Chondrogenic differentiation of Wharton's Jelly mesenchymal stem cells on silk spidroin-fibroin mix scaffold supplemented with L-ascorbic acid and platelet rich plasma.

In this research, hWJ-MSCs were grown on silk scaffolds and induced towards chondrogenesis by supplementation with L-ascorbic acid (LAA) or platelet rich plasma (PRP). Silk scaffolds were fabricated with salt leaching method by mixing silk fibroin (SF) with silk spidroin (SS). The silk fibroin was obtained from Bombyx mori cocoon that had been degummed, and the silk spidroin was obtained from wild-type spider Argiope appensa. The effect of scaffold composition and inducer on cell proliferation was observed through MTT assay. The most optimal treatment then continued to be used to induce hWJ-MSC towards chondrogenic differentiation for 7 and 21 days. Scaffolds characterization showed that the scaffolds produced had 3D structure with interconnected pores, and all were biocompatible with hWJ-MSCs. Scaffold with the addition of 10% SS + 90% SF showed higher compressive strength and better pore interconnectivity in comparison to 100% silk fibroin scaffold. After 48 h, cells seeded on scaffold with spidroin and fibroin mix had flattened morphology in comparison to silk fibroin scaffold which appeared to be more rounded on the scaffold surface. Scaffold with 10% (w/w) of silk spidroin (SS) + 90% (w/w) of silk fibroin (SF) was the most optimal composition for cell proliferation. Immunocytochemistry of integrin ß1 and RGD sequence, showed that scaffold with SS 10% provide better cell attachment with the presence of RGD sequence from the spidroin silk which could explain the higher cell proliferation than SF100% scaffold. Based on Alcian Blue staining and Collagen Type II immunocytochemistry (ICC), cells grown on 10% SS + 90% SF scaffold with 10% PRP supplementation were the most optimal to support chondrogenesis of hWJ-MSCs. These results showed that the addition of spidroin silk from A. appensa. had impact on scaffold compressive strength and chondrogenic differentiation of hWJ-MSC and had the potential for further development of bio-based material scaffold in cartilage tissue engineering.

A hydrogenated black TiO2 coating with excellent effects for photothermal therapy of bone tumor and bone regeneration.

The clinical treatment of bone tumors usually brings about residual tumor cells and large bone defects after tumor removal surgery. To solve this problem, it is imperative to develop a novel implant with bi-functions for eliminating the residual tumor cells and repairing bone defects. In this study, hydrogenated black TiO2 (H-TiO2) coating with hierarchical micro/nano-topographies is fabricated by induction suspension plasma spraying (ISPS). The fabricated H-TiO2 coating possessed excellent and controllable photothermal effect in inhibiting the tumor growth under 808 nm NIR laser irradiation in vitro and in vivo. The hierarchical hybrid micro/nano-structured surface and Ti-OH groups improved the adhesion, proliferation, differentiation and osteogenic gene expressions of rat bone mesenchymal stem cells (rBMSCs). These results demonstrate that the H-TiO2 coating may be a promising implant material for the treatment of bone tumors and bone regeneration.

Surface engineering of titanium-based implants using electrospraying and dip coating methods.

Titanium and its alloys due to their low density, good mechanical and biological properties are of the most common orthopedic metals. One of the main challenges regarding to titanium implants is their loosening after long term implantation in patient's body. Many methods such as alteration in surface topography with focus on improving osseointegration or biocompatibility in overall are supposed to overcome this issue. In this research, titanium surface topography is altered via electrospraying a solution of titanium salt, carrier polymer (polyvinylpyrrolidone) and solvents. The dip coated samples in the same solution are prepared and investigated as control. The electrosprayed or dip coated samples were pyrolysised in furnace at 500 °C to remove polymeric components. Then the stabilized microstructures on the surfaces were evaluated via scanning electron microscopy (SEM), water contact angle (WCA) measurement, X-ray diffraction (XRD) and atomic force microscope (AFM). Also, in order to study the bioactivity of modified samples, they were immersed in simulated body fluid (SBF) and their precipitates were studied. The cellular investigations were done by studying the cell morphology, MTT and alkaline phosphatase (ALP) activity assays. The results showed improvement in bioactivity and cellular response for DP3 and SP15 more than other samples implying the promising potential of these two approaches for titanium implant surface modification.

