Nancy P. Camacho

FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage

Articular cartilage, a connective tissue that provides resistance to compressive forces during joint movements, has not been examined in detail by conventional Fourier transform infrared (FTIR) spectroscopy, microspectroscopy (FTIRM), or imaging (FTIRI). The current study reports FTIRM and FTIRI analyses of normal bovine cartilage and identifies the specific molecular components of cartilage that contribute to its IR spectrum. FTIRM data acquired through the superficial, middle, and deep zones of thin sections of bovine articular cartilage showed a variation in intensities of the absorbance bands that arise from the primary nonaqueous components of cartilage, collagen, and proteoglycan (primarily aggrecan) and thus reflected the differences in quantity of these specific components. The 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. Collagen and aggrecan were then imaged by FTIRI based on markers found in the model compounds. Polarization experiments were also performed to determine the spatial distribution of the collagen orientation in the different zones of cartilage. This study provides a framework in which complex pathological changes in this heterogeneous tissue can be assessed by IR microscopic imaging.

Von Kossa Staining Alone Is Not Sufficient to Confirm that Mineralization In Vitro Represents Bone Formation

Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, electron microscopic analysis (EM), X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR) are among the most common. In this study, we utilized three of these methods to compare the mineralization of cultured fetal rat calvarial cells (FRC) and the osteoblast cell lines 2T3 and MC3T3-E1 with the in vivo mineral of rat calvarial bone. The cells were cultured with or without ascorbic acid (100 µg/ml) and β-glycerophosphate (2.5, 5, or 10 mM βGP), and harvested between 16 and 21 days (FRC cells and 2T3 cells) or at 30 days of culture (MC3T3-E1 cells). In the FRC cultures, maximal von Kossa staining was observed with 2.5 and 5 mM βGP in the presence of 100 µg/ml ascorbate. FRC cells also showed some von Kossa staining when cultured with βGP alone. In contrast, maximal von Kossa staining for MC3T3-E1 cells was observed with 10 mM βGP. Only the cultures of MC3T3-E1 cells that received both ascorbate and βGP produced von Kossa positive structures. The 2T3 cultures produced von Kossa positive staining only upon treatment with ascorbic acid and βGP, which was greatly accelerated by bone morphogenic protein-2 (BMP-2). FTIR was performed on the mineral and matrix generated in FRC, MC3T3, and 2T3 cultures, and the results were compared with spectra derived from 16-day-old rat calvaria. The mineral-to-matrix ratios calculated from FTIR spectra for rat calvaria ranged from 2.97 to 7.44. FRC cells made a bonelike, poorly crystalline apatite, and, with increasing βGP, there was a statistically significant (P ≤ 0.02) dose-dependent increase in the mineral-to-matrix ratio (0.56 ± 0.16, 1.00 ± 0.32, and 2.46 ± 0.76, for 2.5, 5, and 10 mM βGP, respectively). The mean carbonate-to-phosphate ratios of the FRC cultures were 0.015, 0.012, and 0.008, in order of increasing βGP concentration, compared with rat calvaria values of 0.009–0.017. The 2T3 cells treated with BMP-2 also made bonelike crystals, similar to those observed in FRC cultures. In contrast, the cultures of von Kossa positive MC3T3-E1 cells did not display a significant amount of mineral (maximum mineral-to-matrix ratio was 0.4). Thus, although the von Kossa stainings of FRC, 2T3, and MC3T3-E1 were very similar, FTIR analysis indicated that calcium phosphate mineral was not present in the MC3T3 cultures. By EM, the mineral in FRC cell cultures and 2T3 cultures was generally associated with collagen, whereas rare or sparse dystrophic mineralization of unknown chemical origin was evident in the MC3T3-E1 cultures. These studies demonstrate that von Kossa staining alone is not appropriate for the identification and quantitation of bonelike mineral and, hence, other techniques such as X-ray diffraction, EM, or FTIR should be utilized to verify the presence and quality of calcium phosphate phases.

Enzyme Replacement Therapy for Murine Hypophosphatasia

Introduction: Hypophosphatasia (HPP) is the inborn error of metabolism that features rickets or osteomalacia caused by loss‐of‐function mutation(s) within the gene that encodes the tissue‐nonspecific isozyme of alkaline phosphatase (TNALP). Consequently, natural substrates for this ectoenzyme accumulate extracellulary including inorganic pyrophosphate (PPi), an inhibitor of mineralization, and pyridoxal 5′‐phosphate (PLP), a co‐factor form of vitamin B6. Babies with the infantile form of HPP often die with severe rickets and sometimes hypercalcemia and vitamin B6‐dependent seizures. There is no established medical treatment.

Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy

Scanning small angle X-ray scattering (scanning SAXS) and Fourier-transform infrared microspectroscopy (FT-IRM) have previously been utilized independently to characterize the structural properties of bone in an anatomical position-resolved fashion. Whereas SAXS provides a direct measure of the physical characteristics of apatitic crystals, FT-IRM assesses structure of both mineral and organic matrix at the molecular level. In the present study both methods were applied to examine the same developing bone tissue from the L-4 vertebra of a 14-month-old (accidental death). A 200-microm-thick section was processed for examination by scanning electron microscopy and SAXS. Spectra were collected at 200 microm spatial resolution at specific locations in cortical and cancellous bone. Parameters determined included total SAXS intensity, crystal thickness (T), and degree and direction of predominant crystal orientation. For FT-IRM analysis, a section 4 microm thick was cut longitudinally from the top of the sample. Spectra of regions 100 x 100 microm2 were acquired from the same locations as the SAXS spectra. Integrated areas of the phosphate nu(1,3) collagen amide I, and carbonate nu2 absorbances, were calculated to obtain mineral: matrix and carbonate:mineral ratios. The relative quantities of types A, B, and labile carbonate (substituted for apatite hydroxyl, phosphate, and surface positions, respectively) were also evaluated. Polarized FT-IRM data were collected to determine molecular orientation of the apatite and collagen components. The results of this study show that the information obtained from the two techniques is complementary. Both SAXS and FT-IRM data revealed that the crystals were significantly larger in the cancellous region compared with the cortical region, that mineralization was greater in the cortex, and that the crystals were oriented to a larger degree in the cancellous compared with the cortical bone. The scanning SAXS measure of crystal thickness was significantly correlated to the FT-IRM measures of crystallinity, type A carbonate substitution, and crystal orientation. In conclusion, it was found that the combined use of SAXS and FT-IRM provides valuable, unique information on structural changes in bone at both the microstructural and ultrastructural level. Although each method can be used individually, the combination of techniques provides additional insights into the mechanism of bone crystal maturation.

The Material Basis for Reduced Mechanical Properties in oim Mice Bones

Osteogenesis imperfecta (OI), a heritable disease caused by molecular defects in type I collagen, is characterized by skeletal deformities and brittle bones. The heterozygous and homozygous oim mice (oim/+ and oim/oim) exhibit mild and severe OI phenotypes, respectively, serving as controlled animal models of this disease. In the current study, bone geometry, mechanics, and material properties of 1‐year‐old mice were evaluated to determine factors that influence the severity of phenotype in OI. The oim/oim mice exhibited significantly smaller body size, femur length, and moment of area compared with oim/+ and wild‐type (+/+) controls. The oim/oim femur mechanical properties of failure torque and stiffness were 40% and 30%, respectively, of the +/+ values, and 53% and 36% of the oim/+ values. Collagen content was reduced by 20% in the oim/oim compared with +/+ bone and tended to be intermediate to these values for the oim/+. Mineral content was not significantly different between the oim/oim and +/+ bones. However, the oim/oim ash content was significantly reduced compared with that of the oim/+. Mineral carbonate content was reduced by 23% in the oim/oim bone compared with controls. Mineral crystallinity was reduced in the oim/oim and oim/+ bone compared with controls. Overall, for the majority of parameters examined (geometrical, mechanical, and material), the oim/+ values were intermediate to those of the oim/oim and +/+, a finding that parallels the phenotypes of the mice. This provides evidence that specific material properties, such as mineral crystallinity and collagen content, are indicative and possibly predictive of bone fragility in this mouse model, and by analogy in human OI.

Potential Role of Bone Morphogenetic Proteins in Fracture Healing

Since their discovery, bone morphogenetic proteins have held the promise for use in various orthopaedic diseases. One of the largest areas of likely application is the area of fracture repair. Although millions of fractures occur annually and the majority heal satisfactorily, 5% to 10% go on to delayed union or nonunion. Bone morphogenetic proteins may be able to improve bony healing in these conditions and perhaps enhance the healing of fractures that otherwise heal satisfactorily. This study examines the pre-clinical data to support the concept of enhancing bony healing and discusses the preliminary data from clinical trials using bone morphogenetic proteins to augment bony healing. Although the potential clinical uses of bone morphogenetic proteins in fracture healing remain significant, this potential has yet to be realized.

