Evaluation of the in vitro cell-material interactions and in vivo osteo-integration of a spinal acrylic bone cement.

INTRODUCTION: Polymethylmethacrylate bone cements have proven performance in arthroplasty and represent a common bone filler, e.g. in vertebroplasty. However, acrylic cements are still subject to controversy concerning their exothermic reaction and osteo-integration potential. Therefore, we submitted a highly filled acrylic cement to a systematic investigation on the cell-material and tissue-implant response in vitro and in vivo. MATERIALS AND METHODS: Cured Vertecem V+ Cements were characterized by electron microscopy. Human bone marrow-derived mesenchymal stem cell morphology, growth and differentiation on the cured cement were followed for 28 days in vitro. The uncured cement was injected in an ovine cancellous bone defect and analysed 4 and 26 weeks post-implantation. RESULTS: The rough surface of the cement allowed for good stem cells adhesion in vitro. Up-regulation of alkaline phosphatase was detected after 8 days of incubation. No adverse local effects were observed macroscopically and microscopically following 4 and 26 weeks of implantation of the cement into drill-hole defects in ovine distal femoral epiphysis. Direct bone apposition onto the implant surface was observed resulting in extended signs of osteo-integration over time (35.2 ± 24.2% and 88.8 ± 8.8% at week 4 and 26, respectively). CONCLUSION: Contrary to the established opinion concerning bony tissue response to implanted acrylic bone cements, we observed an early cell-implant in vitro interaction leading to cell growth and differentiation and significant signs of osteo-integration for this acrylic cement using standardized methods. Few outlined limitations, such as the use of low cement volumes, have to be considered in the interpretation of the study results.

An In Vitro Investigation of Platelet-Rich Plasma-Gel as a Cell and Growth Factor Delivery Vehicle for Tissue Engineering.

Platelet-rich plasma (PRP) has been used for different applications in human and veterinary medicine. Many studies have shown promising therapeutic effects of PRP; however, there are still many controversies regarding its composition, properties, and clinical efficacy. The aim of this study was to evaluate the influence of different platelet concentrations on the rheological properties and growth factor (GF) release profile of PRP-gels. In addition, the viability of incorporated bone marrow-derived human mesenchymal stem cells (MSCs) was investigated. PRP (containing 1000 × 10(3), 2000 × 10(3), and 10,000 × 10(3) platelets/µL) was prepared from human platelet concentrates. Platelet activation and gelification were achieved by addition of human thrombin. Viscoelastic properties of PRP-gels were evaluated by rheological studies. The release of GFs and inflammatory proteins was measured using a membrane-based protein array and enzyme-linked immunosorbent assay. MSC viability and proliferation in PRP-gels were assessed over 7 days by cell viability staining. Cell proliferation was examined using DNA quantification. Regardless of the platelet content, all tested PRP-gels showed effective cross-linking. A positive correlation between protein release and the platelet concentration was observed at all time points. Among the detected proteins, the chemokine CCL5 was the most abundant. The greatest release appeared within the first 4 h after gelification. MSCs could be successfully cultured in PRP-gels over 7 days, with the highest cell viability and DNA content found in PRP-gels with 1000 × 10(3) platelets/µL. The results of this study suggest that PRP-gels represent a suitable carrier for both cell and GF delivery for tissue engineering. Notably, a platelet concentration of 1000 × 10(3) platelets/µL appeared to provide the most favorable environment for MSCs. Thus, the platelet concentration is an important consideration for the clinical application of PRP-gels.

PDLLA/Bioglass composites for soft-tissue and hard-tissue engineering: an in vitro cell biology assessment.

The aim of this study was to examine the effect of increased content of 45S5 Bioglass (0-40 wt%) in poly(dl-lactic acid) (PDLLA) porous foams on the behaviour of MG-63 (human osteosarcoma cell line) and A549 cells (human lung carcinoma cell line). The ability of these cell lines to grow on bioactive composites was quantitatively investigated in order to assess the potentiality for their use in hard and soft-tissue engineering. Two hours after cell seeding, an increase of cell adhesion according to the increased content of Bioglass((R)) present in the foams for both cell types was observed. Cell proliferation studies performed over a period of 4 weeks showed a better aptitude of the A549 cells to proliferate on PDLLA foams containing 5 wt% Bioglass when compared to the proliferation on foams with 40 wt% Bioglass. A lower proliferation rate was obtained for cells on pure PDLLA. Scanning electron microscopy analysis showed for both cell types the presence of cells inside the porous structure of the foams. These results confirmed the biocompatibility of PDLLA/Bioglass composite foams and the positive effect of Bioglass on MG-63 cell behaviour and also showed for the first time the possibility for human lung epithelial type II cells to adhere and proliferate on these porous scaffolds. In addition, we describe a positive effect of 45S5 Bioglass on A549 cell behaviour in a dose-dependent manner, indicating the potential of using PDLLA/Bioglass composites with an optimal concentration of 45S5 Bioglass not only in bone tissue engineering but also in lung tissue engineering.

Binary CaO-SiO(2) gel-glasses for biomedical applications.

