FT-IR Imaging of Native and Tissue-Engineered Bone and Cartilage

Abstract

Fourier transform Infrared (FT-IR) imaging and microspectroscopy have been extensively applied to the analyses of tissues in health and disease. Spatially resolved mid-infrared data has provided insights into molecular changes that occur in diseases of connective or collagen-based tissues, including osteoarthritis, osteoporosis, osteogenesis imperfecta, osteopetrosis and pathologic calcifications. These techniques have also been used to probe chemical changes associated with load, disuse, and micro-damage in bone, and with degradation and repair in cartilage. This review summarizes the applications of FT-IR microscopy and imaging for analyses of bone and cartilage in healthy and diseased tissues, and illustrates the application of these techniques for the characterization of tissue engineered bone and cartilage.

INTRODUCTION

Vibrational spectroscopy in the mid-infrared (IR) region can provide molecular structure information about mineralized and non-mineralized connective tissues [1–4]. The wavelengths of many IR absorption bands are characteristic of specific types of chemical bonds, and IR spectroscopy is often used to identify compounds. In the infrared, asymmetric vibrations are most intense. Shifts in band intensities and positions are caused by changes in the environment of the molecule, enabling descriptions of variations in these environments. FT-IR microspectroscopy, in which the spectrometer is coupled to a light microscope, first introduced in the 1940s [5], and applied to bone in the 1980s, enabled investigators to examine spectra at discrete points within thin sections of tissues [6]. This point-by-point mapping has been widely applied to the analysis of polymers [7] and to a variety of tissues and individual cells to learn more about spatial variation in tissue and cellular composition [8–11]. It has also been used to identify pathologic inclusions such as foreign matter [12] and unusual soft tissue calcifications [13]. Data collection using point-by-point FT-IR microspectroscopic mapping was relatively slow and tedious, requiring the sample to be moved a specified distance for each point analyzed. The coupling of the FT-IR microscope with an array detector for FT-IR imaging changed the way data was collected and processed, as entire sections of tissue samples could be scanned and analyzed in short periods of time [14,15]. With this technology, the relative amount, molecular nature, distribution and orientation of the components of connective tissues can be evaluated on histological sections at a pixel resolution of 6.25 um and a spatial resolution of 1.3–13 microns depending on the light source, the wavelength being used, and the detector, where the standard thermal globar IR light source has a resolution of ~10 um and a confocal synchrotron unit the better resolution [16]. The data is presented as a “hyperspectral” image where the x and y axes are the spatial coordinates of the tissue section and the z-axis the parameter which is either directly measured (areas or intensities) or calculated (area or intensity ratios). Thus, spatially resolved maps of the molecular environments of tissue components can be obtained.. More recently, integration of this data collection with advanced algorithms has allowed images to be produced and analyzed to provide new molecular insights into spatial variation in tissue properties [17]. In this article we review the application of FT-IR microscopic imaging to bone and cartilage, and indicate how these techniques are being applied to understand material properties of native tissues and tissue engineered constructs.

FT-IR MAPPING OF NATIVE AND TISSUE ENGINEERED BONE

Composition of bone

Bone is a composite material consisting of a mineral phase (hydroxyapatite), collagen, non-collagenous proteins, lipids, and water [18]. The relative amounts of these constituents are variable, depending on animal and tissue age, tissue type, diet, and health status. The functions of these components are reviewed in detail elsewhere [18]. The infrared spectrum of bone (figure 1-a) shows the presence of the major molecular species, phosphate (from the mineral hydroxyapatite), carbonate (from carbonate substitution for hydroxyl and phosphate groups), and amide I, II, and III from the protein constituents of bone (mainly type I collagen). Because water has strong absorbance in the infrared, tissues are generally dehydrated before analyses, thus the water spectrum is not observed. Based on IR analyses of single crystals and model compounds, the origin of the vibrations observed in the IR have been tabulated for the mineral [19–21] and the matrix [22–23] and a number of calculated parameters have been validated for analyses of bone, dentin, and other calcified collagen-based minerals [1]. These parameters (Table I) include the mineral/matrix ratio which is linearly related to the mineral content, or ash weight, of the material being analyzed, the carbonate/phosphate ratio (or carbonate/protein ratio) which is related to the chemically measured carbonate/phosphate content, the crystallinity, proportional to the size of the crystals in their long axis as determined by x-ray diffraction. The type of carbonate substitution [24], the relative acid phosphate content, and a parameter (XLR) indicative of collagen maturity can also be calculated from an individual spectrum. These spectroscopic parameters can be measured in single FT-IR spectrum, in batch files of FT-IR spectra, and from FT-IR spectral images. While spectroscopic parameters obtained from ratios of areas are equivalent when measured either in single spectra or in images, parameters obtained by curve-fitting or deconvolution in single spectra are generally measured based on intensity ratios for images, and provide a related but less sensitive assessment of these values [25].

