Factor In Bone Tissue Engineering Research Articles

Stem Cells and Bone Tissue Engineering.

Segmental bone defects that are caused by trauma, infection, tumor resection, or osteoporotic fractures present significant surgical treatment challenges. Host bone autograft is considered the gold standard for restoring function but comes with the cost of harvest site comorbidity. Allograft bone is a secondary option but has its own limitations in the incorporation with the host bone as well as its cost. Therefore, developing new bone tissue engineering strategies to treat bone defects is critically needed. In the past three decades, the use of stem cells that are delivered with different scaffolds or growth factors for bone tissue engineering has made tremendous progress. Many varieties of stem cells have been isolated from different tissues for use in bone tissue engineering. This review summarizes the progress in using different postnatal stem cells, including bone marrow mesenchymal stem cells, muscle-derived stem cells, adipose-derived stem cells, dental pulp stem cells/periodontal ligament stem cells, periosteum stem cells, umbilical cord-derived stem cells, peripheral blood stem cells, urine-derived stem cells, stem cells from apical papilla, and induced pluripotent stem cells, for bone tissue engineering and repair. This review also summarizes the progress using exosomes or extracellular vesicles that are delivered with various scaffolds for bone repair. The advantages and disadvantages of each type of stem cell are also discussed and explained in detail. It is hoped that in the future, these preclinical results will translate into new regenerative therapies for bone defect repair.

Lysyl oxidase inhibits BMP9-induced osteoblastic differentiation through reducing Wnt/β-catenin via HIF-1a repression in 3T3-L1 cells

BackgroundBone morphogenetic protein 9 (BMP9) is a promising growth factor in bone tissue engineering, while the detailed molecular mechanism underlying BMP9-oriented osteogenesis remains unclear. In this study, we investigated the effect of lysyl oxidase (Lox) on the BMP9 osteogenic potential via in vivo and in vitro experiments, as well as the underlying mechanism.MethodsPCR assay, western blot analysis, histochemical staining, and immunofluorescence assay were used to quantify the osteogenic markers level, as well as the possible mechanism. The mouse ectopic osteogenesis assay was used to assess the impact of Lox on BMP9-induced bone formation.ResultsOur findings suggested that Lox was obviously upregulated by BMP9 in 3T3-L1 cells. BMP9-induced Runx2, OPN, and mineralization were all enhanced by Lox inhibition or knockdown, while Lox overexpression reduced their expression. Additionally, the BMP9-induced adipogenic makers were repressed by Lox inhibition. Inhibition of Lox resulted in an increase in c-Myc mRNA and β-catenin protein levels. However, the increase in BMP9-induced osteoblastic biomarkers caused by Lox inhibition was obviously reduced when β-catenin knockdown. BMP9 upregulated HIF-1α expression, which was further enhanced by Lox inhibition or knockdown, but reversed by Lox overexpression. Lox knockdown or HIF-1α overexpression increased BMP9-induced bone formation, although the enhancement caused by Lox knockdown was largely diminished when HIF-1α was knocked down. Lox inhibition increased β-catenin levels and decreased SOST levels, which were almost reversed by HIF-1α knockdown.ConclusionLox may reduce the BMP9 osteoblastic potential by inhibiting Wnt/β-catenin signaling via repressing the expression HIF-1α partially.Graphical abstract

Inhibition of GSK-3β Enhances Osteoblast Differentiation of Human Mesenchymal Stem Cells through Wnt Signalling Overexpressing Runx2.

