Biological Response to Macroporous Chitosan-Agarose Bone Scaffolds Comprising Mg- and Zn-Doped Nano-Hydroxyapatite

Modification of implantable scaffolds with magnesium and zinc for improvement of bone regeneration is a growing trend in the engineering of biomaterials. The aim of this study was to synthesize nano-hydroxyapatite substituted with magnesium (Mg2+) (HA-Mg) and zinc (Zn2+) (HA-Zn) ions in order to fabricate chitosan-agarose-hydroxyapatite (HA) scaffolds (chit/aga/HA) with improved biocompatibility. Fabricated biomaterials containing Mg2+ or Zn2+ were tested using osteoblasts and mesenchymal stem cells to determine the effect of incorporated metal ions on cell adhesion, spreading, proliferation, and osteogenic differentiation. The study was conducted in direct contact with the scaffolds (cells were seeded onto the biomaterials) and using fluid extracts of the materials. It demonstrated that incorporation of Mg2+ ions into chit/aga/HA structure increased spreading of the osteoblasts, promoted cell proliferation on the scaffold surface, and enhanced osteocalcin production by mesenchymal stem cells. Although biomaterial containing Zn2+ did not improve cell proliferation, it did enhance type I collagen production by mesenchymal stem cells and extracellular matrix mineralization as compared to cells cultured in a polystyrene well. Nevertheless, scaffolds made of pure HA gave better results than material with Zn2+. Results of the experiments clearly showed that modification of the chit/aga/HA scaffold with Zn2+ did not have any positive impact on cell behavior, whereas, incorporation of Mg2+ ions into its structure may significantly improve biocompatibility of the resultant material, increasing its potential in biomedical applications.

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title: Nanosize hydroxyapatite: doping with various ions
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title: Diffraction techniques and vibrational spectroscopy opportunities to characterise bones
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title: Recent advances in the synthesis, functionalization and biomedical applications of hydroxyapatite: a review
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title: Preparation and Comprehensive Characterization of a Calcium Hydroxyapatite Reference Material
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title: Fine structure study on low concentration zinc substituted hydroxyapatite nanoparticles
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title: Characterization and structural analysis of zinc-substituted hydroxyapatites.
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title: Effect of magnesium ion on human osteoblast activity
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title: Mechanisms of magnesium-stimulated adhesion of osteoblastic cells to commonly used orthopaedic implants.
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title: Magnesium ion stimulation of bone marrow stromal cells enhances osteogenic activity, simulating the effect of magnesium alloy degradation.
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title: In vitro prominent bone regeneration by release zinc ion from Zn-modified implant.
abstract: Zinc is one of the trace elements which induce the proliferation and the differentiation of the osteoblast. In the previous study, we found that zinc ions (Zn(2+) ion)-releasing titanium implants had excellent bone fixation using a rabbit femurs model. In this study, we isolated the Zn(2+) ions (eluted Zn(2+) ion; EZ) released from the implant surface, and evaluated the effect of EZ on the osteogenesis of human bone marrow-derived mesenchymal cells (hBMCs). In the result, it was found that the EZ stimulated cell viability, osteoblast marker gene (type I collagen, osteocalcin (OC), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) expressions and calcium deposition in hBMCs.

title: Role of magnesium ions on osteogenic response in bone marrow stromal cells
abstract: Biodegradable magnesium (Mg) alloys have been investigated for craniofacial and orthopedic bone fracture fixation due to their initial mechanical strength and high biocompatibility. Although Mg alloys have been reported to enhance bone regeneration in vivo, and enhanced osteogenic marker expression in human bone marrow stromal cells (hBMSCs) cultured in Mg alloy extract was reported, however, the biological mechanism is not fully understood. Thus, it is important to elucidate which signaling pathway in the hBMSCs are activated by Mg2+ that enhances bone formation. We investigated possible mechanisms underlying effects of Mg2+ on bone regeneration by culturing differentiated and undifferentiated hBMSCs in the presence of culture medium containing 10 mM MgSO4 both with or without osteogenic factors. mRNA expression of osteogenic genes was analyzed using quantitative PCR arrays. Quantitative PCR array data indicated increased mRNA expression of collagen type X and insulin-like growth factor 2, and decreased expression of integrin alpha 3 in the presence of 10 mM MgSO4. Moreover, Western blotting analysis showed enhanced expression of collagen type X, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-2α, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the presence of 10 mM MgSO4. In conclusion, 10 mM of MgSO4 enhanced the production of collagen type X and VEGF by hBMSCs. These results also suggest that Mg2+ released from bone fixation devices may promote bone regeneration by enhancing the production of collagen type X and VEGF of osteogenic cells in bone tissue.