Figure 1.

Typical infrared spectra of bone (a) and cartilage (b). a) Infrared spectra of healthy adult human cortical bone showing the absorbance bands for vibrations of phosphate, carbonate, and protein amide bonds (AmI, AmII, AmIII). Only the frequencies (wavenumbers, cm⁻¹) used in mid-infrared imaging are shown. b) Infrared spectrum of bovine cartilage showing absorbance bands for vibrations of collagen amide bonds (AmI, AmII, AmIII), amide side chain groups, and proteoglycan sugar ring absorbance (PG).

Table I.

Measuring Bone Properties by FT-IR Spectroscopy

Parameter	Definition	Validation	Variation with Age
Mineral-to-Matrix Ratio	Area of 900–1200cm-1 phosphate band/area of amide I band (1585–1720cm−1)	Linearly related to ash content of synthetic collagen and apatite mixtures	Increases with tissue age
Carbonate-to-phosphate ratio	Area of 850–890 carbonate band to phosphate (or amide I) bands	Related to tissue carbonate content as determined by elemental analyses	Increases with tissue age
Crystallinity	Peak area* or intensity ratio of subbands at 1030 and 1020 cm−1	Related to crystal size in c-axis dimension as determined by x-ray diffraction line broadening	Increases with increasing tissue age
XLR	Peak area * or intensity ratio of subbands at 1660 and 1690 cm−1	Ratio changes with photolysis of collagen cross-links	Increases with increasing tissue age
Acid phosphate Content	Peak area or intensity ratio of subbands at 1106 and 1030 or 960 cm−1	Decreases with tissue maturation	Decreases with increasing tissue age

^*determined by curve-fitting

Preparation of Bone Specimens for FT-IR Microscopic and Imaging Analyses

FT-IR measurement of bone is usually performed in transmittance (i.e., light is passed trhough the section), thus requiring the preparation of thin (2–3um thick) transparent sections. To cut such sections the tissue must be embedded in a hard resin such as polymethyl methacrylate or glycol methacrylate [26] Selection of areas to be analyzed may be based on back scatter electron images, micro-computed tomographic images, or routine histology, or in some cases sections from the entire bone or bone biopsy may be examined. While methacrylate can be solubilized from the sections in methanol, when this is done the thin trabeculae have a tendency to wash away, hence spectra are generally obtained with the resin present, and its spectral contribution subtracted from each individual spectrum. This subtraction is most critical for the poorly mineralized tissues where resin penetration is extensive, and less critical for the mineralized bone into which much less resin penetrates.

Imaging spectrometers consisting of a source, a sample handling unit and an focal plane array detector on an infrared microscope are available from a variety of manufacturers, including BioRad (now Varian), Brucker, and Perkin-Elmer. For use of synchrotron radiation a beam line is required, thus most of the data is obtained with thermal globars. The instruments are usually equipped with a photographic device to photographically capture the area of the tissue being examined, and a stage for moving the specimen to the desired areas. Data can then be collected in 20–50 um × 20–50 um points, or in arrays covering a desired area in the tissue.

Age Dependent Changes in FT-IR Spectral Parameters

In healthy bone, the mineral content increases with both animal and tissue age, and the mineral/matrix ratio similarly increases with tissue age, as indicated by images of this parameter in the developing osteons (figure 2-a). Similarly, the carbonate/phosphate ratio increases, the crystallinity increases (figure 2-b), and the XLR increases (figure 2-c) as the tissue age increases. The distribution of each of these parameters is generally described by a Gaussian distribution of pixels. Similar changes are seen in trabecular bone going from the outer surface to the center of the trabeculae [27]. These age related changes have been reported in FT-IR images and point-by-point microscopic data from rats [28,29], chicks [30], fish and cows [31], dogs [32,33], monkeys [34,35], and mice and humans.