Bioactive materials are routinely used in dental and orthopaedic applications. The concept was first introduced in 1971, with the discovery of 45S5 Bioglass, which is known to develop an interfacial bond between the implant and the host tissue. This glass is composed of SiO(2), CaO, P(2)O(5) and Na(2)O. Since then numerous glasses and glass ceramics with similar compositions have been extensively studied for clinical applications. Until 1990 it was accepted that P(2)O(5) and Na(2)O were necessary components for the glass composition to be bioactive. However, calcium silicate glasses with high SiO(2) content are impossible to produce using the traditional melt-quench method. This is due to the liquid-liquid immiscibility region that is present between 0.02 and 0.3 mole fraction of CaO and in terms of bioactivity, high CaO compositions were inferior to those quaternary bioactive glass compositions already in existence. In the last few years several studies have been reported regarding the production of CaO-SiO(2) glasses via the sol-gel processing technique. This report summarises the findings of the past and the present and also outlines potential of these calcium silicate gel-glasses in the field of biomaterials.

Raman micro-spectroscopy as a non-invasive cell viability test.

The number of techniques to identify, quantify and characterise cell death is rapidly increasing as more is known about the complex mechanisms underlying this process. However, most of these techniques are invasive and require preparation steps such as cell fixation, staining or protein extractions. Non-invasive analysis of living cells represents a key point in cell biology, e.g. in toxicology studies or in tissue engineering. In this chapter, we report the usefulness of Raman spectroscopy as a non-invasive method to distinguish cells at different stages of cell cycle and living cells from dead cells. Throughout two examples, we show the performance and the use of Raman spectroscopy as a new non-invasive method to assess cell viability.

The osteogenic differentiation of human osteoprogenitor cells on Anodic-Plasma-Chemical treated Ti6Al7Nb.

Biological integration of an implant to surrounding bone is an important event for its clinical success and is driven by numerous factors, including the attraction of bone forming cells. The implant's surface properties influence the initial cell response at the cell/material interface, ultimately affecting the rate and quality of new tissue formation and the stability of the implant. As a consequence, various surface treatments have been developed to increase the clinical performance of titanium-based implants. Among them, the Anodic Plasma-Chemical (APC) technique allows for the combined chemical and morphological modification of titanium surfaces in a single process step. In the present study, we compared the potential of APC surface treatment of high-strength titanium alloys with vacuum plasma spray treatment and yellow gold anodization in supporting osteogenic differentiation of two different osteoprogenitor cell types. Both human fetal osteoblast cell line (hFOB1.19) and human mesenchymal stromal cells showed extensive cell spreading, faster cell growth and differentiation on APC surfaces compared to vacuum plasma spray treated and yellow gold anodized surfaces. Our findings showed that APC titanium-based surfaces provided an effective substrate for osteoprogenitor cells adhesion, proliferation and differentiation.

Mechanical properties and cytocompatibility of poly(ε-caprolactone)-infiltrated biphasic calcium phosphate scaffolds with bimodal pore distribution.

Biphasic calcium phosphate scaffolds have attracted interest because they have good osteoconductivity and a resorption rate close to that of new bone ingrowth, but their brittleness limits their potential applications. In this study, we show how the infiltration of biphasic calcium phosphate scaffolds with poly(ε-caprolactone) improves their mechanical properties. It was found that the polymer effectively contributes to energy to failure enhancement in bending, compressive and tensile tests. The main toughening mechanism in these composites is crack bridging by polymer fibrils. The presence of fibrils at two different size scales--as found in scaffolds with a bimodal pore distribution--results in a more effective toughening effect as compared to scaffolds with a monomodal pore size distribution, especially in the early stage of mechanical deformation. An optimized infiltration process allowed the preservation of micropore interconnection after infiltration, which is beneficial for cells adhesion. In addition, it is shown that biphasic calcium phosphates infiltrated with poly(ε-caprolactone) are cytocompatible with human bone marrow stromal cells, which makes them good candidates for bone substitution.

Properties of an injectable low modulus PMMA bone cement for osteoporotic bone.

The use of polymethylmethacrylate (PMMA) cement to reinforce fragile or broken vertebral bodies (vertebroplasty) leads to extensive bone stiffening. Fractures in the adjacent vertebrae may be the consequence of this procedure. PMMA with a reduced Young's modulus may be more suitable. The goal of this study was to produce and characterize stiffness adapted PMMA bone cements. Porous PMMA bone cements were produced by combining PMMA with various volume fractions of an aqueous sodium hyaluronate solution. Porosity, Young's modulus, yield strength, polymerization temperature, setting time, viscosity, injectability, and monomer release of those porous cements were investigated. Samples presented pores with diameters in the range of 25-260 microm and porosity up to 56%. Young's modulus and yield strength decreased from 930 to 50 MPa and from 39 to 1.3 MPa between 0 and 56% porosity, respectively. The polymerization temperature decreased from 68 degrees C (0%, regular cement) to 41 degrees C for cement having 30% aqueous fraction. Setting time decreased from 1020 s (0%, regular cement) to 720 s for the 30% composition. Viscosity of the 30% composition (145 Pa s) was higher than the ones received from regular cement and the 45% composition (100-125 Pa s). The monomer release was in the range of 4-10 mg/mL for all porosities; showing no higher release for the porous materials. The generation of pores using an aqueous gel seems to be a promising method to make the PMMA cement more compliant and lower its mechanical properties to values close to those of cancellous bone.

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds.

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.