Small-molecule-inhibitor-based bone differentiation has been recently exploited as a novel approach to regulating osteogenesis-related signaling pathways. In this study, we identified 1-Azakenpaullone, a highly selective inhibitor of glycogen synthase kinase-3β (GSK-3β), as a powerful inducer of osteoblastic differentiation and mineralization of human mesenchymal stem cells (MSCs). GSK-3β is a serine-threonine protein kinase that plays a major role in different disease development. GSK-3β is a key regulator of Runx2 activity in osteoblastic formation. We evaluated alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin Red staining to assess the mineralization of cultured human MSCs. Gene expression profiling was assessed using an Agilent microarray platform, and bioinformatics were performed using Ingenuity Pathway Analysis software. Human MSCs treated with 1-Azakenpaullone showed higher ALP activity, increased in vitro mineralized matrix formation, and the upregulation of osteoblast-specific marker gene expression. Global gene expression profiling of 1-Azakenpaullone-treated human MSCs identified 1750 upregulated and 2171 downregulated mRNA transcripts compared to control cells. It also suggested possible changes in various signaling pathways, including Wnt, TGFβ, and Hedgehog. Further bioinformatics analysis employing Ingenuity Pathway Analysis recognized significant enrichment in the 1-Azakenpaullone-treated cells of genetic networks involved in CAMP, PI3K (Complex), P38 MAPK, and HIF1A signaling and functional categories associated with connective tissue development. Our results suggest that 1-Azakenpaullone significantly induced the osteoblastic differentiation and mineralization of human MSCs mediated by the activation of Wnt signaling and the nuclear accumulation of β-catenin, leading to the upregulation of Runx2, a key transcription factor that ultimately promotes the expression of osteoblast-specific genes. Thus, 1-Azakenpaullone could be used as an osteo-promotor factor in bone tissue engineering.

Osteogenic Induction with Silicon Hydroxyapatite Using Modified Autologous Adipose Tissue-Derived Stromal Vascular Fraction: In Vitro and Qualitative Histomorphometric Analysis.

Large bone defects requiring invasive surgical procedures have long been a problem for orthopedic surgeons. Despite the use of autologous bone grafting, satisfactory results are often not achieved due to associated limitations. Biomaterials are viable alternatives and have lately been used in association with Stromal Vascular Fraction (SVF), stem cells, and signaling factors for bone tissue engineering (BTE). The objective of the current study was to assess the biocompatibility of Silicon Hydroxyapatite (Si-HA) and to improve osteogenic potential by using autologous adipose-derived SVF with Si-HA in a rabbit bone defect model. Si-HA granules synthesized using a wet precipitation method were used. They were characterized using scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and X-ray diffraction (XRD). A hemolysis assay was used to assess the hemolytic effects of Si-HA, while cell viability was assessed through Alamar Blue assay using MC3T3 mouse osteoblasts. The osteogenic potential of Si-HA both alone and with enzymatically/non-enzymatically-derived SVF (modified) was performed by implantation in a rabbit tibia model followed by histomorphometric analysis and SEM of dissected bone after six weeks. The results showed that Si-HA granules were microporous and phase pure and that the addition of Silicon did not influence Si-HA phase composition. Si-HA granules were found to be non-hemolytic on the hemolysis assay and non-toxic to MC3T3 mouse osteoblasts on the Alamar Blue assay. Six weeks following implantation Si-HA showed high biocompatibility, with increased bone formation in all groups compared to control. Histologically more mature bone was formed in the Si-HA implanted along with non-enzymatically-derived modified SVF. Bone formation was observed on and around Si-HA, reflecting osseointegration. In conclusion, Si-HA is osteoconductive and promotes osteogenesis, and its use with SVF enhances osteogenesis.

Synthesis and Evaluation of BMMSC-seeded BMP-6/nHAG/GMS Scaffolds for Bone Regeneration

Bioactive scaffolding materials and efficient osteoinductive factors are key factors for bone tissue engineering. The present study aimed to mimic the natural bone repair process using an osteoinductive bone morphogenetic protein (BMP)-6-loaded nano-hydroxyapatite (nHA)/gelatin (Gel)/gelatin microsphere (GMS) scaffold pre-seeded with bone marrow mesenchymal stem cells (BMMSCs). BMP-6-loaded GMSs were prepared by cross-linking and BMP-6/nHAG/GMS scaffolds were fabricated by a combination of blending and freeze-drying techniques. Scanning electron microscopy, confocal laser scanning microscopy, and CCK-8 assays were carried out to determine the biocompatibility of the composite scaffolds in vitro. Alkaline phosphatase (ALP) activity was measured to evaluate the osteoinductivity of the composite scaffolds. For in vivo examination, critical-sized calvarial bone defects in Sprague-Dawley rats were randomly implanted with BMMSC/nHAG/GMS and BMMSC/BMP-6/nHAG/GMS scaffolds, and compared with a control group with untreated empty defects. The BMP-6-loaded scaffolds showed cytocompatibility by favoring BMMSC attachment, proliferation, and osteogenic differentiation. In radiological and histological analyses, the BMMSC-seeded scaffolds, especially the BMMSC-seeded BMP-6/nHAG/GMS scaffolds, significantly accelerated new bone formation. It is concluded that the BMP-6/nHAG/GMS scaffold possesses excellent biocompatibility and good osteogenic induction activity in vitro and in vivo, and could be an ideal bioactive substitute for bone tissue engineering.