Figure 2.

Infrared images and pixel histograms for osteonal bone in an adult human female femoral neck: (left) mineral/ matrix ratio (M/M), (center) crystallinity (XST), and (right) collagen maturity (XLR). The same area of bone is shown in each figure; note that the crystallinity parameter occupies a lesser area than the collagen maturity parameter, as there is unmineralized matrix (osteoid) in the sample. The mean ± SD for the pixels in this image are 4.1± 0.45 (M/M), 1.30± 0.08 (XST), 2.1 ± 0.6 (XLR).. These pixel histograms can be combined to provide statistics for multiple samples.

The mouse data includes analyses of the effects of gene ablation and overexpression on bone properties. The models [35–47] analyzed by point-by-point FT-IR microspectroscopy and Imaging are listed in Table II. Information on wildtype mice from these studies, while varying in absolute values depending on the background strain, confirms general trends noted in human and other animal models. Specifically in a variety of strains of mice, looking at trabecular or cortical bone, with age there is an increase in mineral content, crystallinity, carbonate/phosphate ratio and collagen maturity [48]. The mutations in many cases have been linked to disease conditions, as noted in the table, and data from the mice can be used to learn more about these disease states.

Table II.

Use of IR Imaging (FTIRI) and Microspectroscopy (FTIRM) to Characterize Changes in Bones of Naturally Occurring Mutant (Mu), Knockout (KO) and Transgenic (TG) Mice

Protein	Model	Model for Human Condition	Method	Reference
Type X collagen	KO	Spondolyloepiphyseal dysplasia	FTIRM	35
Osteocalcin	KO		FTIRM	36
PHEX	Mu	Hypophosphatemic Rickets	FTIRM	37
Type I collagen	Mu	Osteogenesis Imperfecta	FTIRM, FTIRI	38,39
Osteopontin	KO		FTIRM	40
TGF-beta	KO		FTIRI	41
Osteonectin	KO	Vanishing bone disease	FTIRM, FTIRI	25
Vitamin D receptor	TG	Osteoporosis	FTIRM	42
Alkaline Phosphatase	KO	Rickets	FTIRM	43
IGF-binding protein 5	TG		FTIRI	44
Dentin matrix protein 1	KO		FTIRI	45
Biglycan	KO	Osteopenia	FTIRM	46
Beta-galactosidase	KO	Fabry’s Disease	FTIRI	47

In humans, FT-IR spectroscopic imaging and microspectroscopy have been used to characterize disease- related changes (Table III [49–54]). In diseased bones, FT-IR imaging data reveal striking differences in mineral and matrix properties [1,55]. For example, in biopsies of bone from patients with osteoporosis there are increases in crystallinity and in collagen maturity relative to healthy bone. Moreover, while healthy individuals show a wide range of values with maximal values in the center of the trabeculae, comparing the extent of change from the bone surface to the highest value for each of the FTIRI parameters in patients with low and high turnover osteoporosis show minimal change relative to those noted in normal trabeculae (figure 3). Cases of osteopetrosis show increased mineral density with decreased crystal size [54], while spectral imaging and microscopy data from bones of animal models with osteogenesis imperfecta indicate decreases in crystal size, altered crystal acid phosphate content and mineral density [38,39].

Table III.

IR Spectroscopic Imaging (FTIRI) and Microspectroscopic (FTIRM) Studies of Human Diseased Bone

Disease	Method	Reference
Osteoporosis	FTIRM FTIRI	49-52
Osteomalacia	FTIRI	53
Osteopetrosis	FTIRI	54

Figure 3.

Mineral properties in osteoporotic bone show different patterns when the maximum value in the center of the trabeculae is compared to the lowest value (generally at the edge). This figure, summarizing data described in Boskey et al [50], shows the mean (±SD) % change in iliac crest biopsies of both female and male patients with high-(HTOP) and low-(LTOP)- turnover osteoporosis as contrasted with age-matched normal controls for mineral/matrix ratio (top left), carbonate/phosphate ratio (top right), crystallinity (bottom left), and acid phosphate content (bottom right).