title: Promotive Effect of Zinc Ions on the Vitality, Migration, and Osteogenic Differentiation of Human Dental Pulp Cells
abstract: Zinc is an essential trace element for proper cellular function and bone formation. However, its exact role in the osteogenic differentiation of human dental pulp cells (hDPCs) has not been fully clarified before. Here, we speculated that zinc may be effective to regulate their growth and osteogenic differentiation properties. To test this hypothesis, different concentrations (1 × 10−5, 4 × 10−5, and 8 × 10−5 M) of zinc ions (Zn2+) were added to the basic growth culture medium and osteogenic inductive medium. Cell viability and migration were measured by cell counting kit-8 (CCK-8) and transwell migration assay in the basic growth culture medium, respectively. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expression levels of selective osteogenic differentiation markers and zinc transporters. Alkaline phosphatase (ALP) activity analysis and alizarin red S staining were used to investigate the mineralization of hDPCs. Exposure of hDPCs to Zn2+ stimulated their viability and migration capacity in a dose- and time-dependent manner. RT-qPCR assay revealed elevated expression levels of osteogenic differentiation-related genes and zinc transporters genes in various degrees. ALP activity was also increased with elevated Zn2+ concentrations and extended culture periods, but enhanced matrix nodules formation were observed only in 4 × 10−5 and 8 × 10−5 M Zn2+ groups. These findings suggest that specific concentrations of Zn2+ could potentiate the vitality, migration, and osteogenic differentiation of hDPCs. We may combine optimum zinc element into pulp capping materials to improve their biological performance.

title: Zinc-substituted hydroxyapatite: a biomaterial with enhanced bioactivity and antibacterial properties
abstract: Hydroxyapatite (HA) is a synthetic biomaterial and has been found to promote new bone formation when implanted in a bone defect site. However, its use is often limited due to its slow osteointegration rate and low antibacterial activity, particularly where HA has to be used for long term biomedical applications. This work will describe the synthesis and detailed characterization of zinc-substituted HA (ZnHA) as an alternative biomaterial to HA. ZnHA containing 1.6 wt% Zn was synthesized via a co-precipitation reaction between calcium hydroxide, orthophosphoric acid and zinc nitrate hexahydrate. Single-phase ZnHA particles with a rod-like morphology measuring ~50 nm in length and ~15 nm in width, were obtained and characterized using transmission electron microscopy and X-ray diffraction. The substitution of Zn into HA resulted in a decrease in both the a- and c-axes of the unit cell parameters, thereby causing the HA crystal structure to alter. In vitro cell culture work showed that ZnHA possessed enhanced bioactivity since an increase in the growth of human adipose-derived mesenchymal stem cells along with the bone cell differentiation markers, were observed. In addition, antibacterial work demonstrated that ZnHA exhibited antimicrobial capability since there was a significant decrease in the number of viable Staphylococcus aureus bacteria after in contact with ZnHA.

title: Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate
abstract: Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30 μM and 100 μM of ZnCl2. Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth.

