone disease treatment. Biological quantitative analyses were performed for screening osteoinductive and anti-inflammatory properties of injectable drug-loaded systems. Meanwhile, cell morphological features were analyzed through scanning electron microscopy and confocal investigations. The results exhibited that the three systems exerted an osteoinductive effect during later phases of osteogenesis. Simultaneously, all compositions showed an anti-inflammatory activity on inflammation in vitro models.

Inspired by nature: facile design of nanoclay-organic hydrogel bone sealant with multifunctional properties for robust bone regeneration.

Bone repair is a complex process involving the sophisticated interplay of osteogenic stem cells, extracellular matrix, and osteoinductive factors, and it is affected by bacterial toxins and oxidative stress. Inspired by the nature of plant-derived phytochemicals and inorganic-organic analogues of the bone extracellular matrix, we report herein the facile design of a nanoclay-organic hydrogel bone sealant (NoBS) that integrates multiple physico-chemical cues for bone regeneration into a single system. Assembly of phytochemical-modified organic chitosan and silica-rich inorganic nanoclay serves as highly biocompatible and osteoconductive extracellular matrix mimics. The decorated phytochemical exerts inherent bactericidal and antioxidant activities, and acts as an intermolecular networking precursor for gelation with injectable and self-healing capabilities. Moreover, the NoBS exerts osteoinductive effects mediated by the nanoclay, which regulates the Wnt/β-catenin pathway, along with the addition of osteoinductive signals, resulting in bone regeneration in a non-healing cranial defect. Engineering of this integrated bone graft substitute with multifunctional properties inspired by natural materials may suggest a promising and effective approach for creating a favorable microenvironment for optimal bone healing.

Induction of osteogenic differentiation by demineralized and decellularized bovine extracellular matrix derived hydrogels associated with barium titanate

Bone tissue-derive biomaterials have become of great interest to treat diseases of the skeletal system. Biological scaffolds of demineralized and decellularized extracellular matrices (ECM) have been developed and one of these options are ECM hydrogels derived from bovine bone. Nanomaterials may be able to regulate stem cell differentiation due to their unique physical-chemical properties. The present work aimed to evaluate the osteoinductive effects of ECM hydrogels associated with barium titanate nanoparticles (BTNP) on dental pulp cells derived from exfoliated teeth. The addition of BTNP in the ECM derived hydrogel did not affect cell proliferation and the formation of bone nodules. Furthermore, it increased the expression of bone alkaline phosphatase. The results demonstrated that the nanobiocomposites were able to promote the osteogenic differentiation, even in the absence of chemical inducing factors for osteogenic differentiation. In conclusion, bovine bone ECM hydrogel combined with BTNP presented and increased expression of markers of osteogenic differentiation in the absence of chemical inducing factors.

Citrate zinc hydroxyapatite nanorods with enhanced cytocompatibility and osteogenesis for bone regeneration

The development of biomaterials that mimicking the hydroxyapatite nanoparticles existent in the immature bone tissue is crucial, especially to accelerate the bone remodeling and regeneration. In this work, it was developed for the first time, hydroxyapatite nanoparticles (NPs) incorporating citrate and zinc (cit-Zn-Hap) in their composition towards a one-step hydrothermal procedure. For comparison purposes, hydroxyapatite NPs incorporating only zinc (Zn-Hap) or citrate (cit-Hap), as well as hydroxyapatite without any of these elements (Hap) were synthesised. The physicochemical characterization was carried out reveling that, the presence of zinc on hydroxyapatite (cit-Zn-Hap), reduced the size of nanoparticles, changed the phosphate environment and decreased the surface charge when compared with cit-Hap nanoparticles. The osteogenic potential of cit-Zn-Hap NPs was analysed in human bone marrow-derived stromal cells (BMSCs), in the absence of osteoinductive factors. NPs were internalized by endocytosis appearing trapped in endosomes and lysosomes scattered through the cytoplasm. Exposure to these NPs resulted in a significant induction of ALP activity, extracellular matrix mineralization, and gene expression of early and later osteogenic transcription factors, as well as of osteoblastic markers. The osteoinductive effect might be regulated, at least in part, by the increased signalling through the canonical WNT pathway. Evaluation of the cell behaviour following exposure to Zn-Hap and cit-Hap strongly suggested a synergistic effect of citrate and Zn in cit-Zn-Hap NPs towards the induction of the osteogenic commitment and functionality of BMSCs. These findings will allow the design of new biomimetic hydroxyapatite nanoparticles with great potential for bone regeneration.