An important feature of bone is its ability to support load, and adapt to deformations created during normal exposure to stress and strain. This exposure usually causes damage to the tissue, which in the health individual is repaired. A recent study from Burr’s group used synchrotron radiation-based spectroscopy to study canine vertebrae that had been stained with basic fuschin to identify micro-damage. The bone adjacent to the damaged areas had comparable mineral content (mineral/matrix ratio), comparable crystallinity, but decreased carbonate/phosphate ratios, increased acid phosphate content, and increased collagen maturity [56].

FT-IR Imaging of tissue engineered bone structures

Knowledge of bone compositional variation facilitates the application of infrared imaging to the study of tissue engineered products. Implant scaffold compositions are often evaluated as homogenized samples by FT-IR [57–60] and the homogenized mineralized matrix expressed by human stem cells of different origin when plated on these substrates has been shown to be “bone-like” by FT-IR [61]. FT-IR has also been used to characterize the mineral formed on different materials [62] but the spatial distribution of these materials is not often addressed. Yet FT-IR images offer the capability for demonstrating that the tissue engineered construct and the tissue it forms mimics the type of bone it is designed to replace. For example, a comparison of images of materials with different surface roughness seeded with rat calvarial osteoblasts demonstrated that only those cells treated with BMP-2 and plated on a rough surface formed a bone-like matrix, and that the mineral was closely associated with that matrix, while other surfaces did not support the deposition of a bone-like mineral and matrix (figure 4) [63]. The advantages of using FT-IR microspectroscopy for analyses of bone and dental implant materials is reviewed elsewhere [64] and is illustrated by studies of the changes in the spatial distribution of components of dental composites mapped by FT-IR microspectroscopy [65], and the contact of injected bone cement with underlying bone tissues [66]. Another recent study used FT-IR Imaging to localize where hydroxyapatite formed on bioglass templates when they were placed in saline [67].

Figure 4.

Qualitative images of matrix (a) and mineral(b) distribution on surface roughened titanium implants with bmp-2 (20 ng/ml) treated fetal rat calvarial osteoblasts. The cells were cultured on titanium implant materials (PT, pretreated; SLA-course grit blasted and acid etched, and TPS – Ti plasma sprayed). Only SLA and TPS showed close association of mineral and matrix with the greatest yield on the TPS plates. Reprinted with permission from B.D. Boyan et al [61].

FT-IR Imaging of Native and Tissue-Engineered Cartilage

Composition of Cartilage

Articular cartilage is a specialized connective tissue that lines the ends of the bones and provides a smooth, frictionless surface for weight-bearing during joint loading and motion [68]. The framework of cartilage is comprised of a network of type II collagen, a triple helical fibrillar protein, that is associated with other collagens, non-collagenous proteins, and proteoglycan (PG) components [69]. Aggrecan, the primary cartilage PG, contains covalently attached side chains of the glycosaminoglycans chondroitin sulfate and keratan sulfate linked to a protein core. Cartilage displays zonal heterogeneity with respect to its molecular composition and structure. The superficial surface layer of cartilage has densely packed type II collagen fibrils that are oriented parallel to the surface, a small amount of PG and a high water content [70;71] The middle transitional zone has a lower water content, a higher concentration of PG, and a lower concentration of collagen fibrils that are randomly oriented. The deep zone contains radially oriented collagen fibrils, has the highest concentration of PG, and lowest percentage of water compared to the other zones. Pathological changes in the macromolecular structure and content of the primary cartilage components are hallmarks of cartilage degeneration [72–74]. Such changes are characteristic of osteoarthritis (OA), a progressively disabling musculoskeletal disease that affects millions of individuals in the United States [75], with prevalence directly correlated to age. When articular cartilage is damaged as a result of OA or injury, the tissue has limited capacity for repair and regeneration, and as such, advanced stages of OA are typically characterized by extensive cartilage damage that can eventually lead to joint arthroplasty.