title: Incorporation of zinc oxide nanoparticles into chitosan-collagen 3D porous scaffolds: Effect on morphology, mechanical properties and cytocompatibility of 3D porous scaffolds.
abstract: The zinc oxide nanoparticles (particles size <50nm) incorporated into chitosan-collagen 3D porous scaffolds and investigated the effect of zinc oxide nanoparticles incorporation on microstructure, mechanical properties, biodegradation and cytocompatibility of 3D porous scaffolds. The 0.5%, 1.0%, 2.0% and 4.0% zinc oxide nanoparticles chitosan-collagen 3D porous scaffolds were fabricated via freeze-drying technique. The zinc oxide nanoparticles incorporation effects consisting in chitosan-collagen 3D porous scaffolds were investigated by mechanical and swelling tests, and effect on the morphology of scaffolds examined microscopically. The biodegradation and cytocompatibility tests were used to investigate the effects of zinc oxide nanoparticles incorporation on the ability of scaffolds to use for tissue engineering application. The mean pore size and swelling ratio of scaffolds were decreased upon incorporation of zinc oxide nanoparticles however, the porosity, tensile modulus and biodegradation rate were increased upon incorporation of zinc oxide nanoparticles. In vitro culture of human fibroblasts and keratinocytes showed that the zinc oxide nanoparticles facilitated cell adhesion, proliferation and infiltration of chitosan-collagen 3D porous scaffolds. It was found that the zinc oxide nanoparticles incorporation enhanced porosity, tensile modulus and cytocompatibility of chitosan-collagen 3D porous scaffolds.

title: The material and biological characteristics of osteoinductive calcium phosphate ceramics
abstract: The discovery of osteoinductivity of calcium phosphate (Ca-P) ceramics has set an enduring paradigm of conferring biological regenerative activity to materials with carefully designed structural characteristics. The unique phase composition and porous structural features of osteoinductive Ca-P ceramics allow it to interact with signaling molecules and extracellular matrices in the host system, creating a local environment conducive to new bone formation. Mounting evidence now indicate that the osteoinductive activity of Ca-P ceramics is linked to their physicochemical and three-dimensional structural properties. Inspired by this conceptual breakthrough, many laboratories have shown that other materials can be also enticed to join the rank of tissue-inducing biomaterials, and besides the bones, other tissues such as cartilage, nerves and blood vessels were also regenerated with the assistance of biomaterials. Here, we give a brief historical recount about the discovery of the osteoinductivity of Ca-P ceramics, summarize the underlying material factors and biological characteristics, and discuss the mechanism of osteoinduction concerning protein adsorption, and the interaction with different types of cells, and the involvement of the vascular and immune systems.

title: The summary of the most important cell-biomaterial interactions that need to be considered during in vitro biocompatibility testing of bone scaffolds for tissue engineering applications.
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title: Zinc stimulates osteoblastogenesis and suppresses osteoclastogenesis by antagonizing NF-κB activation
abstract: Zinc is essential for numerous biochemical pathways in the body and is known to significantly affect the skeleton. Zinc has a profound effect on bone turnover promoting bone formation and mineralization, but paradoxically inhibiting osteoclastic bone breakdown (resorption). How zinc regulates these disparate effects on bone cells, however, is poorly understood. We recently characterized several pharmacological and nutritional factors that are likewise endowed with the capacity to promote bone formation and suppress bone resorption and demonstrated that a common centralized mechanism for achieving such actions is through suppression of NF-κB activation. NF-κB is a pathway required for osteoclastogenesis, but suppresses osteoblast differentiation. In this study, we investigated the actions of zinc on NF-κB activation in osteoclast and osteoblast precursors in vitro. Our data show that zinc suppressed osteoclast differentiation and promoted osteoblast mineralization and did indeed act as a potent NF-κB activation antagonist in both osteoclast and osteoblast precursors. Importantly, zinc antagonized NF-κB activation driven by TNFα, a potent inflammatory mediator or bone resorption and suppressor of bone formation in vitro and in vivo. Zinc further alleviated the suppressive effect of TNFα on Smad activation induced by TGFβ and BMP2, cytokines that play critical roles in osteoblast commitment, differentiation, and recruitment to sites of bone remodeling. In conclusion, our data reveal for the first time that a major mechanism by which zinc promotes osteoblastogenesis and suppresses osteoclastogenesis may center on the antagonism of NF-κB activation.

