Stem Cells for Bone Regeneration: Role of Trophic Factors

Stem cells play a critical role in tissue regeneration and repair, maintenance and turnover and the control of haematopoiesis in the various tissues. These cells have an incredible ability to differentiate into specific cell types like osteoblasts, chondrocytes or myocytes and to develop bone, cartilage or muscle tissues. Now it is believed that the cells do not differentiate by themselves but rather the secretion of the bioactive (trophic) factors which are responsible for the functional outcome of the tissue. Stem cells reside in complicated and dynamic three-dimensional (3D) microenvironments in vivo known as stem cell niches. The niches are composed of extracellular matrix (ECM), soluble and tethered proteins and supporting cells, which have a profound influence on the functionality of the cells, including differentiation and trophic factor release. In this chapter, we review and emphasize the influence of stem cell microenvironment on the secretion of trophic factors and their perspective application for bone regeneration

Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 μM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 μM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.NHMRC Project grant 63265

The effects of bioactive akermanite on physiochemical, drug-delivery, and biological properties of poly(lactide-co-glycolide) beads

Poly(lactide-co-glycolide) (PLGA) beads have been widely studied as a potential drug/protein carrier. The main shortcomings of PLGA beads are that they lack bioactivity and controllable drug-delivery ability, and their acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Akermanite (AK) (Ca(2) MgSi(2) O(7) ) is a novel bioactive ceramic which has shown excellent bioactivity and degradation in vivo. This study aimed to incorporate AK to PLGA beads to improve the physiochemical, drug-delivery, and biological properties of PLGA beads. The microstructure of beads was characterized by SEM. The effect of AK incorporating into PLGA beads on the mechanical strength, apatite-formation ability, the loading and release of BSA, and the proliferation, and differentiation of bone marrow stromal cells (BMSCs) was investigated. The results showed that the incorporation of AK into PLGA beads altered the anisotropic microporous structure into homogenous one and improved their compressive strength and apatite-formation ability in simulated body fluids (SBF). AK neutralized the acidic products from PLGA beads, leading to stable pH value of 7.4 in biological environment. AK led to a sustainable and controllable release of bovine serum albumin (BSA) in PLGA beads. The incorporation of AK into PLGA beads enhanced the proliferation and alkaline phosphatase activity of BMSCs. This study implies that the incorporation of AK into PLGA beads is a promising method to enhance their physiochemical and biological property. AK/PLGA composite beads are a potential bioactive drug-delivery system for bone tissue repair

Hydroxyapatite reinforcement of different starch-based polymers affects osteoblast-like cells adhesion/spreading and proliferation

The aim of this study was to determine which, from a range of the starch-based biomaterials, would be more suitable to be used in orthopaedic applications. This included blends of corn starch and ethylene vinyl alcohol (SEVA-C), corn starch and cellulose acetate (SCA), corn starch and polycaprolactone (SPCL) and its composites with increasing percentages of hydroxyapatite (HA). Osteoblast-like cells (SaOs-2) were cultured in direct contact with the polymers and composites and the effect of the incorporation and of increasing percentages of the ceramic in osteoblast adhesion/proliferation was assessed. In the evaluation of cell adhesion and proliferation rate, two variables were considered; cells adhered to the bottom of the tissue culture polystyrene wells (TCPS) and cells adhered to the surface of the materials, in order to distinguish, respectively: (i) the effect of possible degradation products released from the materials to the culture medium and (ii) the effect of the surface properties on the osteoblast-like cells. In addition, the morphology of cells adherent to the surface of the starch-based polymers was analysed and correlated with their topography and with other chemical properties previously evaluated. The proliferation rate was found to differ from blend to blend as well as with the time of culture and with the presence of HA depending on the material. SEVA-C and respective composites systematically presented the higher number of cells comparatively to the other two blends. SPCL composites were found to be less suitable for cell proliferation. The amount of cells quantified after 7 days of culture, both on the surface and on the wells showed a delay in the proliferation of the cells cultured with SPCL composites comparatively to other materials and to TCPS. SCA composites, however, did support cell adhesion but also induce a slight level of toxicity, which results in delayed proliferation on the cells adhered to the wells. Cell morphology on the surface of the materials was also, in almost every case, found to be appropriate. In fact, cells were well adhered and spread on the majority of the surfaces. Thus, starch-based biomaterials can be seen as good substrates for osteoblast-adhesion and proliferation that demonstrates their potential to be used in orthopaedic applications and as bone tissue engineering scaffolds.Fundação para a Ciência e a Tecnologia (FCT

