Absence Of Osteogenic Factors Research Articles

Chitosan in association with osteogenic factors as a cell-homing platform for dentin regeneration: Analysis in a pulp-in-a-chip model

ObjectiveIn this paper we propose the association of β-glycerophosphate (βGP) and calcium-hydroxide with chitosan (CH) to formulate a porous bioactive scaffold suitable as a cell-homing platform for dentin regeneration. MethodsCalcium hydroxide and βGP solutions were incorporated into chitosan to modulate scaffold architecture and composition by a phase separation technique. Architecture, chemical composition, and degradability were evaluated, and biological characterizations were performed by the seeding of dental pulp cells (DPCs) onto scaffolds, or by cultivating them in contact with leachable components (extracts), to determine cytocompatibility and odontoblastic differentiation. Cell-free scaffolds were then positioned in intimate contact with a 3D culture of DPCs in a pulp-in-a-chip platform under simulated pulp pressure. Cell mobilization and odontoblastic marker expression were evaluated. Deposition of mineralized matrix was assessed in direct contact with dentin, in the absence of osteogenic factors. ResultsIncorporation of calcium hydroxide and βGP generated a stable porous chitosan scaffold containing Ca-P nanoglobule topography (CH-Ca-βGP), which favored cell viability, alkaline phosphatase activity, and mineralized matrix deposition by cells seeded onto the scaffold structure and at a distance. The pulp-in-a-chip assay denoted its chemotactic and bioactive potential, since dentin sialoprotein-positive DPCs from 3D culture adhered to CH-Ca-βGP more than to plain chitosan. The higher deposition of mineralized matrix onto the scaffold and surrounding dentin was also observed. SignificanceA CH-Ca-βGP scaffold creates a microenvironment capable of mobilizing DPC migration toward its structure, harnessing the odontogenic potential and culminating in the expression of a highly mineralizing phenotype, key factors for a cell-homing strategy.

Lotus root and osteons-inspired channel structural scaffold mediate cell biomineralization and vascularized bone tissue regeneration.

The interconnectivity of porous scaffold is vital for cell and tissue infiltration, and vascular networks formation, determining the successful bone regeneration in large bone defects. Here, inspired by the lotus-root and Haversian system of natural bone, a nano-hydroxyapatite/polyurethane (n-HA/PU) lotus root-like scaffold inlaid with micro holes on the wall of the adjacent channel was utilized to mediate cell biomineralization and vascularized bone tissue regeneration. Such a particular lotus-type structure remarkably promoted cells to proliferate and infiltrate into the center of the entire scaffold, serving as a clue to account for regulating cell alignment and differentiation physically. In vitro studies suggested that apatite accumulated dramatically on the channel wall in the lotus-type scaffold, probably promoting specific osteogenic differentiation of cells by the orientated channels, even in the absence of osteogenic factors. In vivo creation of critical bone defects (15-mm segments) was done in the radius of rabbits and implanted with the scaffold of different geometry to assess the structural parameters on the efficacy of new bone regeneration. The more extensive positive staining of BMP-2, more considerable amount of infiltrated capillary, more robust new bone formation, particularly the biomechanical strength of lotus-type scaffold group could reach the level of the control group without surgery, indicating that the lotus-type scaffold was more favorable for new bone tissue formation along tube-like channels. These results highlighted the potential of this biomimetic scaffold for cell and tissue infiltration and thus repair large bony defects.

The inhibitory effects of vancomycin on rat bone marrow-derived mesenchymal stem cell differentiation.

The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow-derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%-60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

Improving the Osteogenicity of PCL Fiber Substrates by Surface-Immobilization of Bone Morphogenic Protein-2.