There has been extensive research into techniques to repair damaged cartilage, including the use of microfracture techniques [76–80], autograft and allograft transplants [81–85] and autologous chondrocyte implantation procedures [86–90]. Emerging tissue engineering techniques include bioreactor growth of cartilagenous tissue [91–94] and the use of three-dimensional scaffolds, such as hydrogels, in combination with cell seeding [95–98]. In general, the goal of these tissue engineering strategies is to produce a cartilage-like tissue that has a normal organization and molecular structure, and can sustain load. Since cartilage is spatially heterogeneous, quantification of both native articular cartilage and engineered tissues at the macroscopic, microscopic and molecular levels is critical.

FT-IR Parameters Utilized to Monitor Normal and Degenerative Cartilage

FT-IR spectroscopy is a powerful tool to assess degenerative cartilage structure. Although there have been several conventional FT-IR studies on collagen molecular changes and hydration [99–101], and on extracted proteoglycans from various tissues [102–104], until very recently, infrared spectroscopic studies on articular cartilage were non-existent. The first IR studies on cartilage were imaging spectroscopy (FT-IRIS) studies from our group [105] followed closely by Potter et al [106]. In our initial studies, FT-IR imaging data acquired through the superficial, middle, and deep zones of thin, histological sections of bovine articular cartilage showed variation in the absorbance bands that arise from the primary non-aqueous components of cartilage, collagen, and proteoglycans (primarily aggrecan), and thus reflected the differences in quantity of these specific components (figure 1-b). In addition, spectra of mixtures of model compounds, which had varying proportions of type II collagen and aggrecan, were analyzed to identify spectral markers that could be used to quantitatively analyze these components in cartilage. It was found that the integrated area of the amide I absorbance (1590 – 1720 cm⁻¹) correlated with collagen concentration, and the ratio of the integrated area of the PG sugar ring C-O absorbance (985–1140 cm⁻¹) to the amide I absorbance area correlated to the quantity of PG. The quantification of PG was further validated in studies of tissue-engineered cartilage where biochemical assessment of glycosaminoglycan (GAG) content correlated with the FT-IR-derived spectral parameter [107]. Polarization experiments were also performed to determine the spatial distribution of the collagen orientation in articular cartilage, and the ratio of the collagen amide I/amide II absorbance was utilized as a spectral marker of collagen orientation [105;108].

Another spectral parameter that has been utilized to assess normal and degenerative cartilage structure is the 1338 cm⁻¹ absorbance, a feature that arises from collagen CH₂ side chain vibrations. In earlier studies, this absorbance band was shown to decrease in intensity as collagen denatures [109]. Initial cartilage studies found that the integrated area of this band (ratioed to the amide II collagen band) was reduced in human osteoarthritic tissues compared to normal tissues [4]. Further work to elucidate the molecular origin of this change confirmed that the reduction in the 1338 cm⁻¹ absorbance was linked to collagen degradation [110]. Figure 5 shows an example of collagen and PG distribution, collagen integrity (based on the 1338 cm⁻¹ absorbance), and collagen orientation in articular cartilage from a rabbit surgical model of osteoarthritis. Differences in these parameters in the OA compared to the normal control tissue reflect compositional and structural changes that arise at an early stage of disease, the most obvious being loss of PG (~35% decrease) and changes in collagen orientation as evidenced by an almost complete loss of deep zone collagen fibrils orientated perpendicular to the articular surface. Collagen integrity was slightly higher in the OA tissues at this early stage ( ~ 20 % increase), a finding consistent with increased collagen synthesis early in the development of OA, possibly indicative of an active repair response.

Figure 5.

FT-IR images of collagen content (amide I area) (i), PG content (PG sugar ring C-O absorbance (985–1140 cm⁻¹) normalized to amide I area) (ii), collagen integrity (1338 cm⁻¹ normalized to amide II area ratio) (iii), and collagen orientation (amide I/amide II ratio in polarized images) (iv) from the articular surface (superficial zone, SZ) down to the tidemark (lower boundary of deep zone, DZ), acquired from the medial femoral condyle of an OA (right panel) and non-surgical control (left panel) rabbit at two weeks post-surgery. The color scale indicates the pixel values for collagen and PG content, and collagen integrity index, where red and dark blue represent highest and lowest values, respectively. For the polarized data, the boundaries on the color bar indicate the three collagen fibril orientation categories with respect to the articular surface, corresponding to amide I / amide II peak area ratios ≥ 2.7 (parallel fibrils), between 2.7 and 1.7 (random fibrils), and ≤ 1.7 (perpendicular fibrils), respectively. A marked reduction in PG content is noted in the OA compared to Control cartilage.