Highly sensitive quantification of hydrogen-transmitting coenzyme in physiological pH using silver nanoparticles dispersed on nitrogen doped graphene quantum dots

The good mechanical and biological performance of natural biopolymers is important factor in biomedical science. In metabolism, nicotinamide adenine dinucleotide (NAD) is involved in redox reactions, carrying electrons from one reaction to another. On such a basis, much attention has been devoted to developing procedures to monitoring the presence of NAD at very low concentrations in a physiological environment and biological fluids. The good mechanical and biological performance of natural biopolymers is important factor in bone tissue engineering. Herein, a new platform based on silver nanoparticles (Ag NPs) and nitrogen doped graphene quantum dots (N-GQDS) is developed for sensitive voltammetric detection of NAD in human plasma samples. N-GQDS-Ag NPs film were prepared by the full electrochemically method onto the surface of GCE. Ag NPs were covalently bonded to N-GQDS films and used to bind of NAD biomarker. The novel detection scheme is a suitable electrochemical assay, which makes the NAD detection simple and rapid. The engineered electrochemical sensor showed a linear dynamic range and low limit of quantification limit of 1.45–7.9 μM and 1.45 μM, respectively. The proposed sensor showed excellent analytical performance for the detection of NAD with high sensitivity and reproducibility. The prepared sensor detected NAD ion human plasma samples with high recovery values. We hope that this platform has the potential to be used in clinical applications and point-of-care diagnosis of disease.

In vitro effect of graphene structures as an osteoinductive factor in bone tissue engineering: A systematic review.

Graphene and its derivatives have been well-known as influential factors in differentiating stem/progenitor cells toward the osteoblastic lineage. However, there have been many controversies in the literature regarding the parameters effect on bone regeneration, including graphene concentration, size, type, dimension, hydrophilicity, functionalization, and composition. This study attempts to produce a comprehensive review regarding the given parameters and their effects on stimulating cell behaviors such as proliferation, viability, attachment and osteogenic differentiation. In this study, a systematic search of MEDLINE database was conducted for in vitro studies on the use of graphene and its derivatives for bone tissue engineering from January 2000 to February 2018, organized according to the PRISMA statement. According to reviewed articles, different graphene derivative, including graphene, graphene oxide (GO) and reduced graphene oxide (RGO) with mass ratio ≤1.5 wt % for all and concentration up to 50 μg/mL for graphene and GO, and 60 μg/mL for RGO, are considered to be safe for most cell types. However, these concentrations highly depend on the types of cells. It was discovered that graphene with lateral size less than 5 µm, along with GO and RGO with lateral dimension less than 1 µm decrease cell viability. In addition, the three-dimensional structure of graphene can promote cell-cell interaction, migration and proliferation. When graphene and its derivatives are incorporated with metals, polymers, and minerals, they frequently show promoted mechanical properties and bioactivity. Last, graphene and its derivatives have been found to increase the surface roughness and porosity, which can highly enhance cell adhesion and differentiation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2284-2343, 2018.

A three-dimensional porous hydroxyapatite nanocomposite scaffold with shape memory effect for bone tissue engineering

It is known that scaffold is a key factor in bone tissue engineering. The aim of this study was to improve the design of scaffold in order to achieve an effect of precisely matching the irregular boundaries of bone defects as well as facilitate clinical application. In this study, controllable three-dimensional porous shape memory polyurethane/nano-hydroxyapatite composite scaffolds were successfully fabricated. Detailed studies were performed to evaluate its structure, porosities, and mechanical properties, emphasizing the effect of different apertures of scaffolds on shape recovery behaviors and biological performance in vitro. Results showed its compression recovery ratios and shape recovery ratios of all scaffolds could reach more than 99 and 90%, respectively, which could let it more accurately match the irregular boundaries of bone defects. And also its cell proliferation ability was improved with the increase in the apertures. Thus, these scaffolds have potential applications for the bone tissue engineering.

The effect of hydroxyapatite nanoparticles on mechanical behavior and biological performance of porous shape memory polyurethane scaffolds.