Biofabrication of gold nanoparticles with bone remodeling potential: an in vitro and in vivo assessment

Recent thrust in the biomaterials research has shifted towards developing bioactive and resorbable scaffolds with nanotechnological interventions. Next-generation bone scaffolds need to be multifunctional with desired osteo-integration ability, conformed resorption rate and in vivo imaging potential. In this study, we synthesized biogenic multifunctional gold nanoparticles (AuNPs) using immobilized crude enzyme (ImCE) of Bacillus licheniformis. The multifunctional characteristics of AuNPs and reusability of ImCE for AuNP synthesis can make the process and application cost-effective. ImCE catalyzed the bioreduction of gold ions to AuNPs of 18.4 ± 3.3 nm. AuNPs showed good hemocompatibility and antioxidant property without any toxic effects. Further, AuNPs demonstrated the increase in alkaline phosphatase activity and calcium deposition through MC3T3-E1 murine preosteoblast cell line and rat bone marrow stromal cells (BMSCs). The osteoinductive effect of AuNPs was also investigated in vivo wherein the composite cryogel scaffold functionalized with bone morphogenic protein-2 (rhBMP-2), zoledronic acid (ZA), and AuNPs was implanted in a muscle pouch of Wistar rats. AuNPs had synergistic effect on bone formation along with rhBMP-2 and ZA as seen from micro-computed tomography and histological analysis. The AuNPs were found to exhibit autofluorescence property and enhanced the degradation of scaffold at the implanted site in vivo. The developed process of using ImCE presents a novel approach for biogenic fabrication of AuNPs with the potential orthopedic application.

Enhancement of local bone formation on titanium implants in osteoporotic rats by biomimetic multilayered structures containing parathyroid hormone (PTH)-related protein

Osteoporosis is a severe health problem causing bone fragility and consequent fracture. Titanium (Ti) implants, used in patients with osteoporotic fractures, are prone to failure because of the decreased bone mass and strength. Therefore, it is of utmost importance to fabricate implants possessing osteogenic properties to improve implant osseointegration. To improve the long-term survival rate of Ti implants in osteoporotic patients, hyaluronic acid/ϵ-polylysine multilayers containing the parathyroid hormone (PTH)-related protein (PTHrP) were deposited on Ti implants by a layer-by-layer (LBL) electro assembly technique. The murine pre-osteoblast cell line MC3T3-E1, possessing a high potential of osteoblast differentiation, was used to evaluate the osteo-inductive effects of Ti-LBL-PTHrP in vitro. In addition, the performance of the Ti (Ti-LBL-PTHrP) implant was evaluated in vivo in a femoral intramedullary implantation in Sprague Dawley rats. The Ti-LBL-PTHrP implant regulated the release of the loaded PTHrP to increase bone formation in the early stage of implantation. The in vitro results revealed that cells on Ti-LBL-PTHrP did not show any evident proliferation, but a high level of alkaline phosphatase activity and osteoblast-related protein expression was found, compared to the uncoated Ti group (p < 0.05). In addition, in vivo micro-CT and histological analysis demonstrated that the Ti-LBL-PTHrP implants could significantly promote the formation and remodeling of new bone in osteoporotic rats at 14 d after implantation. Overall, this study established a profound and straightforward methodology for the manufacture of biofunctional Ti implants for the treatment of osteoporosis.

Biofabrication of SDF-1 Functionalized 3D-Printed Cell-Free Scaffolds for Bone Tissue Regeneration.