Taken together, these parameters provide a framework in which complex pathological changes in articular cartilage and engineered tissues can be assessed by FT-IR imaging spectroscopy. Recent studies that have applied FT-IR imaging to normal and degenerative cartilage include work that addresses the contribution of collagen and PG composition and structure to the mechanical properties of cartilage in compression [111], evaluation of molecular contributions to stresses in the chondrocyte microenvironment [112] and imaging of human osteoarthritic cartilage[113,114].

Preparation of Cartilage Specimens for FT-IR Microscopic and Imaging Analyses

Soft tissues such as cartilage may be cryosectioned without embedding for FT-IR imaging analysis, but typically are paraffin-embedded to ensure optimal preservation of the tissue structure. If bone is attached to the cartilaginous tissue, samples are decalcified prior to paraffin processing. The processing sequence includes fixation/dehydration in ethanol or formalin with cetyl pyridinium choloride (CPC) added to preserve proteoglycans, decalcification with EDTA, and embedding in paraffin. For FT-IR imaging data acquisition in transmittance, sections 5 to 10 microns in thickness are cut onto an infrared-transmissive substance such as a BaF₂ window. It is also possible to sample the tissues in reflectance mode using a reflective substrate such as MirrIR low-e microscope slides (Kevley Technologies, Chesterl and, OH).

FT-IR Imaging of Tissue-Engineered Cartilage

The composition of tissue-engineered cartilage can be considered to be predictive of its in vivo performance. Therefore, the ability to monitor changes in the tissue matrix components during engineered cartilage development, including collagen, PG, and water, would be a key element in determining whether a protocol is viable. A previous FT-IR imaging study evaluated PG content in cartilage grown from avian chondrocytes within a hollow fiber bioreactor (HFBR) system, where a gradient of PG content was found, with the highest content in the tissue center near the nutritive fibers and the lowest near the interior surface of the HFBR [107]. Recent studies have utilized FT-IR imaging to assess tissue matrix changes during development of mammalian cartilage in a custom-designed single-fiber HFBR [115]. Immature bovine chondrocytes were inoculated (100 million/0.7mL) into a HFBR and cultured for a period of 8 weeks. Figure 6 shows FT-IR images based on collagen and PG content obtained from histological sections of the HFBR cartilage grown for 2, 4 and 8 weeks in culture, and comparisons made to type II collagen immunohistochemistry and PG distribution determined from alcian blue histology. The FT-IR imaging data show qualitative temporal changes in collagen quantity, increased at 4 and 8 weeks, and PG distribution. The highest amount of PG was near the fiber (center) at 2 and 4 weeks, but was nearer the tissue edges at 8 weeks. Based on the FT-IR imaging data, it is likely that both increased collagen and redistribution of PG around the tissue periphery contribute to improved mechanical properties noted at 8 weeks of growth.

Figure 6.

Images from cross-sections of engineered mammalian cartilage grown in a hollow fiber bioreactor for 2, 4 and 8 weeks. Data include immunohistochemical type II collagen distribution, histological PG distribution (Alcian Blue stain), and FT-IR-determined collagen (amide I area) and proteoglycan ((PG sugar ring C-O absorbance (985–1140 cm⁻¹) normalized to amide I area) distribution. Qualitatively, increased collagen and a change in the distribution of PG can be observed at 4 and 8 weeks compared to 2 weeks of growth.