The scaffold which provides space for cell growth, proliferation, and differentiation, is a key factor in bone tissue engineering. However, improvements in scaffold design are needed to precisely match the irregular boundaries of bone defects as well as facilitate clinical application. In this study, controllable three-dimensional (3D) porous shape memory polyurethane/nano-hydroxyapatite (SMPU/nHAP) composite scaffold was successfully fabricated for bone defect reparation. Detailed studies were performed to evaluate its structure, apparent density, porosity, and mechanical properties, emphasizing the contribution of nHAP particles on shape recovery behaviors and biological performance in vitro. The effect of nHAP particles in porous SMPU/nHAP composite scaffold was found to enhance the compression resistance by 37%, shorten the compression recovery time by 41%, reduce the tensile resistance by 78%, reach the shape recovery ratio of 99%, and promote the cell proliferation by 13% after 7 days of culture. These results revealed that the 3D structure and aperture of as-prepared scaffold were controllable. And in minimally invasive surgery and bone repair surgery, this porous composite scaffold could significantly reduce the operative time and promote the bone cell growth. Therefore, this porous SMPU/nHAP composite scaffold design has potential applications for the bone tissue engineering. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 244-254, 2018.

Design of Hierarchical Three-Dimensional Printed Scaffolds Considering Mechanical and Biological Factors for Bone Tissue Engineering

Computational approaches have great potential for aiding clinical product development by finding promising candidate designs prior to expensive testing and clinical trials. Here, an approach for designing multilevel bone tissue scaffolds that provide structural support during tissue regeneration is developed by considering mechanical and biological perspectives. Three key scaffold design properties are considered: (1) porosity, which influences potential tissue growth volume and nutrient transport, (2) surface area, which influences biodegradable scaffold dissolution rate and initial cell attachment, and (3) elastic modulus, which influences scaffold deformation under load and, therefore, tissue stimulation. Four scaffold topology types are generated by patterning beam or truss-based unit cells continuously or hierarchically and tuning the element diameter, unit cell length, and number of unit cells. Parametric comparisons suggest that structures with truss-based scaffolds have higher surface areas but lower elastic moduli for a given porosity in comparison to beam-based scaffolds. Hierarchical scaffolds possess a large central pore that increases porosity but lowers elastic moduli and surface area. Scaffold samples of all topology types are 3D printed with dimensions suitable for scientific testing. A hierarchical scaffold is fabricated with dimensions and properties relevant for a spinal interbody fusion cage with a maximized surface-volume ratio, which illustrates a potentially high performing design configured for mechanical and biological factors. These findings demonstrate the merit in using multidisciplinary and computational approaches as a foundation of tissue scaffold development for regenerative medicine.

Immobilization and Application of Electrospun Nanofiber Scaffold-based Growth Factor in Bone Tissue Engineering.

Electrospun nanofibers have been extensively used in growth factor delivery and regenerative medicine due to many advantages including large surface area to volume ratio, high porosity, excellent loading capacity, ease of access and cost effectiveness. Their relatively large surface area is helpful for cell adhesion and growth factor loading, while storage and release of growth factor are essential to guide cellular behaviors and tissue formation and organization. In bone tissue engineering, growth factors are expected to transmit signals that stimulate cellular proliferation, migration, differentiation, metabolism, apoptosis and extracellular matrix (ECM) deposition. Bolus administration is not always an effective method for the delivery of growth factors because of their rapid diffusion from the target site and quick deactivation. Therefore, the integration of controlled release strategy within electrospun nanofibers can provide protection for growth factors against in vivo degradation, and can manipulate desired signal at an effective level with extended duration in local microenvironment to support tissue regeneration and repair which normally takes a much longer time. In this review, we provide an overview of growth factor delivery using biomimetic electrospun nanofiber scaffolds in bone tissue engineering. It begins with a brief introduction of different kinds of polymers that were used in electrospinning and their applications in bone tissue engineering. The review further focuses on the nanofiber-based growth factor delivery and summarizes the strategies of growth factors loading on the nanofiber scaffolds for bone tissue engineering applications. The perspectives on future challenges in this area are also pointed out.