Large segmental bone defects occurring after trauma, bone tumors, infections or revision surgeries are a challenge for surgeons. The aim of our study was to develop a new biomaterial utilizing simple and cheap 3D-printing techniques. A porous polylactide (PLA) cylinder was printed and functionalized with stromal-derived factor 1 (SDF-1) or bone morphogenetic protein 7 (BMP-7) immobilized in collagen type I. Biomechanical testing proved biomechanical stability and the scaffolds were implanted into a 6 mm critical size defect in rat femur. Bone growth was observed via x-ray and after 8 weeks, bone regeneration was analyzed with µCT and histological staining methods. Development of non-unions was detected in the control group with no implant. Implantation of PLA cylinder alone resulted in a slight but not significant osteoconductive effect, which was more pronounced in the group where the PLA cylinder was loaded with collagen type I. Addition of SDF-1 resulted in an osteoinductive effect, with stronger new bone formation. BMP-7 treatment showed the most distinct effect on bone regeneration. However, histological analyses revealed that newly formed bone in the BMP-7 group displayed a holey structure. Our results confirm the osteoinductive character of this 3D-biofabricated cell-free new biomaterial and raise new options for its application in bone tissue regeneration.

Surface Functionalization with Proanthocyanidins Provides an Anti-Oxidant Defense Mechanism That Improves the Long-Term Stability and Osteogenesis of Titanium Implants.

PurposeAseptic loosening is a major complication after total joint replacement. Reactive oxygen species generated by local tissue cells and liberated from implant surfaces have been suggested to cause implant failures. Surface modification of titanium (Ti)-based implants with proanthocyanidins (PAC) is a promising approach for the development of anti-oxidant defense mechanism to supplement the mechanical functions of Ti implants. In this study, a controlled PAC release system was fabricated on the surface of Ti substrates using the layer-by-layer (LBL) assembly.Materials and MethodsPolyethyleneimine (PEI) base layer was fabricated to enable layer-by-layer (LBL) deposition of hyaluronic acid/chitosan (HA/CS) multi-layers without or with the PAC. Surface topography and wettability of the fabricated HA/CS-PAC substrates were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR) and contact angle measurement. PAC release profiles were investigated using drug release assays. MC3T3-E1 pre-osteoblast cells were used to assess the osteo-inductive effects of HA/CS-PAC substrates under conditions H2O2-induced oxidative stress in vitro. A rat model of femoral intramedullary implantation evaluated the osseo-integration and osteo-inductive potential of the HA/CS-PAC coated Ti implants in vivo.ResultsSEM, AFM, FTIR and contact angle measurements verified the successful fabrication of Ti surfaces with multi-layered HA/CS-PAC coating. Drug release assays revealed controlled and sustained release of PAC over 14 days. In vitro, cell-based assays showed high tolerability and enhanced the osteogenic potential of MC3T3-E1 cells on HA/CS-PAC substrates when under conditions of H2O2-induced oxidative stress. In vivo evaluation of femoral bone 14 days after femoral intramedullary implantation confirmed the enhanced osteo-inductive potential of the HA/CS-PAC coated Ti implants.ConclusionMulti-layering of HA/CS-PAC coating onto Ti-based surfaces by the LBL deposition significantly enhances implant osseo-integration and promotes osteogenesis under conditions of oxidative stress. This study provides new insights for future applications in the field of joint arthroplasty.

A novel fluorescent hydroxyapatite based on iron quantum cluster template to enhance osteogenic differentiation.

Template-mediated self-assembly synthesis has produced a diverse range of biomimetic materials with unique physicochemical properties. Here, we fabricated novel fluorescent three-dimensional (3-D) hydroxyapatite (HAP) nanorod-assembled microspheres using iron quantum cluster (FeQC) as a hybrid template, containing three organic components: hemoglobin chains, piperidine, and iron clusters. The material characterization indicated that the synthesized HAP possessed a uniform rod-like morphology, ordered 3-D architecture, high crystallinity, self-activated fluorescence, and remarkable photostability. Our study proposed that this FeQC template is a promising regulating agent to fabricate fluorescent self-assembled HAP microspheres with a controlled morphology. The effect of HAP on stem cell fate and their osteogenic differentiation was investigated by culturing human bone marrow-derived mesenchymal stromal/stem cells (BMSCs) with HAP microspheres. Significant increases in collagen matrix production and gene expression of osteogenic markers, including osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP) and alkaline phosphatase (ALP), were observed compared to the controls after 21days of culture. Taken together, our data suggest that synthetic HAP nanorod-assembled microspheres represent a promising new biomaterial which exhibits enhanced fluorescent properties and osteoinductive effects on human BMSCs.

