Elisabetta Cenni

In vitro evaluation of cell/biomaterial interaction by MTT assay

The tetrazolium-based colorimetric assay (MTT test) measures only in vitro living cells and the results are directly related to the number of viable cultured cells. It has been adopted in immunological investigations, cancer research and, recently, biocompatibility evaluation. We used the MTT method with minor modifications to fit it to an in vitro study of biomaterial-cell interactions. The MTT assay was confirmed to be feasible, rapid and reproducible. Moreover, it showed a good correlation with other in vitro proliferation assays, such as the 3H-thymidine uptake assay. By using the MTT method and the ASTM procedure for extracting biomaterials, we quantified the in vitro cell compatibility of different metals and polymers.

Osteoblast growth and function in porous poly ε-caprolactone matrices for bone repair: a preliminary study

Abstract Current methods for the replacement of skeletal tissue involve the use of autografts, allografts and, recently, synthetic substitutes, which provide a proper amount of material to repair large bone defects. Engineered bone seems a promising approach, but a number of variables have to be set prior to any clinical application. In this study, four different poly caprolactone-based polymers (PCL) were prepared and tested in vitro using osteoblast-like Saos-2 cells. Differences among three-dimensional polymers include porosity, addition of hydroxyapatite (HA) particles, and treatment with simulated body fluid. Biochemical parameters to assess cell/material interactions include viability, growth, alkaline phosphatase release, and mineralization of osteoblastic cells seeded onto three-dimensional samples, while their morphology was observed using light microscopy and SEM. Preliminary results show that the polymers, though degrading in the medium, have a positive interaction with cells, as they support cell growth and functions. In the short-term culture (3–7 days) of Saos-2 on polymers, little differences were found among PCL samples, with the presence of HA moderately improving the number of cells onto the surfaces. In the long term (3–4 weeks), it was found that the HA-added polymers obtained the best colonization by cells, and more mineral formation was observed after coating with SBF. It can be concluded that PCL is a promising material for three-dimensional scaffold for bone formation, and the presence of bone-like components improves osteoblast activity.

Bone regeneration and stem cells

•  Introduction •  Bone fracture healing and healing problems •  Biomaterial scaffolds and tissue engineering in bone formation ‐  Bone tissue engineering ‐  Biomaterial scaffolds ‐  Synthetic scaffolds ‐  Micro‐ and nanostructural properties of scaffolds ‐  Conclusion •  Mesenchymal stem cells and osteogenesis ‐  Bone tissue ‐  Origin of osteoblasts ‐  Isolation and characterization of bone marrow derived MSC ‐  In vitro differentiation of MSC into osteoblast lineage cells ‐  In vivo differentiation of MSC into bone ‐  Factors and pathways controlling osteoblast differentiation of hMSC ‐  Defining the relationship between osteoblast and adipocyte differentiation from MSC ‐  MSC and sex hormones ‐  Effect of aging on osteoblastogenesis ‐  Conclusion •  Embryonic, foetal and adult stem cells in osteogenesis ‐  Cell‐based therapies for bone ‐  Specific features of bone cells needed to be advantageous for clinical use ‐  Development of therapeutic biological agents ‐  Clinical application concerns ‐  Conclusion •  Platelet‐rich plasma (PRP), growth factors and osteogenesis ‐  PRP effects in vitro on the cells involved in bone repair ‐  PRP effects on osteoblasts ‐  PRP effects on osteoclasts ‐  PRP effects on endothelial cells ‐  PRP effects in vivo on experimental animals ‐  The clinical use of PRP for bone repair ‐  Non‐union ‐  Distraction osteogenesis ‐  Spinal fusion ‐  Foot and ankle surgery ‐  Total knee arthroplasty ‐  Odontostomatology and maxillofacial surgery ‐  Conclusion •  Molecular control of osteogenesis ‐  TGF‐β signalling ‐  FGF signalling ‐  IGF signalling ‐  PDGF signalling ‐  MAPK signalling pathway ‐  Wnt signalling pathway ‐  Hedgehog signalling ‐  Notch signalling ‐  Ephrin signalling ‐  Transcription factors regulating osteoblast differentiation ‐  Conclusion •  Summary

Enhanced tibial osteotomy healing with use of bone grafts supplemented with platelet gel or platelet gel and bone marrow stromal cells.