The effects of platelet-rich plasma on the osteogenic induction of bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells. Finding methods to improve the osteogenic potential of these cells is a key factor in bone tissue engineering. Platelet-rich plasma (PRP) contains powerful growth factors that produce changes in a variety of cell types. The purpose of this study was to explore the effects of PRP on the osteogenic differentiation of BMSCs in vitro. Rabbit BMSCs were harvested and cultured in vitro in control media or in media enhanced with PRP. BMSCs began to attach 12–24 hours after seeding. A MTT assay demonstrated that PRP-induced BMSCs grew rapidly compared with the control group. The PRP group also showed strongly positive staining of alkaline phosphatase and mineralized nodules whereas the control group showed negative staining. However, the alkaline phosphatase activity and the mRNA level of the osteogenic markers (osteocalcin and osteopontin) remained higher in the PRP group. These results confirmed that PRP could enhance the proliferation of BMSCs and effectively promote the osteogenic differentiation of BMSCs in vitro.

Application of strontium doped calcium polyphosphate bioceramic as scaffolds for bone tissue engineering

The critical success factors for bone tissue engineering in clinical applications are scaffolds. Ion doping is one of the most important methods to modify the properties of bioceramics for better angiogenesis abilities, biomechanical properties, and biocompatibility. This paper presents a novel ion doping method applied in calcium polyphosphate (CPP)-based bioceramic scaffolds substituted by strontium ions to form (SCPP) scaffolds for bone tissue regeneration. The microstructure and crystallization of the scaffolds were detected by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Degradation tests were assessed to evaluate the mechanical and chemical stabilities of SCPP in vitro. The cell biocompatibility was measured with respect to the cytotoxicity of the extractions of scaffolds. Bone implantation was performed to evaluate the biodegradability and osteoconductivity of the scaffolds, and the bone formation examined by using X-ray radiography. The results indicated that the obtained SCPP scaffolds had a single CPP phase. The SCPP scaffolds yielded a better degradation property than the pure CPP scaffold. The MTT assay and in vivo results reveal that the SCPP scaffolds exhibited a better cell biocompatibility and tissue biocompatibility than CPP and hydroxyapatite (HA) scaffolds. The in vivo immunohistochemistry staining for VEGF also showed that SCPP had a potential to promote the formation of angiogenesis and the regeneration of bone. SCPP scaffold could serve as a potential biomaterial with stimulating angiogenesis in bone tissue engineering and bone repair.

Effect of Hydrostatic Pressure on Bone Regeneration Using Human Mesenchymal Stem Cells

Mechanics is increasingly being recognized as the fourth essential factor in bone tissue engineering next to cell, scaffold, and growth factors. The development of bioprocessors has made it possible to simulate the in vivo mechanics that are needed to generate three-dimensional (3D) bone constructs. However, although hydrostatic pressure (HP) is a dominant and constant mechanical strain on bone cells in vivo, little is known about the effect of HP applied via perfusion bioprocessors on in vitro human bone marrow-derived mesenchymal stem cell (hMSC) behavior. hMSCs underwent primary culture for three passages before being seeded into hydroxyapatite (HA) scaffolds. The scaffolds were incubated for 3 weeks in an automated bioprocessor under cyclic HP. Scaffolds exposed to atmospheric pressure (AP) served as the comparator. Osteogenic differentiation medium was employed for both the HP and AP groups. Immediately before and 1, 2, and 3 weeks after incubation, the scaffolds were harvested for histological, immunohistochemical, and gene expression analyses. Cells were only found in the AP scaffold surfaces, whereas in the HP group, they were distributed evenly throughout the scaffolds. Immunohistochemical analysis revealed that the HP group expressed higher levels of osteocalcin (OC), osteopontin (OP), osteonectin (ON), and collagen type 1 (Col1) than the AP group during the 3-week process. Gene expression analysis revealed that the HP group expressed higher levels of ON, Col1, alkaline phosphatase, and integrin β5 than the AP group at the 1-, 2-, and 3-week timepoints. The HP group also expressed higher levels of core-binding factor α-1 (Cbfa1) at the 2- and 3-week timepoints and higher levels of OP and OC at the 1-week timepoint. Their proliferating cell nuclear antigen levels were lower at the 1- and 2-week timepoints. HP enhances cellular viability and improves osteogenic differentiation and maturation, although somewhat at the expense of proliferation and self-renewal of MSCs. Possible negative effects of the bioprocessor-induced HP on bone regeneration were not observed. Further, the mechanotransductive molecule integrin β5 was expressed at high levels after HP stimulation and may enhance migration, promote differentiation, and inhibit osteoclast maturation during HP-driven osteogenesis in vitro.

