In situ non‐invasive spectral discrimination between bone cell phenotypes used in tissue engineering

Raman micro‐spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast‐like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non‐tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non‐tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non‐tumour bone cells. A supervised classification model (LDA) was built and showed high cross‐validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non‐tumour cells, with 96% of the cells being correctly classified either as tumour or non‐tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells. © 2004 Wiley‐Liss, Inc.

[1]  L. Choo-Smith,et al.  Discriminating Vital Tumor from Necrotic Tissue in Human Glioblastoma Tissue Samples by Raman Spectroscopy , 2002, Laboratory Investigation.

[2]  A. Boccaccini,et al.  In vitro evaluation of novel bioactive composites based on Bioglass-filled polylactide foams for bone tissue engineering scaffolds. , 2003, Journal of biomedical materials research. Part A.

[3]  T. B. Bakker Schut,et al.  Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy. , 2002, The Journal of investigative dermatology.

[4]  J. Polak,et al.  In situ Characterisation of Living Cells by Raman Spectroscopy , 2002 .

[5]  L L Hench,et al.  Osteoblast attachment and mineralized nodule formation on rough and smooth 45S5 bioactive glass monoliths. , 2004, Journal of biomedical materials research. Part A.

[6]  M. Gowen,et al.  Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype? , 1994, Bone.

[7]  K. A. Hartman,et al.  Raman studies of nucleic acids. 8. Estimation of RNA secondary structure from Raman scattering by phosphate-group vibrations. , 1973, Biochimica et biophysica acta.

[8]  Ioan Notingher,et al.  In situ spectroscopic study of nucleic acids in differentiating embryonic stem cells , 2004 .

[9]  D. B. Evans,et al.  PCR phenotyping of cytokines, growth factors and their receptors and bone matrix proteins in human osteoblast-like cell lines. , 1996, Bone.

[10]  J. Mourant,et al.  Raman Spectroscopy and Factor Analysis of Tumorigenic and Non-Tumorigenic Cells , 2002 .

[11]  B D Boyan,et al.  Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells. , 1996, Journal of biomedical materials research.

[12]  P. O'Brien,et al.  Prevention of cyanide-induced cytotoxicity by nutrients in isolated rat hepatocytes. , 1994, Toxicology and applied pharmacology.

[13]  W. Campbell,et al.  Raman microspectroscopy of intracellular cholesterol crystals in cultured bovine coronary artery endothelial cells. , 1997, Journal of lipid research.

[14]  D. Naumann,et al.  Prospective Study of the Performance of Vibrational Spectroscopies for Rapid Identification of Bacterial and Fungal Pathogens Recovered from Blood Cultures , 2003, Journal of Clinical Microbiology.

[15]  H. Bruining,et al.  Raman spectroscopic method for identification of clinically relevant microorganisms growing on solid culture medium. , 2000, Analytical chemistry.

[16]  Anthony T. Tu,et al.  Raman spectroscopy in biology: Principles and applications , 1982 .

[17]  Michael D Morris,et al.  Mineralization of Developing Mouse Calvaria as Revealed by Raman Microspectroscopy , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[18]  R. Dasari,et al.  Raman microspectroscopic model of human breast tissue: implications for breast cancer diagnosis in vivo , 2002 .

[19]  D. Beruto,et al.  A bone substitute composed of polymethylmethacrylate and alpha-tricalcium phosphate: results in terms of osteoblast function and bone tissue formation. , 2002, Biomaterials.

[20]  L L Hench,et al.  Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells. , 2003, Biopolymers.

[21]  L L Hench,et al.  In situ monitoring of cell death using Raman microspectroscopy. , 2004, Biopolymers.

[22]  B. Boyan,et al.  Titanium surface roughness alters responsiveness of MG63 osteoblast-like cells to 1 alpha,25-(OH)2D3. , 1998, Journal of biomedical materials research.

[23]  H. Barr,et al.  Raman Spectroscopy for Early Detection of Laryngeal Malignancy: Preliminary Results , 2000, The Laryngoscope.

[24]  R. Dasari,et al.  Identifying microcalcifications in benign and malignant breast lesions by probing differences in their chemical composition using Raman spectroscopy. , 2002, Cancer research.

[25]  H. Gremlich,et al.  Infrared and Raman Spectroscopy of Biological Materials , 2000 .

[26]  J. Greve,et al.  Studying single living cells and chromosomes by confocal Raman microspectroscopy , 1990, Nature.

[27]  Abigail S Haka,et al.  Model‐based biological Raman spectral imaging , 2002, Journal of cellular biochemistry. Supplement.

[28]  B. Komm,et al.  Development and characterization of a conditionally transformed adult human osteoblastic cell line , 1996, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[29]  Christoph Krafft,et al.  Mapping of single cells by near infrared Raman microspectroscopy , 2003 .

[30]  J. Greve,et al.  Laser irradiation and Raman spectroscopy of single living cells and chromosomes: sample degradation occurs with 514.5 nm but not with 660 nm laser light. , 1991, Experimental cell research.

[31]  J. A. López del Val,et al.  Principal Components Analysis , 2018, Applied Univariate, Bivariate, and Multivariate Statistics Using Python.

[32]  N Stone,et al.  The use of Raman spectroscopy to identify and grade prostatic adenocarcinoma in vitro , 2003, British Journal of Cancer.

[33]  Hugh Barr,et al.  Near‐infrared Raman spectroscopy for the classification of epithelial pre‐cancers and cancers , 2002 .

[34]  J. Wergedal,et al.  Characterization of Cells Isolated and Cultured from Human Bone , 1984, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[35]  Gerwin J. Puppels,et al.  Raman Spectroscopic Methods for In Vitro and In Vivo Tissue Characterization , 1999 .

[36]  J Greve,et al.  Nonresonant confocal Raman imaging of DNA and protein distribution in apoptotic cells. , 2003, Biophysical journal.

[37]  D. Naumann,et al.  Investigating Microbial (Micro)colony Heterogeneity by Vibrational Spectroscopy , 2001, Applied and Environmental Microbiology.

[38]  Hugh Barr,et al.  Raman spectroscopy, a potential tool for the objective identification and classification of neoplasia in Barrett's oesophagus , 2003, The Journal of pathology.

[39]  B Willekens,et al.  Nonresonant Raman imaging of protein distribution in single human cells. , 2003, Biopolymers.

[40]  H. Takeuchi,et al.  Lipid structure of cytotoxic granules in living human killer T lymphocytes studied by Raman microspectroscopy. , 1997, Biochimica et biophysica acta.

[41]  J. Jeng,et al.  Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanisms , 1998, Cell Biology and Toxicology.

[42]  N Stone,et al.  Raman Spectral Mapping in the Assessment of Axillary Lymph Nodes in Breast Cancer , 2003, Technology in cancer research & treatment.

[43]  M. O'hare,et al.  Retroviral transduction of alveolar bone cells with a temperature-sensitive SV40 large T antigen , 2001, Cell and Tissue Research.