Platelet-rich concentrate in serum-free medium enhances cartilage-specific extracellular matrix synthesis and reduces chondrocyte hypertrophy of human mesenchymal stromal cells encapsulated in alginate.

Platelet-rich concentrate (PRC), used in conjunction with other chondroinductive growth factors, have been shown to induce chondrogenesis of human mesenchymal stromal cells (hMSC) in pellet culture. However, pellet culture systems promote cell hypertrophy and the presence of other chondroinductive growth factors in the culture media used in previous studies obscures accurate determination of the effect of platelet itself in inducing chondrogenic differentiation. Hence, this study aimed to investigate the effect of PRC alone in enhancing the chondrogenic differentiation potential of human mesenchymal stromal cells (hMSC) encapsulated in three-dimensional alginate constructs. Cells encapsulated in alginate were cultured in serum-free medium supplemented with only 15% PRC. Scanning electron microscopy was used to determine the cell morphology. Chondrogenic molecular signature of hMSCs was determined by quantitative real-time PCR and verified at protein levels via immunohistochemistry and enzyme-linked immunosorbent assay. Results showed that the cells cultured in the presence of PRC for 24 days maintained a chondrocytic phenotype and demonstrated minimal upregulation of cartilaginous extracellular matrix (ECM) marker genes (SOX9, TNC, COL2, ACAN, COMP) and reduced expression of chondrocyte hypertrophy genes (Col X, Runx2) compared to the standard chondrogenic medium (p < 0.05). PRC group had correspondingly higher levels of glycosaminoglycan and increased concentration of chondrogenic specific proteins (COL2, ACAN, COMP) in the ECM. In conclusion, PRC alone appears to be very potent in inducing chondrogenic differentiation of hMSCs and offers additional benefit of suppressing chondrocyte hypertrophy, rendering it a promising approach for providing abundant pool of chondrogenic MSCs for application in cartilage tissue engineering.

Descemet's Membrane Biomimetic Microtopography Differentiates Human Mesenchymal Stem Cells Into Corneal Endothelial-Like Cells.

PURPOSE: Loss of corneal endothelial cells (CECs) bears disastrous consequences for the patient, including corneal clouding and blindness. Corneal transplantation is currently the only therapy for severe corneal disorders. However, the worldwide shortages of corneal donor material generate a strong demand for personalized stem cell-based alternative therapies. Because human mesenchymal stem cells are known to be sensitive to their mechanical environments, we investigated the mechanotransductive potential of Descemet membrane-like microtopography (DLT) to differentiate human mesenchymal stem cells into CEC-like cells. METHODS: Master molds with inverted DLT were produced by 2-photon lithography (2-PL). To measure the mechanotransductive potential of DLT, mesenchymal stem cells were cultivated on silicone or collagen imprints with DLT. Changes in morphology were imaged, and changes in gene expression of CEC typical genes such as zonula occludens (ZO-1), sodium/potassium (Na/K)-ATPase, paired-like homeodomain 2 (PITX2), and collagen 8 (COL-8) were measured with real-time polymerase chain reaction. At least immunofluorescence analysis has been conducted to confirm gene data on the protein level. RESULTS: Adhesion of MSCs to DLT molded in silicone and particularly in collagen initiates polygonal morphology and monolayer formation and enhances not only transcription of CEC typical genes such as ZO-1, Na/K-ATPase, PITX2, and COL-8 but also expression of the corresponding proteins. CONCLUSIONS: Artificial reproduction of Descemet membrane with respect to topography and similar stiffness offers a potential innovative way to bioengineer a functional CEC monolayer from autologous stem cells.

Soybean Lecithin-Mediated Nanoporous PLGA Microspheres with Highly Entrapped and Controlled Released BMP-2 as a Stem Cell Platform.