Osteoinductivity of polycaprolactone nanofibers grafted functionalized with carboxymethyl chitosan: Synergic effect of β-carotene and electromagnetic field

In this study, carboxymethyl chitosan (CMC) was grafted on polycaprolactone (PCL) nanofibers to fabricate scaffolds for bone tissue engineering. The electrospun PCL nanofibers were treated by cold atmospheric plasma (CAP) of helium to generate the reactive functions necessary for CMC grafting. β-carotene (βC) as a biochemical clue and electromagnetic field (EMF, 31.4 μT, 1 h per day) as a biophysical stimulator were used to promote the proliferation and osteodifferentiation of adipose mesenchymal stem cells (ADSCs). Alizarin red staining and calcium content results indicated the generation of nodal calcium on the CMC30%-g-PCL scaffold after 14 days of incubation in the presence or absence of external stimulation factors. Immunocytochemistry (ICC) results confirmed the expression of osteonectin protein for the stem cells seeded on CMC30%-g-PCL with or without using βC or EMF. These results suggest that the fabricated CMC-grafted scaffolds have the ability to self-differentiate stem cells to osteoblasts due to the osteoinductive effects of the grafted CMC. Furthermore, the osteodifferentiation of ADSCs is promoted by using an external stimulation factor such as βC or EMF.

Reduction of inflammation in a chronic periodontitis model in rats by TNF-α gene silencing with a topically applied siRNA-loaded calcium phosphate paste

We developed a calcium phosphate-based paste containing siRNA against TNF-α and investigated its anti-inflammatory and bone-healing effects in vitro and in vivo in a rat periodontitis model. The bioactive spherical CaP/PEI/siRNA/SiO2 nanoparticles had a core diameter of 40–90 nm and a positive charge (+23 mV) that facilitated cellular uptake. The TNF- α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. CaP/PEI/siRNA/SiO2 nanoparticles cancelled the suppression of alkaline phosphatase (ALP) activity in LPS-stimulated bone marrow-derived cells. In vivo, ALP mRNA was up-regulated, TNF-α mRNA was down-regulated, and the amount of released TNF-α was significantly reduced after topical application of the calcium phosphate-based paste containing siRNA-loaded nanoparticles. The number of TNF-α-positive cells in response to CaP/PEI/siRNA/SiO2 nanoparticle application was lower than that observed in the absence of siRNA. Elevated ALP activity and numerous TRAP-positive cells (osteoclasts) were observed in response to the application of all calcium phosphate pastes. These results demonstrate that local application of a paste consisting of siRNA-loaded calcium phosphate nanoparticles successfully induces TNF-α silencing in vitro and in vivo and removes the suppression of ALP activity stimulated by inflammation. Statement of significanceWe developed a calcium phosphate-based paste containing nanoparticles loaded with siRNA against TNF-α. The nanoparticles had a core diameter of 40–90 nm and positive charge (+23 mV). The anti-inflammatory and osteoinductive effects of the paste were investigated in vitro and in vivo in a rat periodontitis model. In vitro, the TNF-α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. The ALP activity of bone marrow-derived cells was recovered. In vivo, TNF-α mRNA was down-regulated and the amount of released TNF-α was significantly reduced, whereas the ALP mRNA was up-regulated. Elevated ALP activity and TRAP-positive cells were observed by immunohistochemistry.

A biocompatible vascularized graphene oxide (GO)-collagen chamber with osteoinductive and anti-fibrosis effects promotes bone regeneration in vivo.