BACKGROUND There is great interest in the use of bone substitutes to improve bone repair. We compared the osteogenic potential of lyophilized bone chips combined with platelet gel, or with platelet gel and bone marrow stromal cells, with that of lyophilized bone chips alone in the healing of a high tibial osteotomy. METHODS A prospective, randomized, controlled study was performed, and a standardized clinical model was applied. Thirty-three patients undergoing high tibial osteotomy to treat genu varum were enrolled and assigned to three groups. During the osteotomy, lyophilized bone chips with platelet gel were implanted into eleven patients (Group A), lyophilized bone chips with platelet gel and bone marrow stromal cells were implanted in twelve patients (Group B), and lyophilized bone chips without gel were placed in ten patients as controls (Group C). Six weeks after surgery, computed tomography-guided biopsies of the grafted areas were performed and the specimens were analyzed by histomorphometry. Clinical and radiographic evaluation was performed at six weeks, twelve weeks, six months, and one year after surgery. RESULTS Histomorphometry at six weeks showed significantly increased osteoblasts and osteoid areas in both Group A (p = 0.006 and p = 0.03, respectively) and Group B (p = 0.009 and p = 0.001) in comparison with controls, as well as increased bone apposition on the chips (p = 0.007 and p = 0.001, respectively), which was greater in Group B than in Group A (p < 0.05). Group B showed significantly higher revascularization than the controls (p = 0.004). Radiographs revealed a significantly higher rate of osseointegration in Groups A and B than in the controls at six weeks (p < 0.005 and p < 0.0001, respectively). At the final evaluation at one year, the osseointegration was still better in Groups A and B than in Group C; however, all patients had complete clinical and functional evidence of healing. CONCLUSIONS Adding a platelet gel or a platelet gel combined with bone marrow stromal cells to lyophilized bone chips increases the osteogenetic potential of the lyophilized bone chips and may be a useful tool in the treatment of patients with massive bone loss.

Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use

The biocompatibility of two cyanoacrylate surgical glues (Glubran and Glubran 2), supplied by General Enterprise Marketing, Viareggio, Lucca, Italy, was tested through cytotoxicity and blood compatibility tests and the evaluation of antimicrobial activity. Cytotoxicity and blood compatibility tests were performed on the polymerized glues. Using the neutral red uptake test, the extracts from Glubran and Glubran 2 after polymerization were non-toxic to L929 cells only when diluted 1: 10 with culture medium. Glubran and Glubran 2 induced a significant decrease of activated partial thromboplastin time (APTT), which is favourable with regard to the desired haemostasis. The APTT shortening determines a haemostatic effect and therefore contribute to the tissue adhesion induced by the glues. Otherwise, no significant variation of prothrombin activity, fibrinogen, platelet number, total and differential leukocyte count was induced by the glues, which, in addition, did not show haemolytic effect. There was no difference between Glubran and Glubran 2 regarding haemocompatibility. The antimicrobial ability of the unpolymerized glues was tested onto Bacillus subtilis var. niger for 3 weeks: neither Glubran nor Glubran 2 were found effective in this respect. In conclusion, we can assume that cytotoxicity was severe with the undiluted glues, but was acceptable when glues were diluted. On the contrary, blood compatibility was acceptable for the intended use of the glues. No difference was found between Glubran and Glubran 2 after polymerization.

Cell culture methods for testing biocompatibility.

Cell culture systems may be of value in testing the biocompatibility of prosthetic materials before they are introduced into clinical use. In recent years, in vitro methods for assaying biomaterials have gained in importance owing to the growing concern over the use of animals for biomaterials testing. Significant effort is therefore being focused toward developing predictive and quantitative, but also simple and reliable, methods of testing using cultured cells. At present, a number of methods for measuring both the cytotoxicity and the specific cytocompatibility of different materials are available. The usefulness of these systems is no longer confined to screening new materials; they can be used to study the mechanisms of action of various materials during tissue/material interaction. This paper reviews the published literature on the use of cell culture models in evaluating biocompatibility and reports on the personal experience of the authors, who have been using cell culture systems for many years and for different purposes.