Enhanced bone tissue formation by alginate gel‐assisted cell seeding in porous ceramic scaffolds and sustained release of growth factor

Increasing cell seeding efficiency in a tissue engineering construct can enhance cellular activity and tissue formation in vivo. Here, we demonstrate the use of alginate gel as a secondary phase material in 3D porous β-tricalcium phosphate scaffolds to improve cell seeding and provide controlled release of growth factors for bone tissue engineering. Cells were seeded in scaffolds in three ways: conventional seeding (CS), alginate gel-assisted seeding (GS), and alginate GS with bone morphogenetic protein-2 (BMP-2, GSB). In vitro study with MG-63 cells showed that cell seeding efficiency and cell population 1 week after seeding were significantly elevated in GS and GSB samples compared to CS samples. The GSB system demonstrated a sustained, steady release of BMP-2 over 2 weeks. In vivo, scaffolds seeded with rat mesenchymal stem cells were implanted ectopically into Sprague-Dawley rats for 8 weeks. GS and GSB samples exhibited improved osteogenic activity, with the GSB samples inducing the greatest osteocalcin and osteoid deposition. This study suggests that the alginate gel-assisted cell seeding increases seeding efficiency and allows for sustained release of growth factors. The use of the secondary phase polymer bolsters bone formation in vivo and has the potential for improving outcome in other tissue engineering applications.

Response of a Preosteoblastic Cell Line to Cyclic Tensile Stress Conditioning and Growth Factors for Bone Tissue Engineering

Bone regeneration can be accelerated by utilizing mechanical stress and growth factors (GFs). However, a limited understanding exists regarding the response of preosteoblasts to tensile stress alone or with GFs. We measured cell proliferation and expression of heat-shock proteins (HSPs) and other bone-related proteins by preosteoblasts following cyclic tensile stress (1%-10% magnitude) alone or in combination with bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1). Tensile stress (3%) with GFs induced greater gene upregulation of osteoprotegerin (3.3 relative fold induction [RFI] compared to sham-treated samples), prostaglandin E synthase 2 (2.1 RFI), and vascular endothelial growth factor (VEGF) (11.5 RFI), compared with samples treated with stimuli alone or sham-treated samples. The most significant increases in messenger RNA expression occurred with GF addition to either static-cultured or tensile-loaded (1% elongation) cells for the following genes: HSP47 (RFI=2.53), cyclooxygenase-2 (RFI=72.52), bone sialoprotein (RFI=11.56), and TGF-β1 (RFI=8.05). Following 5% strain with GFs, VEGF secretion increased 64% (days 3-6) compared with GF alone and cell proliferation increased 23% compared with the sham-treated group. GF addition increased osteocalcin secretion but decreased matrix metalloproteinase-9 significantly (days 3-6). Tensile stress and GFs in combination may enhance bone regeneration by initiating angiogenic and anti-osteoclastic effects and promote cell growth.