The survival of transplanted cells and tissues in bone regeneration requires a microenvironment with a vibrant vascular network. A tissue engineering chamber can provide this in vivo. However, the commonly used silicone chamber is biologically inert and can cause rejection reactions and fibrous capsule. Studies have revealed that collagen is highly biocompatible and graphene oxide (GO) could regulate osteogenic activity in vivo. Besides, GO can be cross-linked with natural biodegradable polymers to construct scaffolds.Methods: A vascularized GO-collagen chamber model was built by placing vessels traversing through the embedded tissue-engineered grafts (osteogenic-induced bone mesenchymal stem cells -gelatin) in the rat groin area. Osteogenic activity and inflammatory reactions were assessed using different methods including micro-CT scanning, Alizarin red staining, and immunohistochemical staining.Results: After one month, in vivo results showed that bone mineralization and inflammatory responses were significantly pronounced in the silicone model or no chamber (control) groups. Vascular perfusion analysis confirmed that the GO-collagen chamber improved the angiogenic processes. Cells labeled with EdU revealed that the GO-collagen chamber promoted the survival and osteogenic differentiation of bone mesenchymal stem cells.Conclusion: Overall, the novel biocompatible GO-collagen chamber exhibited osteoinductive and anti-fibrosis effects which improved bone regeneration in vivo. It can, therefore, be applied to other fields of regenerative medicine.

DEVELOPMENT OF OSTEOPLASTIC MATERIAL IMPREGNATED WITH PLASMID ENCODING BONE MORPHOGENETIC PROTEIN-2

A new gene-activated osteoplastic based on chitosan material impregnated with plasmid with the BMP-2 gene has been developed. The material had an osteoinductive effect on mesenchymal stromal cells (MSC).

PI3K/Akt Signaling Involved with Osteoinductive Effects of Achatina fulica Mucus

Abstract Mesenchymal stem cells and osteoblasts play important roles in bone formation. Achatina fulica mucus presented the property of osteoinduction. This study aimed to examine the effects of A. fulica mucus on human mesenchymal stem cell (hMSC) and human fetal osteoblastic cell line (HFOB) differentiation. The integrated effects of A. fulica mucus and polycaprolactone (PCL) on the differentiation of hMSCs were tested. The cell viability of hMSCs treated with A. fulica mucus was investigated by the MTT assay. The cell mineralization was observed by Alizarin Red S staining, the gene expression was investigated using RT-PCR, and the PI3K activation was studied using flow cytometry. The results indicated that A. fulica mucus induced osteogenic differentiation in hMSCs and HFOBs by upregulation of the osteogenic markers; osteopontin (OPN) and osteocalcin (OCN). The results of the Alizarin Red S staining indicated that A. fulica mucus supported mineralization in both hMSCs and HFOBs. The hMSCs cultured on PCL supplemented with A. fulica mucus showed significantly increased RUNX2 and OPN expressions. A. fulica mucus was observed to increase PI3K activation in hMSCs. The findings of this study suggested that A. fulica mucus and biomaterials could be applied together for use in bone regeneration in the future.

Osteoinductive Material to Fine-Tune Paracrine Crosstalk of Mesenchymal Stem Cells With Endothelial Cells and Osteoblasts.

While stem cell/biomaterial studies provide solid evidences that biomaterial intrinsic cues deeply affect cell fate, current strategies tend to neglect their effects on mesenchymal stem cells (MSCs) secretory activities and resulting cell-crosstalks. The present study aims to investigate the impact of bone-mimetic material (B-MM), with intrinsic osteoinductive property, on MSCs mediator secretions; and to explore underlying effects on cells involved in bone regeneration. Human MSCs were cultured, on B-MM, made from inorganic calcium phosphate supplemented with chitosan and hyaluronic acid biopolymers. Collected MSCs culture media were assessed for mediators release quantification and used further to stimulate endothelial cells (ECs) and alveolar bone derived osteoblasts (OBs). Without osteogenic supplements, MSCs committed into bone lineage forming thus 3D bone-like nodules after 21 days. Despite a weak percentage of cell commitment, our data elucidate new aspects of osteoinductive material effect on MSCs functions through the regulation of the secretion of mediators involved in bone regeneration and subsequently the MSCs/ECs indirect crosstalk with osteogenesis-boosting effect. Using MSCs culture media, we demonstrate a large potential of osteoinductive materials and MSCs in bone regenerative medicine. Such strategies could help to address some insights in cell-free therapies using MSCs derived media.