FT-IR Imaging of Cartilage Repair Tissue

Many studies have utilized animal models to assess therapeutic modalities for cartilage repair [116–118]. In general, such studies require the harvested repair tissue to be subjected to several different analyses to obtain a full picture of the quality of the repair tissue formed. These would include histology for PG evaluation, immunohistochemistry for type I and type II collagen, and polarized light microscopy to assess collagen fibril organization. The use of FT-IR imaging to assess harvested repair tissue enables one unstained tissue section to be evaluated for several features simultaneously, including collagen organization. One recent study investigated repair tissue formed in a rabbit osteochondral defect treated with TP508, a synthetic peptide that contains a receptor-binding domain of thrombin [119]. TP508 binds to high affinity non-proteolytically activated thrombin receptors and modulates thrombin-mediated chemotaxis and mitogenesis. The study investigated whether application of this peptide in a sustained release delivery vehicle to a full-thickness osteochondral defect would result in neochondrogenesis in an articular cartilage defect site. FT-IR imaging data obtained from histological sections of the defect site and adjacent cartilage at 6 weeks post-treatment showed that there was new tissue formation in the defect (figure 7). In the data shown, the tissue filled the defect site. Compared to the adjacent cartilage, the repair tissue had a lower collagen content (~30% reduction), but also displayed regions where the PG content was similar to that in the native tissue. In addition, there was some orientation of the collagen fibrils in the repair tissue as evidenced by the parallel layer of fibrils observed at the tissue surface.

Figure 7.

FT-IR images of rabbit osteochondral defect at 6 weeks post-repair with TP 508 protein. The defect repair cartilage has lower collagen content (amide I area) compared to the adjacent articular cartilage, but also has regions with PG content ((PG sugar ring C-O absorbance (985–1140 cm⁻¹)) of a similar magnitude to that in the native cartilage. There is some orientation present in the repair cartilage, as evidenced by the layer of fibrils parallel to the surface. For the orientation data, the boundaries on the color bar indicate the three collagen fibril orientation categories with respect to the articular surface, corresponding to amide I / amide II peak area ratios ≥ 2.7 (parallel fibrils), between 2.7 and 1.7 (random fibrils), and ≤ 1.7 (perpendicular fibrils), respectively.

FT-IR Imaging of Cartilage Scaffolds

A final illustration of the application of FT-IR imaging to cartilage tissue engineering involves the assessment of a tissue-engineered porous hydrogel scaffold for filling cartilage defects. As with other tissue engineering strategies, the ultimate goal is for the synthetic material to recover the initial composition and function of cartilage and to integrate into adjacent normal cartilage. In our recent study, a combination of non-degradable and degradable polymers, polyvinyl-alcohol (PVA) and poly (lactic co-glycolic acid) (PLGA), were evaluated. FT-IR imaging spectroscopy was utilized to assess PLGA degradation-induced changes in hydrogel structure and self-mobilized chondrocyte migration via porous channels [120]. Infrared absorbance bands unique to PLGA (1756 cm⁻¹), and PVA (1092 cm⁻¹), were utilized to monitor the distribution of these components within thin sections of the hydrogel (figure 8-a). The morphology of the hydrogels was altered with increasing quantity of PLGA, a feature that can be utilized to optimize the hydrogel structure. Factor Analysis, a multivariate technique recently applied to IR imaging determination of protein structure and distribution in skin [121], was utilized here to evaluate the matrix formed by infiltration of chondrocytes into the hydrogel (figure 8-b). The presence of matrix formation is clearly distinguished from the hydrogel, supporting the utility of such analyses for assessment of tissue integration into cartilage scaffolds.

Figure 8.

(i). FT-IR and SEM images of PVA/PLGA hydrogels comprised of 10, 25, or 50% PLGA. Infrared absorbance peaks unique to PLGA and PVA are at 1756 cm⁻¹ and 1092 cm⁻¹, respectively, and the ratio of the peaks was used to monitor the quantity and distribution of the PLGA within the hydrogel. The hydrogel morphology changed with increaseing PLGA content.

(ii). Extracellular matrix (ECM) produced by chondrocytic infiltration into the hydrogel was evident on Alcian Blue stained hydrogel sections, and was monitored by FT-IR imaging of the protein Amide I absorbance.

CONCLUSIONS

In conclusion, the data presented demonstrate the utility and flexibility of infrared imaging for investigations into the micro and molecular structure of bone and cartilage and related engineered constructs. The ability to utilize one unstained histological or thick section to obtain a multitude of information on the content and distribution of native and engineered molecular components makes this an exceedingly powerful technique. Although IR imaging is currently limited to assessment of harvested tissues, the development of related techniques that can be utilized in a minimally-invasive environment, such as with a fiber optic probe [122], are emerging.