Injectable polymer microsphere-based stem cell delivery systems have a severe problem that they do not offer a desirable environment for stem cell adhesion, proliferation, and differentiation because it is difficult to entrap a large number of hydrophilic functional protein molecules into the core of hydrophobic polymer microspheres. In this work, soybean lecithin (SL) is applied to entrap hydrophilic bone morphogenic protein-2 (BMP-2) into nanoporous poly(lactide-co-glycolide) (PLGA)-based microspheres by a two-step method: SL/BMP-2 complexes preparation and PLGA/SL/BMP-2 microsphere preparation. The measurements of their physicochemical properties show that PLGA/SL/BMP-2 microspheres had significantly higher BMP-2 entrapment efficiency and controlled triphasic BMP-2 release behavior compared with PLGA/BMP-2 microspheres. Furthermore, the in vitro and in vivo stem cell behaviors on PLGA/SL/BMP-2 microspheres are analyzed. Compared with PLGA/BMP-2 microspheres, PLGA/SL/BMP-2 microspheres have significantly higher in vitro and in vivo stem cell attachment, proliferation, differentiation, and matrix mineralization abilities. Therefore, injectable nanoporous PLGA/SL/BMP-2 microspheres can be potentially used as a stem cell platform for bone tissue regeneration. In addition, SL can be potentially used to prepare hydrophilic protein-loaded hydrophobic polymer microspheres with highly entrapped and controlled release of proteins.

In vitro design of mesenchymal to epithelial transition of prostate cancer metastasis using 3D nanoclay bone-mimetic scaffolds.

Nanocomposite scaffolds show extensive applications in regenerative medicine and have shown promise as in vitro analogues of human tissue that can be used for the study of diseases. The complex nature of cancer metastasis is recently investigated using several 3D scaffold models. Herein, we report a polymer-nanoclay-based in vitro tumour model that recapitulates early stage of prostate cancer (PCa) colonization during skeletal metastasis on bone mimetic scaffolds. A unique cell culture system termed as "sequential culture (SC)" has been applied to create a bone-mimetic niche for colonization of PCa cells. Human mesenchymal stem cells (MSCs) were seeded on the bone-mimetic scaffolds, where they differentiated into bone cells and then formed mineralized bone matrix without osteogenic supplements. Further, PCa was seeded on MSCs-seeded scaffolds. Sequentially cultured PCa cells with MSCs formed self-organized multicellular tumoroids with distinct tight cellular junctions and hypoxic core regions. Extensive quantitative reverse transcription-polymerase chain reaction experiments were performed to evaluate the expressions of genes related to osteotropic bone metastasis of PCa. On the nanoclay scaffolds, the MSCs differentiated to mature osteoblasts and epithelial to mesenchymal transition was inhibited whereas mesenchymal to epithelial transition was enhanced, as also the hypoxia increased angiogenesis, and finally, PCa cells initiated osteoblastic lesion. Further, the SC technique has significant effects on expression of key metastasis-related genes. Therefore, the SC-based tumour model can be applied to recapitulate more consistent osteotropic cancer cell behavior in understanding tumour biology. This model also can be implemented for drug screening to target colonization stage of PCa cells in the bone microenvironment.

Functional quantum dot-siRNA nanoplexes to regulate chondrogenic differentiation of mesenchymal stem cells.

SOX9 plays an important role in mesenchymal condensations during the early development of embryonic skeletons. However, its function in the chondrogenic differentiation of adult mesenchymal stem cells (MSCs) has not been fully investigated because SOX9 RNA interference in adult MSCs has seldom been studied. This study used SOX9 gene as the target gene and the quantum dot (QD)-based nanomaterial QD-NH2 (ZnS shell and poly-ethylene glycol (PEG) coating) with a fluorescent tracer function as the gene carrier to transfect siSOX9 into MSCs after sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) activation in vitro and in vivo. The results showed that QD-SMCC could effectively bind and deliver siRNAs into the MSCs, followed by efficient siRNA escape from the endosomes. The siRNAs released from QD-SMCC retained their structural integrity and could effectively inhibit the targeted gene expression, leading to reduced chondrogenic differentiation of MSCs and delayed cartilage repair. QDs were excreted from living cells instead of dead cells, and the ZnS shell and PEG coating layer greatly reduced the cytotoxicity of the QDs. The transfection efficiency of QD-SMCC was superior to that of polyethylenimine (PEI). In addition, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection. STATEMENT OF SIGNIFICANCE: In this study, we developed functional quantum dot (QD) nanoplexes by sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) activation of PEG-coated CdSe/ZnS QDs as the gene carrier of siRNA to study the effect of SOX9 RNA interference on the chondrogenic differentiation of MSCs. This study confirmed the importance of SOX9 in chondrogenesis, as evidenced by the findings that SOX9 knockdown significantly inhibited the expression of cartilage-specific markers including acan and col2a1 in MSCs and further delayed cartilage repair. Moreover, QD-SMCC has an intrinsic signal for noninvasive imaging of siRNA transport. The results indicate that SOX9 is imperative for the chondrogenesis of MSCs and QD-SMCC has great potential for real-time tracking of transfection.