Polycaprolactone (PCL) fiber scaffolds are attractive, albeit inert, substrates for ligament regeneration, that may be improved by incorporating trophic factors to guide tissue remodeling in vivo. In particular, immobilization of bone morphogenic protein-2 (BMP-2) to the scaffold surface may facilitate rapid and robust integration of the scaffold with adjacent bone tissues. As a first step toward testing this, model PCL surfaces were modified by the addition of heparin (Hep) and BMP-2 to facilitate osteoblastic differentiation. Specifically, Hep was combined with PCL at 0, 0.5, and 1wt% (denoted as PCL, PCL-0.5Hep, and PCL-1Hep), cast into films, and then BMP-2 was immobilized to surfaces by either adsorption and covalent conjugation. Here, BMP-2 concentration increased systematically with incorporation of Hep, and higher concentrations were achieved by covalent conjugation. Next, blends were electrospun to form thin meshes with fiber diameters of 0.92, 0.62, and 0.54μm for PCL, PCL-0.5Hep, and PCL-1Hep, respectively. Mesenchymal stem cells (MSCs) had no difficulty attaching to and proliferating on all meshes. Lastly, PCL-1Hep meshes were prepared with adsorbed or covalently conjugated BMP-2 and cultured with MSCs in the absence of osteogenic factors. Under these conditions, alkaline phosphatase activity and deposition of bone sialoprotein, osteopontin, and calcium minerals-markers of osteoblastic differentiation-were significantly higher on surfaces with immobilized BMP-2. Together, these data indicate that covalent immobilization of trophic factors confers bioactivity to scaffolds, which may be applied in aspatially controlled manner for ligament regeneration and bone integration.

Combinatorial Effect of Silicon and Calcium Release from Starch-Based Scaffolds on Osteogenic Differentiation of Human Adipose Stem Cells.

Instructive materials able to drive cells, in particular the differentiation of stem cells toward osteoblastic lineages, have been investigated as a promising strategy for bone tissue engineering. Inorganic ions, such as phosphorus, calcium, silicon, and strontium, have been used in bone regeneration strategies as instructive ions for material-based approaches. The use of effective inorganic ions is being investigated as a promising approach for bone regeneration applications, mainly because they are highly available and cost-effective and thus reducing the need to use expensive and less-stable growth factors. The aim of the present study is to investigate the effect of the release of silicon (Si) and calcium (Ca) ions from a blend of starch and poly caprolactone (SPCL) scaffolds on the osteogenic behavior of human adipose stem cells (hASCs). The scaffolds were developed by a wet-spinning technique and two different solutions were used as coagulation bath, one containing Ca and Si ions and other one containing only Si ions. The composition of the scaffolds as well as their mechanical properties was also evaluated. Our study showed that both scaffolds were able to sustain cell attachment and induce their differentiation into the osteogenic lineage in basal medium, i.e., in the absence of osteogenic factors. The scaffolds containing both ions, Si and Ca, had a stronger influence on the osteogenic differentiation of hASCs than the scaffolds containing only Si ion. Thus, the present work highlights the importance of combining Si and Ca ions in the control of cellular response, namely, cell differentiation and/or in stem cells recruitment upon implantation of a cell-free scaffold, and thus, avoiding the use of costly growth factors.

Trabecular titanium can inducein vitroosteogenic differentiation of human adipose derived stem cells without osteogenic factors

Trabecular Titanium (TT) is an innovative highly porous structure that imitates the morphology of trabecular bone with good mechanical properties. Adipose-derived stem cells are a multipotent cell population that can be used in regenerative medicine, in particular, for bone therapeutic applications. The ability of TT to induce the osteogenic differentiation of human adipose derived stem cells (hASCs) in the absence of osteogenic factors was evaluated using molecular biological, biochemical, and immunohistochemical methods. At 7 and 21 days from differentiation, the hASCs grown on TT scaffolds showed similar expressions of alkaline phosphatase (ALP) and Runx-2 both in the presence and in the absence of osteogenic factors, as well as at transcript and protein levels. hASCs cultured on monolayer in the presence of the medium obtained from the wells where hASCs/scaffold constructs were cultured in the absence of osteogenic factors differentiated towards the osteogenic phenotype: their gene and protein expression of ALP and Runx-2 was similar to that of the same cells cultured in the presence of osteogenic factors, and significantly higher than that of the ones cultured in growth medium.