S100A8 and S100A9 in experimental osteoarthritis

INTRODUCTION: The objective was to evaluate the changes in S100A8 S100A9, and their complex (S100A8/S100A9) in cartilage during the onset of osteoarthritis (OA) as opposed to inflammatory arthritis. METHODS: S100A8 and S100A9 protein localization were determined in antigen-induced inflammatory arthritis in mice, mouse femoral head cartilage explants stimulated with interleukin-1 (IL-1), and in surgically-induced OA in mice. Microarray expression profiling of all S100 proteins in cartilage was evaluated at different times after initiation of degradation in femoral head explant cultures stimulated with IL-1 and surgically-induced OA. The effect of S100A8, S100A9 or the complex on the expression of aggrecan (Acan), collagen II (Col2a1), disintegrin and metalloproteases with thrombospondin motifs (Adamts1, Adamts 4 &Adamts 5), matrix metalloproteases (Mmp1, Mmp3, Mmp13 &Mmp14) and tissue inhibitors of metalloproteinases (Timp1, Timp2 &Timp3), by primary adult ovine articular chondrocytes was determined using real time quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Stimulation with IL-1 increased chondrocyte S100a8 and S100a9 mRNA and protein levels. There was increased chondrocyte mRNA expression of S100a8 and S100a9 in early but not late mouse OA. However, loss of the S100A8 staining in chondrocytes occurred as mouse OA progressed, in contrast to the positive reactivity for both S100A8 and S100A9 in chondrocytes in inflammatory arthritis in mice. Homodimeric S100A8 and S100A9, but not the heterodimeric complex, significantly upregulated chondrocyte Adamts1, Adamts4 and Adamts 5, Mmp1, Mmp3 and Mmp13 gene expression, while collagen II and aggrecan mRNAs were significantly decreased. CONCLUSIONS: Chondrocyte derived S100A8 and S100A9 may have a sustained role in cartilage degradation in inflammatory arthritis. In contrast, while these proteins may have a role in initiating early cartilage degradation in OA by upregulating MMPs and aggrecanases, their reduced expression in late stages of OA suggests they do not have an ongoing role in cartilage degradation in this non-inflammatory arthropathy

The Role of Surface Characteristics in the Initial Adhesion of Human Bone-Derived Cells on Ceramics

The tissue/biomaterial interactions of three biomaterials of potential use in bone implants were studied in vitro. The mechanism of cell adherence to various ceramic substrata has been investigated by measurement of short term (90 minutes) cell attachment and spreading rate. We have determined the effect of two serum glycoproteins, fibronectin and vitronectin, on the adhesion of human bone derived cells (HBD-cells) cultured on three different types of ceramics {alumina (Al2O3), hydroxyapatite (HAP) and yttria-doped tetragonal zirconia polycrystal (Y-TZP)} . The attachment of HBD-cells to alumina and hydroxyapatite was approximately 60% of that to Y-TZP. Furthermore, the requirement for serum adhesive glycoproteins vitronectin and fibronectin, for HBD-cell attachment to Al2O3, HAP and Y-TZP reveals a dependence upon serum vitronectin for the initial attachment of HBD-cells. There was no difference in the mechanism of initial adhesion between bioactive hydroxyapatite, as compared to the bioinert ceramics, alumina and Y-TZP, so the mechanism of the initial attachment of HBD-cells appears not to be dependent on the ceramic composition. The effect of surface roughness of alumina in the order of 8.3-70.7 nm on the adhesion of HBD-cells was also investigated. The Al2O3 disks with a root mean square surface roughness (roughness assessment: Ra) value of 8.3 nm had significantly fewer cells attached than those with an Ra of 70.7 nm

A Novel Bone Substitute with High Bioactivity, Strength, and Porosity for Repairing Large and Load-Bearing Bone Defects.

Achieving adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autografts or allografts has many drawbacks. A bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicate-based ceramic) is developed, which when 3D-printed combines high strength with outstanding bone regeneration ability. In this study, the performance of purely synthetic, 3D-printed Sr-HT-Gahnite scaffolds is assessed in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with bone autografts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, and histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy shows scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D-printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects and overcome the limitations of bone graft transplantation

Controlling the Bureaucracy of the Antipoverty Program

Rapid progress made in various areas of regenerative medicine in recent years occurred both at the cellular level, with the Nobel prize-winning discovery of reprogramming (generation of induced pluripotent stem (iPS) cells) and also at the biomaterial level. The use of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4 (called commonly "Yamanaka factors") for the conversion of differentiated cells, back to the pluripotent/embryonic stage, has opened virtually endless and ethically acceptable source of stem cells for medical use. Various types of stem cells are becoming increasingly popular as starting components for the development of replacement tissues, or artificial organs. Interestingly, many of the transcription factors, key to the maintenance of stemness phenotype in various cells, are also overexpressed in cancer (stem) cells, and some of them may find the use as prognostic factors. In this review, we describe various methods of iPS creation, followed by overview of factors known to interfere with the efficiency of reprogramming. Next, we discuss similarities between cancer stem cells and various stem cell types. Final paragraphs are dedicated to interaction of biomaterials with tissues, various adverse reactions generated as a result of such interactions, and measures available, that allow for mitigation of such negative effects

In vitro kinetic study of growth and mineralization of osteoblast-like cells (Saos-2) on titanium surface coated with a RGD functionalized bisphosphonate

Osteoconduction and osseointegration are the critical stages for implantation success. Peptides containing RGD (Arg-Gly-Asp) adhesive sequence are known to promote cell adhesion and consequently to favor osseointegration of medical devices. In this study, RGD peptides were coupled to a bisphosphonate used as an anchor system and chemically adsorbed on polished titanium discs. Two different concentrations, 10−10 mol/L (RGD 10−10) and 10−4 mol/L (RGD 10−4) were compared to non coated discs (RGD 0). Adhesion, spreading, and mineralization of osteoblast-like cells (Saos-2) were assessed. Mineralization kinetic was done at 3, 6, 10, 14, and 18 days of culture; the extent of mineral deposits was quantified by image analysis. Histogram repartitions of nuclear area, characterizing cell spreading, showed a shift to higher values in cells cultured on RGD coated titanium disks. Mineralization started at day 3 in the three groups, but had a faster development in the RGD 10−10 group from day 6 to day 18 compared to RGD 0 and RGD 10−4. At day 18, the percentage of mineralized area was significantly higher for RGD 10−10 compared to RGD 0 (p < 0.05). In the present study, this new method was found suitable to anchor RGD containing species on titanium: this favored adhesion and spreading of osteoblast-like cells and mineralization compared to noncoated titanium