Advances in vibrational spectroscopy have propelled new insights into the molecular composition and structure of biological tissues. In this review, we discuss common modalities and techniques of vibrational spectroscopy, and present key examples to illustrate how they have been applied to enrich the assessment of connective tissues. In particular, we focus on applications of Fourier transform infrared (FTIR), near infrared (NIR) and Raman spectroscopy to assess cartilage and bone properties. We present strengths and limitations of each approach and discuss how the combination of spectrometers with microscopes (hyperspectral imaging) and fiber optic probes have greatly advanced their biomedical applications. We show how these modalities may be used to evaluate virtually any type of sample (ex vivo, in situ or in vivo) and how “spectral fingerprints” can be interpreted to quantify outcomes related to tissue composition and quality. We highlight the unparalleled advantage of vibrational spectroscopy as a label-free and often nondestructive approach to assess properties of the extracellular matrix (ECM) associated with normal, developing, aging, pathological and treated tissues. We believe this review will assist readers not only in better understanding applications of FTIR, NIR and Raman spectroscopy, but also in implementing these approaches for their own research projects.

Bone mineral crystallinity is an important factor determining bone quality and strength. The gold standard method to quantify crystallinity is X-ray diffraction (XRD), but vibrational spectroscopic methods present powerful alternatives to evaluate a greater variety of sample types. We describe original approaches by which transmission Fourier transform infrared (FT-IR), attenuated total reflection (ATR) FT-IR, and Raman spectroscopy can be confidently used to quantify bone mineral crystallinity. We analyzed a range of biological and synthetic apatite nanocrystals (10–25 nm) and found strong correlations between different spectral factors and the XRD determination of crystallinity. We highlight striking differences between FT-IR spectra obtained by transmission and ATR. In particular, we show for the first time the absence of the 1030 cm ⁻¹ crystalline apatite peak in ATR FT-IR spectra, which excludes its use for analyzing crystallinity using the traditional 1030/1020 cm ⁻¹ ratio. The ν ₄ PO ₄ splitting ratio was also not adequate to evaluate crystallinity using ATR FT-IR. However, we established original approaches by which ATR FT-IR can be used to determine apatite crystallinity, such as the 1095/1115 and 960/1115 cm ⁻¹ peak ratios in the second derivative spectra. Moreover, we found a simple unified approach that can be applied for all three vibrational spectroscopy modalities: evaluation of the ν ₁ PO ₄ peak position. Our results allow the recommendation of the most reliable analytical methods to estimate bone mineral crystallinity by vibrational spectroscopy, which can be readily implemented in many biomineralization, archeological and orthopedic studies. In particular, we present a step forward in advancing the use of the increasingly utilized ATR FT-IR modality for mineral research.

Right angle magnetron sputtering (RAMS) was used to produce hydroxyapatite (HA) film coatings on pure titanium substrates and oriented silicon wafer (Si(0 0 1)) substrates with flat surfaces as well as engineered surfaces having different forms. Analyses using synchrotron XRD, AFM, XPS, FTIR and SEM with EDS showed that as-sputtered thin coatings consist of highly crystalline hydroxyapatite. The HA coatings induced calcium phosphate precipitation when immersed in simulated body fluid, suggesting in vivo bioactive behavior. In vitro experiments, using murine osteoblasts, showed that cells rapidly adhere, spread and proliferate over the thin coating surface, while simultaneously generating strong in-plane stresses, as observed on SEM images. Human osteoblasts were seeded at a density of 2500 cells cm(-2) on silicon and titanium HA coated substrates by RAMS. Uncoated glass was used as a reference substrate for further counting of cells. The highest proliferation of human osteoblasts was achieved on HA RAMS-coated titanium substrates. These experiments demonstrate that RAMS is a promising coating technique for biomedical applications.

Bone mineral development has been described to proceed through an amorphous precursor prior to apatite crystallization. However, further analytical approaches are necessary to identify specific markers of amorphous mineral components in bone. Here, we establish an original Fourier transform infrared (FTIR) spectroscopy approach to allow the specific identification of the amorphous and/or crystalline nature of bone mineral. Using a series of standards, our results demonstrate that obtaining the second derivative of the FTIR spectra could reveal a peak specifically corresponding to amorphous calcium phosphate (ACP) at ∼992 cm⁻¹. The intensity of this peak was strongly correlated to ACP content in standard mixtures. The analysis of a variety of bones showed that a clear ACP peak could be identified as a specific marker of the existence of an amorphous mineral component in developing bones. In contrast, the ACP peak was not detected in the mature bones. Moreover, subjecting developing bones to ex vivo crystallization conditions led to a clear reduction of the ACP peak, further substantiating the conversion of amorphous mineral precursor into mature apatite crystals. Analysis of mineralization in osteogenic cell cultures corroborated our observations, showing the presence of ACP as a major transient component in early mineralization, but not in the mature matrix. Additionally, FTIR imaging revealed that ACP was present in areas of matrix development, distributed around the edges of mineralizing nodules. Using an original analytical approach, this work provides strong evidence to support that bone mineral development is initiated by an amorphous precursor prior to apatite crystallization.

Tissue engineering (TE) approaches strive to regenerate or replace an organ or tissue. The successful development and subsequent integration of a TE construct is contingent on a series of in vitro and in vivo events that result in an optimal construct for implantation. Current widely used methods for evaluation of constructs are incapable of providing an accurate compositional assessment without destruction of the construct. In this review, we discuss the contributions of vibrational spectroscopic assessment for evaluation of tissue engineered construct composition, both during development and post-implantation. Fourier transform infrared (FTIR) spectroscopy in the mid and near-infrared range, as well as Raman spectroscopy, are intrinsically label free, can be non-destructive, and provide specific information on the chemical composition of tissues. Overall, we examine the contribution that vibrational spectroscopy via fiber optics and imaging have to tissue engineering approaches.