A thermosensitive low molecular weight hydrogel as scaffold for tissue engineering

Hydrogels that are non-toxic, easy to use, cytocompatible, injectable and degradable are valuable biomaterials for tissue engineering and tissue repair. However, few compounds currently fulfil these requirements. In this study, we describe the biological properties of a new type of thermosensitive hydrogel based on low-molecular weight glycosyl-nucleosyl-fluorinated (GNF) compound. This gel forms within 25 min by self-assembly of monomers as temperature decreases. It degrades slowly in vitro and in vivo. It induces moderate chronic inflammation and is progressively invaded by host cells and vessels, suggesting good integration to the host environment. Although human adult mesenchymal stem cells derived from adipose tissue (ASC) cannot adhere on the gel surface or within a 3D gel scaffold, cell aggregates grow and differentiate normally when entrapped in the GNF-based gel. Moreover, this hydrogel stimulates osteoblast differentiation of ASC in the absence of osteogenic factors. When implanted in mice, gel-entrapped cell aggregates survive for several weeks in contrast with gel-free spheroids. They are maintained in their original site of implantation where they interact with the host tissue and adhere on the extracellular matrix. They can differentiate in situ into alkaline phosphatase positive osteoblasts, which deposit a calcium phosphate-rich matrix. When injected into subcutaneous sites, gel-encapsulated cells show similar biological properties as implanted gel-cells complexes. These data point GNF-based gels as a novel class of hydrogels with original properties, in particular osteogenic potential, susceptible of providing new therapeutic solutions especially for bone tissue engineering applications.

Odontoblast marker gene expression is enhanced by a CC-chemokine family protein MIP-3α in human mesenchymal stem cells

Objective Macrophage inflammatory protein-3 alpha (MIP-3α) is a major CC-chemokine family protein, which serves as a differentiation factor for mesenchymal cells, including osteoblasts and dental pulp cells. The purpose of this study was to investigate the influence of MIP-3α on human mesenchymal stem cell differentiation in vitro. Design Human mesenchymal stem cells were maintained in Dulbecco's modified Eagle's medium in the presence or absence of MIP-3α and the presence or absence of osteogenic factors (dexamethasone, β-glycerophoshate and ascorbic acid). Alkaline phosphatase (ALP) activity was measured, and expression of odontoblast and osteoblast markers were examined by RT-PCR and Western blotting. Results MIP-3α alone did not increase ALP activity, as compared to controls. The combination of MIP-3α and osteogenic factors increased ALP activity beyond increases observed with osteogenic factors alone. mRNA expression of the odontoblast marker dspp was only detectable when MIP-3α was added together with osteogenic factors at day 7 in three out of four samples. DSP protein level was increased only in the samples treated with both MIP-3α and osteogenic factors until day 5. In contrast, MIP-3α did not influence levels of the osteoblast markers CBFA1 or BSP. Conclusions The present study demonstrated that MIP-3α enhanced gene expression and protein levels of odontoblast-related genes, without affecting levels of the osteogenic proteins CBFA1 or BSP.

Enhanced differentiation and mineralization of human fetal osteoblasts on PDLLA containing Bioglass® composite films in the absence of osteogenic supplements

This study investigates the cellular response of fetal osteoblasts to bioactive resorbable composite films consisting of a poly-D,L-lactide (PDLLA) matrix and bioactive glass 45S5 Bioglass (BG) particles at three different concentrations (0% (PDLLA), 5% (P/BG5), and 40% (P/BG40)). Using scanning electron microscopy (SEM) we observed that cells were less spread and elongated on PDLLA and P/BG5, whereas cells on P/BG40 were elongated but with multiple protrusions spreading over the BG particles. Vinculin immunostaining revealed similar distribution of focal adhesion contacts on all cells independent of substratum, indicating that all materials permitted cell adhesion. However, when differentiation and maturation of fetal osteoblasts was examined, incorporation of 45S5 BG within the PDLLA matrix was found to significantly (p < 0.05) enhance alkaline phosphatase enzymatic activity and osteocalcin protein synthesis compared to tissue culture polystyrene controls and PDLLA alone. Alizarin red staining indicated extracellular matrix mineralization on both P/BG5 and P/BG40, with significantly more bone nodules formed than on PDLLA. Real time RT-PCR revealed that expression of bone sialoprotein was also affected by the BG containing films compared to controls, whereas expression of Collagen Type I was not influenced. By performing these investigations in the absence of osteogenic factors it appears that the incorporation of BG stimulates osteoblast differentiation and mineralization of the extracellular matrix, demonstrating the osteoinductive capacity of the composite.