Growth factors in bone repair

The role of growth factors (GF) in bone repair is widely recognised, particularly for bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF). GF are usually stored in the extracellular matrix (ECM), but after injury are actively released by ECM, cells and platelets. In this paper, the use of different recombinant GF for bone repair stimulation is summarised in experimental research and clinical applications. Drug delivery systems, including carriers, cell or gene therapy, are needed to ensure a sustained local release of the factors, but efficacy and potential side effects of such systems require additional research prior to clinical applications. Current sources for delivery of a GF mixture into the site of bone repair are platelet gel and demineralised bone matrix. Nevertheless, the levels of GF in such preparations are affected by variability among donors and differences in preparation. Autogenous GF, produced by the patient himself during the bone repair process, potentially interfere with prosthetic devices or even have a role in implant loosening due to the periprosthetic tissue reaction. In conclusion, GF are key components of functional bone regeneration: screening of basic research results and controlled clinical trials are accelerating the development of GF in orthopaedic surgery.

Biocompatibility of poly(d,l-lactide-co-glycolide) nanoparticles conjugated with alendronate

Nanoparticles made of a conjugate of poly(D,L-lactide-co-glycolide) with alendronate (PLGA-ALE NPs), were prepared by emulsion/solvent evaporation technique. The conjugation yield, determined by MALDI TOF analysis, was 30-35%. PLGA-ALE NPs size, evaluated by photon correlation spectroscopy, was 198.7+/-0.2 nm. Haemocompatibility studies using different concentrations of PLGA-ALE NPs did not show any significant effect on haemolysis, leukocyte number, platelet activation, APTT and complement consumption, in comparison with blood incubated with phosphate buffered saline (PBS). A significant reduction of the prothrombin activity was demonstrated after incubation with 560 microg/ml of PLGA-ALE NPs; a significant increase was observed at the highest dilutions. The viability of human umbilical vein endothelial cells and bone marrow stromal cells (BMSC), evaluated through the neutral red test, was not affected by PLGA-ALE NPs. There were no significant differences in cell-associated alkaline phosphatase between BMSC incubated with PLGA-ALE NP- and PBS-added media. These results demonstrated that PLGA-ALE NPs had an acceptable degree of blood compatibility and were not cytotoxic; therefore, they may be considered suitable for intravenous administration.

Metal hypersensitivity testing in patients undergoing joint replacement: A systematic review

We report a systematic review and meta-analysis of the peer-reviewed literature focusing on metal sensitivity testing in patients undergoing total joint replacement (TJR). Our purpose was to assess the risk of developing metal hypersensitivity post-operatively and its relationship with outcome and to investigate the advantages of performing hypersensitivity testing. We undertook a comprehensive search of the citations quoted in PubMed and EMBASE: 22 articles (comprising 3634 patients) met the inclusion criteria. The frequency of positive tests increased after TJR, especially in patients with implant failure or a metal-on-metal coupling. The probability of developing a metal allergy was higher post-operatively (odds ratio (OR) 1.52 (95% confidence interval (CI) 1.06 to 2.31)), and the risk was further increased when failed implants were compared with stable TJRs (OR 2.76 (95% CI 1.14 to 6.70)). Hypersensitivity testing was not able to discriminate between stable and failed TJRs, as its predictive value was not statistically proven. However, it is generally thought that hypersensitivity testing should be performed in patients with a history of metal allergy and in failed TJRs, especially with metal-on-metal implants and when the cause of the loosening is doubtful.

Cytotoxicity testing of cyanoacrylates using direct contact assay on cell cultures

The use of a tissue adhesive for surgical procedures has prompted a large number of clinical and experimental studies. Alkyl-2-cyanoacrylate esters constitute a family of adhesives with good mechanical properties but their biological compatibility has to be assessed. In this study the cytotoxicity of three commercially available cyanoacrylates and one of unknown composition has been determined. The first part of the study deals with direct contact testing procedures using L 929 cells challenged with drops of adhesives: cell morphology, cell growth and bacterial growth inhibition were assayed. Testing methods included cell viability assay using vital dyes, cell growth measurement using crystal violet staining uptake and bacterial growth assay using S. aureus growth inhibition. All the cyanoacrylate adhesives tested were found to be cytotoxic and to inhibit cell proliferation: differences between the cyanoacrylates were found.