Dmitriev et al. respond

We appreciate the interest in our work by Salisbury et al. The role of bone morphogenetic protein-2 (BMP-2) in bone formation has been extensively characterized over the last 30 years [ 1 Boden S.D. Bioactive factors for bone tissue engineering. Clin Orthop Relat Res. 1999; : S84-S94 Crossref PubMed Scopus (136) Google Scholar , 2 Axelrad T.W. Einhorn T.A. Bone morphogenetic proteins in orthopaedic surgery. Cytokine Growth Factor Rev. 2009; 20: 481-488 Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar ]. The neuroscience field has also found significant involvement of BMPs in development, maturation, and repair of the peripheral nervous system (PNS) and central nervous system (CNS) [ 3 Hall A.K. Miller R.H. Emerging roles for bone morphogenetic proteins in central nervous system glial biology. J Neurosci Res. 2004; 76: 1-8 Crossref PubMed Scopus (61) Google Scholar , 4 Setoguchi T. Yone K. Matsuoka E. et al. Traumatic injury-induced BMP7 expression in the adult rat spinal cord. Brain Res. 2001; 921: 219-225 Crossref PubMed Scopus (105) Google Scholar , 5 Fuller M.L. Dechant A.K. Rothstein B. et al. Bone morphogenetic proteins promote gliosis in demyelinating spinal cord lesions. Ann Neurol. 2007; 62: 288-300 Crossref PubMed Scopus (105) Google Scholar ]. Although the direct influence of the PNS on bone homeostasis is well described, the interactions through which peripheral neurons may influence bone formation or repair remain to be fully defined [ 6 Togari A. Arai M. Pharmacological topics of bone metabolism: the physiological function of the sympathetic nervous system in modulating bone resorption. J Pharmacol Sci. 2008; 106: 542-546 Crossref PubMed Scopus (69) Google Scholar , 7 Li J. Kreicbergs A. Bergström J. et al. Site-specific CGRP innervation coincides with bone formation during fracture healing and modeling: a study in rat angulated tibia. J Orthop Res. 2007; 25: 1204-1212 Crossref PubMed Scopus (69) Google Scholar , 8 Li J. Ahmed M. Bergstrom J. Ackermann P. et al. Occurrence of substance P in bone repair under different load comparison of straight and angulated fracture in rat tibia. J Orthop Res. 2010; 28: 1643-1650 Crossref PubMed Scopus (20) Google Scholar ]. We would agree that further study of neuron-bone interaction could be highly significant for the orthopedic world. However, the potential role of BMPs in this interaction is largely unexplored. Adverse events and bone morphogenetic protein-2The Spine JournalVol. 11Issue 8PreviewRecently, a number of adverse events have been reported in the use of recombinant bone morphogenetic protein-2 (BMP-2) in spinal fusion [1–4]. We have been studying new bone formation after delivery of physiologic levels of BMP-2 using a cell therapy approach and have found this to rely on very selective neurogenic inflammation. We would like to suggest that this is actually a normal physiologic event in bone formation and fracture repair rather than an adverse event associated with delivery of this protein. Full-Text PDF

Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells

Casein phosphopeptides (CPPs), originating by in vitro and/or in vivo casein digestion, are characterized by the ability to complex and solubilize calcium ions preventing their precipitation. Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines, are able to re-mineralize carious lesions in a dental enamel, and, as components of a diet, affect bone weight and calcium content in rats. The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization. Primary human osteoblast-like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery. Commercial mixtures of bovine casein phosphopeptides were used. The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2-fluorescence assays. Results show that CPPs: (i) stimulate calcium uptake by primary human osteoblast-like cells; (ii) increase the expression and activity of alkaline phosphatase, a marker of human osteoblast differentiation; (iii) affect the cell proliferation rate and the apoptotic level; (iv) enhance nodule formation by human SaOS-2. Taken together these results confirm the possibility that CPPs play a role as modulator of bone cell activity, probably sustained by their ability as calcium carriers. Although the exact mechanism by which CPPs act remains not completely clarified, they can be considered as potential anabolic factors for bone tissue engineering.

Effect of lactoferrin-embedded collagen membrane on osteogenic differentiation of human osteoblast-like cells

Lactoferrin (LF) has the ability to promote the proliferation and differentiation of osteoblasts, suggesting its potential utility as an osteogenic growth factor in bone tissue engineering. However, this type of application requires improved drug delivery system (DDS) technology at the target site. In this study, we report enhanced calcium deposition and alkaline phosphatase (ALP) activity using the type I collagen membrane during osteogenic differentiation of MG63 human osteoblast-like cells, indicating that type I collagen not only acts as a site for calcification but also promotes the expression of differentiated phenotypes. We also used this membrane as a drug delivery carrier for bovine LF. Approximately 27% of LF embedded on the type I collagen membrane was released within the first hour in cell-free condition. This initial burst release of LF was followed by a slower release from the collagen membrane. Bovine LF embedded in the type I collagen membrane promoted its calcification during osteogenic differentiation of MG63 cells without the loss of LF bioactivity. Taken together, ALP activity and osteocalcin production were enhanced in the MG63 cells plated on the LF-embedded collagen membrane, suggesting that LF incorporated in the collagen membrane promoted bone-like tissue formation by MG63 cells. These observations suggest that the type I collagen membrane is useful as a drug delivery carrier for LF in bone tissue engineering.

Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2- N, 6- O-sulfated chitosan in vitro and in vivo

Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6- O-sulfated chitosan (6SCS), while 2- N-sulfate was subsidiary group with less activation. Low dose of 2- N, 6- O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.