The impact of silica encapsulated cobalt zinc ferrite nanoparticles on DNA, lipids and proteins of rat bone marrow mesenchymal stem cells.

Nanomaterials are currently the subject of intense research due to their wide variety of potential applications in the biomedical, optical and electronic fields. We prepared and tested cobalt zinc ferrite nanoparticles (Co0.5Zn0.5Fe2O4+γ [CZF-NPs]) encapsulated by amorphous silica in order to find a safe contrast agent and magnetic label for tracking transplanted cells within an organism using magnetic resonance imaging (MRI). Rat mesenchymal stem cells (rMSCs) were labeled for 48 h with a low, medium or high dose of CZF-NPs (0.05; 0.11 or 0.55 mM); silica NPs (Si-NPs; 0.11 mM) served as a positive control. The internalization of NPs into cells was verified by transmission electron microscopy. Biological effects were analyzed at the end of exposure and after an additional 72 h of cell growth without NPs. Compared to untreated cells, Annexin V/Propidium Iodide labeling revealed no significant cytotoxicity for any group of treated cells and only a high dose of CZF-NPs slowed down cell proliferation and induced DNA damage, manifested as a significant increase of DNA-strand breaks and oxidized DNA bases. This was accompanied by high concentrations of 15-F2t-isoprostane and carbonyl groups, demonstrating oxidative injury to lipids and proteins, respectively. No harmful effects were detected in cells exposed to the low dose of CZF-NPs. Nevertheless, the labeled cells still exhibited an adequate relaxation rate for MRI in repeated experiments and ICP-MS confirmed sufficient magnetic label concentrations inside the cells. The results suggest that the silica-coated CZF-NPs, when applied at a non-toxic dose, represent a promising contrast agent for cell labeling.

The synergistic effect of micro-topography and biochemical culture environment to promote angiogenesis and osteogenic differentiation of human mesenchymal stem cells.

Critical failures associated with current engineered bone grafts involve insufficient induction of osteogenesis of the implanted cells and lack of vascular integration between graft scaffold and host tissue. This study investigated the combined effects of surface microtextures and biochemical supplements to achieve osteogenic differentiation of human mesenchymal stem cells (hMSCs) and revascularization of the implants in vivo. Cells were cultured on 10µm micropost-textured polydimethylsiloxane (PDMS) substrates in either proliferative basal medium (BM) or osteogenic medium (OM). In vitro data revealed that cells on microtextured substrates in OM had dense coverage of extracellular matrix, whereas cells in BM displayed more cell spreading and branching. Cells on microtextured substrates in OM demonstrated a higher gene expression of osteoblast-specific markers, namely collagen I, alkaline phosphatase, bone sialoprotein, and osteocalcin, accompanied by substantial amount of bone matrix formation and mineralization. To further investigate the osteogenic capacity, hMSCs on microtextured substrates under different biochemical stimuli were implanted into subcutaneous pockets on the dorsal aspect of immunocompromised mice to study capacity for ectopic bone formation. In vivo data revealed greater expression of osteoblast-specific markers coupled with increased vascular invasion on microtextured substrates with hMSCs cultured in OM. Together, these data represent a novel regenerative strategy that incorporates defined surface microtextures and biochemical stimuli to direct combined osteogenesis and re-vascularization of engineered bone scaffolds for musculoskeletal repair and relevant bone tissue engineering applications.

Biomineralized hydroxyapatite nanoclay composite scaffolds with polycaprolactone for stem cell-based bone tissue engineering.