title: Osteogenic activity and antibacterial effect of zinc ion implanted titanium.
abstract: Titanium (Ti) and its alloys are widely used as orthopedic and dental implants. In this work, zinc (Zn) was implanted into oxalic acid etched titanium using plasma immersion ion implantation technology. Scanning electron microscopy and X-ray photoelectron spectroscopy were used to investigate the surface morphology and composition of Zn-implanted titanium. The results indicate that the depth profile of zinc in Zn-implanted titanium resembles a Gaussian distribution, and zinc exists in the form of ZnO at the surface whereas in the form of metallic Zn in the interior. The Zn-implanted titanium can significantly stimulate proliferation of osteoblastic MC3T3-E1 cells as well as initial adhesion, spreading activity, ALP activity, collagen secretion and extracellular matrix mineralization of the rat mesenchymal stem cells. The Zn-implanted titanium presents partly antibacterial effect on both Escherichia coli and Staphylococcus aureus. The ability of the Zn-implanted titanium to stimulate cell adhesion, proliferation and differentiation as well as the antibacterial effect on E. coli can be improved by increasing implantation time even to 2 h in this work, indicating that the content of zinc implanted in titanium can easily be controlled within the safe concentration using plasma immersion ion implantation technology. The Zn-implanted titanium with excellent osteogenic activity and partly antibacterial effect can serve as useful candidates for orthopedic and dental implants.

title: Calcium phosphate cements for bone engineering and their biological properties
abstract: Calcium phosphate cements (CPCs) are frequently used to repair bone defects. Since their discovery in the 1980s, extensive research has been conducted to improve their properties, and emerging evidence supports their increased application in bone tissue engineering. Much effort has been made to enhance the biological performance of CPCs, including their biocompatibility, osteoconductivity, osteoinductivity, biodegradability, bioactivity, and interactions with cells. This review article focuses on the major recent developments in CPCs, including 3D printing, injectability, stem cell delivery, growth factor and drug delivery, and pre-vascularization of CPC scaffolds via co-culture and tri-culture techniques to enhance angiogenesis and osteogenesis.

title: Extracellular matrix stiffness controls osteogenic differentiation of mesenchymal stem cells mediated by integrin α5
abstract: BackgroundHuman mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs.MethodshMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young’s modulus of 13–16 kPa and 62–68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1typeIcollagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis.ResultsOsteogenic differentiation was increased on 62–68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62–68 kPa ECM.ConclusionsTaken together, our results provide evidence that matrix stiffness (62–68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.

title: Osteogenesis-Related Behavior of MC3T3-E1 Cells on Substrates with Tunable Stiffness
abstract: Osteogenic differentiation of cells has considerable clinical significance in bone defect treatment, and cell behavior is linked to extracellular matrix stiffness. This study aimed to determine how matrix stiffness affects cell morphology and subsequently regulates the osteogenic phenotype of osteogenesis precursor cells. Four PDMS substrates were prepared with stiffness corresponding to the elastic modulus ranging from 0.6 MPa to 2.7 MPa by altering the Sylgard 527 and Sylgard 184 concentrations. MC3T3-E1 cells were cultured on the matrices. Cell morphology, vinculin expression, and key osteogenic markers, Col I, OCN, OPN, and calcium nodule, were examined. The activity and expression level of Yes-associated protein (YAP) were evaluated. Results showed that cell spreading exhibited no correlation with the stiffness of matrix designed in this paper, but substratum stiffness did modulate MC3T3-E1 osteogenic differentiation. Col I, OPN, and OCN proteins were significantly increased in cells cultured on soft matrices compared with stiff matrices. Additionally, cells cultured on the 1:3 ratio matrices had more nodules than those on other matrices. Accordingly, cells on substrates with low stiffness showed enhanced expression of the osteogenic markers. Meanwhile, YAP expression was downregulated on soft substrates although the subcellular location was not affected. Our results provide evidence that matrix stiffness (elastic modulus ranging from 0.6 MPa to 2.7 MPa) affects the osteogenic differentiation of MC3T3-E1, but it is not that “the stiffer, the better” as showed in some of the previous studies. The optimal substrate stiffness may exist to promote osteoblast differentiation. Cell differentiation triggered by the changes in substrate stiffness may be independent of the YAP signal. This study has important implications for biomaterial design and stem cell-based tissue engineering.