Sustained Osteomalacia of Long Bones Despite Major Improvement in Other Hypophosphatasia- Related Mineral Deficits in Tissue Nonspecific Alkaline Phosphatase/Nucleotide Pyrophosphatase Phosphodiesterase 1 Double-Deficient Mice

We have shown previously that the hypomineralization defects of the calvarium and vertebrae of tissue nonspecific alkaline phosphatase (TNAP)-deficient (Akp2-/-) hypophosphatasia mice are rescued by simultaneous deletion of the Enpp1 gene, which encodes nucleotide pyrophosphatase phosphodiesterase 1 (NPP1). Conversely, the hyperossification in the vertebral apophyses typical of Enpp1-/- mice is corrected in [Akp2-/-; Enpp1-/-] double-knockout mice. Here we have examined the appendicular skeletons of Akp2-/-, Enpp1-/-, and [Akp2-/-; Enpp1-/-] mice to ascertain the degree of rescue afforded at these skeletal sites. Alizarin red and Alcian blue whole mount analysis of the skeletons from wild-type, Akp2-/-, and [Akp2-/-; Enpp1-/-] mice revealed that although calvarium and vertebrae of double-knockout mice were normalized with respect to mineral deposition, the femur and tibia were not. Using several different methodologies, we found reduced mineralization not only in Akp2-/- but also in Enpp1-/- and [Akp2-/-; Enpp1-/-] femurs and tibias. Analysis of calvarial- and bone marrow-derived osteoblasts for mineralized nodule formation in vitro showed increased mineral deposition by Enpp1-/- calvarial osteoblasts but decreased mineral deposition by Enpp1-/- long bone marrow-derived osteoblasts in comparison to wild-type cells. Thus, the osteomalacia of Akp2-/- mice and the hypomineralized phenotype of the long bones of Enpp1-/- mice are not rescued by simultaneous deletion of TNAP and NPP1 functions.

Novel assessment of bone using time-resolved transcutaneous Raman spectroscopy

With fragility fractures increasing as the population ages, there is a need for improved means to estimate risk of fracture. We recorded Raman spectra of both the mineral and organic phases of bone transcutaneously, a technology with potential to enhance bone quality and fracture risk assessment.

Fourier transform infrared spectral analysis of degenerative cartilage: an infrared fiber optic probe and imaging study.

A preliminary investigation into the diagnostic potential of an infrared fiber optic probe (IFOP) for evaluating degenerative human articular cartilage is described. Twelve arthritic human tibial plateaus obtained during arthroplasty were analyzed using the IFOP. Infrared spectra were obtained from IFOP contact with articular surface sites visually graded normal or degraded (Collins Scale grade 1 and grade 3, respectively). Comparisons of infrared spectral parameters (peak heights and areas) were made to elucidate spectral indicators of surface degeneration. IFOP spectral analysis revealed subtle but consistent changes between grades 1 and 3 sites. Infrared absorbance bands arising from type II collagen were observed to change with degradation. More degraded tissues exhibited increased amide II (1590–1480 cm−1)/1338 cm−1 area ratio (p = 0.034) and decreased 1238/1227 cm−1 peak ratio (p = 0.017); similar changes were seen with Fourier transform infrared imaging spectroscopy (FT-IRIS) analysis. Grades 1 and 3 cartilage showed consistent spectral differences in the amide II, III, and 1338 cm−1 regions that are likely related to type II collagen degradation that accompanies cartilage degeneration. These results suggest that it may be possible to monitor subtle changes related to early cartilage degeneration, allowing for IFOP use during arthroscopy for in situ determination of cartilage integrity.

Mineral Changes in a Mouse Model of Osteogenesis Imperfecta Detected by Fourier Transform Infrared Microscopy

Osteogenesis imperfecta (OI) is a heritable disease characterized by skeletal deformities and brittle bones. In the current study, the nature of the mineral in long bones of a mouse model of OI (oim/oim, a mutant which produces an alpha 1(I) collagen homotrimer) was examined by Fourier transform infrared microscopy. The mineral:matrix ratio of oim/oim cortical bone was greater than that of the heterozygous oim/+ and of the normal +/+ bones, probably as a result of reduced collagen content. The molecular environments of the apatitic phosphates differed among the oim/oim and the oim/+ and the +/+ bones. This was attributable to several factors, including dissimilar mineral-matrix interactions and differences in the chemical composition of the mineral. It was concluded from these data that the defective collagen matrix leads to abnormal mineral formation at the molecular level and thus results in tissues with reduced mechanical properties.