Nanoclay modified with unnatural amino acid was used to design a nanoclay-hydroxyapatite (HAP) hybrid by mineralizing HAP in the nanoclay galleries mimicking biomineralization. This hybrid (in situ HAPclay) was used to fabricate polycaprolactone (PCL)/in situ HAPclay films and scaffolds for bone regeneration. Cell culture assays and imaging were used to study interactions between human mesenchymal stem cells (hMSCs) and PCL/in situ HAPclay composites (films and scaffolds). SEM imaging indicated MSC attachment, formation of mineralized extracellular (ECM) on PCL/in situ HAPclay films, and infiltration of MSCs to the interior of PCL/in situ HAPclay scaffolds. Mineralized ECM was formed by MSCs without use of osteogenic supplements. AFM imaging performed on this in vitro generated mineralized ECM on PCL/in situ HAPclay films revealed presence of components (collagen and mineral) of hierarchical organization reminiscent of natural bone. Cellular events observed during two-stage seeding experiments on PCL/in situ HAPclay films indicated similarities with events occurring during in vivo bone formation. PCL/in situ HAPclay films showed significantly increased (100-595% increase in elastic moduli) nanomechanical properties and PCL/in situ HAPclay scaffolds showed increased degradation. This work puts forth PCL/in situ HAPclay composites as viable biomaterials for bone tissue engineering.

Fabrication of injectable, cellular, anisotropic collagen tissue equivalents with modular fibrillar densities.

Technological improvements in collagen gel fabrication are highly desirable as they may enable significant advances in the formation of tissue-equivalent biomaterials for regenerative medicine, three-dimensional (3D) in vitro tissue models, and injectable scaffolds for cell and drug delivery applications. Thus, strategies to modulate collagen gel fibrillar density and organization in the mesostructure have been pursued to fabricate collagenous matrices with extracellular matrix-like features. Herein, we introduce a robust and simple method, namely gel aspiration-ejection (GAE), to engineer 3D, anisotropic, cell seeded, injectable dense collagen (I-DC) gels with controllable fibrillar densities, without the use of crosslinking. GAE allows for the hybridization of collagen gels with bioactive agents for increased functionality and supports highly aligned homogenous cell seeding, thus providing I-DC gels with distinct properties when compared to isotropic DC gels of random fibrillar orientation. The hybridization of I-DC with anionic fibroin derived polypeptides resulted in the nucleation of carbonated hydroxyapatite within the aligned nanofibrillar network upon exposure to simulated body fluid, yielding a 3D, anisotropic, mineralized collagen matrix. In addition, I-DC gels accelerated the osteoblastic differentiation of seeded murine mesenchymal stem cells (m-MSCs) when exposed to osteogenic supplements, which resulted in the cell-mediated, bulk mineralization of the osteoid-like gels. In addition, and upon exposure to neuronal transdifferentiation medium, I-DC gels supported and accelerated the differentiation of m-MSCs toward neuronal cells. In conclusion, collagen GAE presents interesting opportunities in a number of fields spanning tissue engineering and regenerative medicine to drug and cell delivery.

Zinc in calcium phosphate mediates bone induction: in vitro and in vivo model.

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP - up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material.

The effects of Ca2SiO4-Ca3(PO4)2 ceramics on adult human mesenchymal stem cell viability, adhesion, proliferation, differentiation and function.

Bioceramic samples with osteogenic properties, suitable for use in the regeneration of hard tissue, were synthesized. The materials consisting of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5 wt.% or 3.0 wt.% of dicalcium silicate (C2S) in the system Dicalcium Silicate-Tricalcium Phosphate (C2S-TCP) were obtained by solid state reaction. All materials were composed of a single phase, αTCP in the case of a pure material, or solid solution of C2S in αTCP (αTCPss) for the doped αTCP. Viability, proliferation and in vitro osteoinductive capacity were investigated by seeding, adult mesenchymal stem cells of human origin (ahMSCs) which were CD73(+), CD90(+), CD105(+), CD34(-) and CD45(-) onto the 3 substrates for 30 days. Results show a non-cytotoxic effect after applying an indirect apoptosis test (Annexin V/7-AAD staining), so ahMSCs adhered, spread, proliferated and produced extracellular matrix (Heparan-sulfate proteoglycan (HS) and osteopontin (OP)) on all the ceramics studied. Finally, the cells lost the cluster differentiation marker expression CD73, CD90 y CD105 characteristic of ahMSCs and they showed an osteoblastic phenotype (Alkaline phosphatase activity (ALP), Osteocalcin production (OC), Collagen type I expression (Col-I), and production of mineralization nodules on the extracellular matrix). These observations were more evident in the αTCP ceramic doped with 1.5 wt.% C2S, indicating osteoblastic differentiation as a result of the increased concentration of solid solution of C2S in αTCP (αTCPss). Overall, these results suggest that the ceramics studied are cytocompatible and they are able to induce osteoblastic differentiation of undifferentiated ahMSCs.