title: Comprehensive study on the roles of released ions from biodegradable Mg–5 wt% Ca–1 wt% Zn alloy in bone regeneration
abstract: We report here the effect of micro‐environmental changes from biodegradable magnesium alloys on the activities of cells – osteoblasts, osteoclasts and macrophages – which play critical roles in each phase of the bone‐regeneration process. Despite positive bone formation effects from several in vivo studies, minimal progress has been made in identifying underlying mechanisms through in vitro studies, which are currently concentrated on osteoblastic activities. The observed in vitro and in vivo results indicated that alkaline pH and released magnesium and zinc ions derived from Mg–5 wt% Ca–1 wt% Zn alloy biodegradation promote the progress of bone formation. In contrast, alkaline pH and magnesium ions remarkably suppressed osteoclastic activities and pro‐inflammatory cytokine production, closely related to osteolysis and prosthesis failure. Findings from the present study conclude that the degradation of Mg–5 wt% Ca–1 wt% Zn alloys can promote new bone formation by simultaneously affecting the complex combination of variable cellular activities and phases.

title: Fabrication and physicochemical characterization of porous composite microgranules with selenium oxyanions and risedronate sodium for potential applications in bone tumors
abstract: Nanocrystalline hydroxyapatite containing selenite ions (SeHA; 9.6 wt.% of selenium) was synthesized using wet method and subject to careful physicochemical analysis by powder X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, solid-state nuclear magnetic resonance, wavelength dispersive X-ray fluorescence, and inductively coupled plasma optical emission spectrometry. SeHA was then used to develop the selenium-containing hydroxyapatite/alginate (SeHA/ALG) composite granules. Risedronate sodium (RIS) was introduced to the obtained spherical microgranules of a size of about 1.1–1.5 mm in 2 ways: during the granules’ preparation (RIS solution added to a suspension of ALG and SeHA), and as a result of SeHA/ALG granules soaking in aqueous RIS solution. The analysis made using 13C and 31P cross-polarization magic angle spinning nuclear magnetic resonance confirmed the presence of RIS and its interaction with calcium ions. Then, the release of selenium (inductively coupled plasma optical emission spectrometry) and RIS (high-performance liquid chromatography) from microgranules was examined. Moreover, cytotoxicity of fabricated granules was assessed by MTT test. Selenium release was biphasic: the first stage was short and ascribed to a “burst release” probably from a hydrated surface layer of SeHA crystals, while the next stage was significantly longer and ascribed to a sustained release of selenium from the crystals’ interior. The study showed that the method of obtaining microgranules containing RIS significantly affects its release profile. Performed cytotoxicity test revealed that fabricated granules had high antitumor activity against osteosarcoma cells. However, because of the “burst release” of selenium during the first 10 h, the granules significantly reduced viability of normal osteoblasts as well.

title: Enhanced differentiation of osteoblastic cells on novel chitosan/β-1,3-glucan/bioceramic scaffolds for bone tissue regeneration.
abstract: Bone scaffolds for regenerative medicine applications should have the ability to promote adhesion, proliferation and differentiation of osteoblast cells. Osteoconductive, osteoinductive and osteopromotive properties of the material are essential for rapid bone regeneration and new bone formation. In this study, the osteogenic potential of two novel tri-component scaffolds composed of krill chitosan, bacterial β-1,3-glucan and bioceramics (HAp or a mix of HAp/β-TCP granules) was investigated. The typical markers of the first (type I collagen), second (bone alkaline phosphatase) and third stages (osteocalcin) of the osteoblast differentiation process were evaluated during in vitro experimentation. The study was carried out using three various osteoblastic cell lines (normal human fetal osteoblast cells hFOB 1.19, human osteoblast-like cells derived from osteosarcoma Saos-2 and mouse calvarial preosteoblast cells MC3T3-E1 Subclone 4). The bone alkaline phosphatase (bALP) and osteocalcin (OC) were determined quantitatively using enzyme-linked immunosorbent assays, and type I collagen (Col I) was evaluated qualitatively using the direct immunofluorescence (DIF) method. The data obtained clearly prove that novel scaffolds have the ability to increase bALP activity, to enhance extracellular matrix synthesis (Col I and OC) and to induce mineralized nodule formation during osteogenic differentiation. In conclusion, novel tri-component materials have osteoconductive and osteopromotive properties, and thus are promising materials in bone tissue engineering applications to accelerate the bone regeneration process.