Tissue-engineered bone formation in vivo for artificial laminae of the vertebral arch using ß-tricalcium phosphate bioceramics seeded with mesenchymal stem cells.

STUDY DESIGN: A rabbit laminectomy model was used to evaluate the efficacy of artificial laminae of vertebral arch using bone marrow-derived mesenchymal stem cells (MSCs) transplanted in porous beta-calcium phosphates (ß-TCP) bioceramics. OBJECTIVE: The aim of this study was to establish artificial lamina of the vertebral arch for bone tissue engineering using ß-TCP bioceramics seeded with MSCs in a rabbit model of decompressive laminectomy. SUMMARY OF BACKGROUND DATA: Decompressive laminectomy may induce various degrees of scar tissue and adhesion formation in the epidural space, and thus is the most common cause of failed back surgery syndrome. However, there is no effective method of bone defect treatment to control and reduce the scar tissue formation. METHODS: MSCs were harvested from New Zealand rabbits (2-week old) by femoral bone marrow extraction. These cells were seeded into porous ß-TCP bioceramics and cultivated for up to 3 weeks in the presence of osteogenic supplements. Segmental defects (20 × 8 mm) were created in 48 adult New Zealand rabbits that underwent laminectomy at the L5 to L6 levels. The animals were transplanted with cell media (control), ß-TCP bioceramics (group I), or MSC-loaded ß-TCP bioceramics (group II). Bone formation was evaluated after operation using scanning electron microscopy, computed tomography, magnetic resonance imaging, histomorphometry, and immunohistochemistry. RESULTS: Scanning electron microscopy showed that MSCs filled the pores and surfaces of bioceramics in MSC-loaded ß-TCP. In addition, significant increases in bone formation were observed in group II compared with other groups. Computed tomography and magnetic resonance imaging at 16 weeks showed that the artificial lamina of the vertebral arch was successfully formed. Hematoxylin-eosin and Masson trichrome staining were used to show the artificial laminae of the vertebral arch and the degraded bioceramics. In addition, immunohistochemistry results showed that the expression of bone morphogenetic protein-2 increased significantly in group II compared with group I at 2,4, and 8 weeks after implantation (P < 0.05). CONCLUSION: ß-TCP bioceramics seeded with MSCs are a promising source of tissue-engineered bone for the artificial lamina of the vertebral arch.

Osteogenic activity and antibacterial effects on titanium surfaces modified with Zn-incorporated nanotube arrays.

Titanium implants having enhanced osteogenic activity and antibacterial property are highly desirable for the prevention of implant associated infection and promotion of osseointegration. In this study, coatings containing titania nanotubes (NTs) incorporated with zinc (NT-Zn) are produced on Ti implants by anodization and hydrothermal treatment in Zn containing solutions. The amount of incorporated Zn can be adjusted by varying the structural parameters such as the nanotube diameter and length as well as hydrothermal treatment time. The suitable NT-Zn coatings with good intrinsic antibacterial properties can prevent post-operation infection. Excellent osteogenesis inducing ability in the absence of extraneous osteogenic supplements is demonstrated and the ERK1/2 signaling is found to be involved. The NT-Zn structure which is simple, stable, and easy to produce and scale up has immense potential in bone implant applications.

Effects of micropitted/nanotubular titania topographies on bone mesenchymal stem cell osteogenic differentiation.

Micro/nanotopographical modification of biomaterials constitutes a promising approach to direct stem cell osteogenic differentiation to promote osseointegration. In this work, titania nanotubes (NTs) 25 and 80 nm in size with the acid-etched Ti topography (AcidTi) and hierarchical hybrid micropitted/nanotubular topographies (Micro/5VNT and Micro/20VNT) are produced to mimic the structure of the natural bone extracellular matrix (ECM). The effects on bone mesenchymal stem cell (MSC) osteogenic differentiation are studied systematically by various microscopic and biological characterization techniques. Cell adhesion is assayed by nucleus fluorescence staining and cell proliferation is studied by CCK-8 assay and flow cytometry. Osteogenic differentiation is assayed by alkaline phosphatase (ALP) expression, collagen secretion, matrix mineralization, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis on the osteogenesis related gene expression. All the topographies are observed to induce MSC osteogenic differentiation in the absence of osteogenic supplements. The nanotube surfaces significantly promote cell attachment and spread, collagen secretion and ECM mineralization, as well as osteogenesis-related gene expression. Among them, Micro/20VNT shows the best ability to simultaneously promote MSC proliferation and osteogenic differentiation. Our results unambiguously demonstrate their excellent ability to support MSC proliferation and induce MSC osteogenic differentiation, especially those with the micropitted topography.

Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds without supplemental osteogenic growth factors.

The development of a new family of implantable bioinspired materials is a focal point of bone tissue engineering. Implant surfaces that better mimic the natural bone extracellular matrix, a naturally nano-composite tissue, can stimulate stem cell differentiation towards osteogenic lineages in the absence of specific chemical treatments. Herein we describe a bioactive composite nanofibrous scaffold, composed of poly-caprolactone (PCL) and nano-sized hydroxyapatite (HA) or beta-tricalcium phosphate (TCP), which was able to support the growth of human bone marrow mesenchymal stem cells (hMSCs) and guide their osteogenic differentiation at the same time. Morphological and physical/chemical investigations were carried out by scanning, transmission electron microscopy, Fourier-transform infrared (FTIR) spectroscopy, mechanical and wettability analysis. Upon culturing hMSCs on composite nanofibers, we found that the incorporation of either HA or TCP into the PCL nanofibers did not affect cell viability, meanwhile the presence of the mineral phase increases the activity of alkaline phosphatase (ALP), an early marker of bone formation, and mRNA expression levels of osteoblast-related genes, such as the Runt-related transcription factor 2 (Runx-2) and bone sialoprotein (BSP), in total absence of osteogenic supplements. These results suggest that both the nanofibrous structure and the chemical composition of the scaffolds play a role in regulating the osteogenic differentiation of hMSCs.

Optimization of culture conditions for osteogenically-induced mesenchymal stem cells in ß-tricalcium phosphate ceramics with large interconnected channels.

The aim of this study was to optimize culture conditions for human mesenchymal stem cells (hMSCs) in ß-tricalcium phosphate ceramics with large interconnected channels. Fully interconnected macrochannels comprising pore diameters of 750 µm and 1400 µm were inserted into microporous ß-tricalcium phosphate (ß-TCP) scaffolds by milling. Human bone marrow-derived MSCs were seeded into the scaffolds and cultivated for up to 3 weeks in both static and perfusion culture in the presence of osteogenic supplements (dexamethasone, ß-glycerophosphate, ascorbate). It was confirmed by scanning electron microscopic investigations and histological staining that the perfusion culture resulted in uniform distribution of cells inside the whole channel network, whereas the statically cultivated cells were primarily found at the surface of the ceramic samples. It was also determined that perfusion with standard medium containing 10% fetal calf serum (FCS) led to a strong increase (seven-fold) of cell numbers compared with static cultivation observed after 3 weeks. Perfusion with low-serum medium (2% FCS) resulted in moderate proliferation rates which were comparable to those achieved in static culture, although the specific alkaline phosphatase (ALP) activity increased by a factor of more than 3 compared to static cultivation. Gene expression analysis of the ALP gene also revealed higher levels of ALP mRNA in low-serum perfused samples compared to statically cultivated constructs. In contrast, gene expression of the late osteogenic marker bone sialoprotein II (BSPII) was decreased for perfused samples compared to statically cultivated samples.

The role of nanostructured mesoporous silicon in discriminating in vitro calcification for electrospun composite tissue engineering scaffolds.

The impact of mesoporous silicon (PSi) particles-embedded either on the surface, or totally encapsulated within electrospun poly (ε-caprolactone) (PCL) fibers-on its properties as a tissue engineering scaffold is assessed. Our findings suggest that the resorbable porous silicon component can sensitively accelerate the necessary calcification process in such composites. Calcium phosphate deposition on the scaffolds was measured via in vitro calcification assays both at acellular and cellular levels. Extensive attachment of fibroblasts, human adult mesenchymal stem cells, and mouse stromal cells to the scaffold were observed. Complementary cell differentiation assays and ultrastructural measurements were also carried out; the levels of alkaline phosphatase expression, a specific biomarker for mesenchymal stem cell differentiation, show that the scaffolds have the ability to mediate such processes, and that the location of the Si plays a key role in levels of expression.