Non‐invasive identification of proteoglycans and chondrocyte differentiation state by Raman microspectroscopy

Proteoglycans (PGs) are crucial extracellular matrix (ECM) components that are present in all tissues and organs. Pathological remodeling of these macromolecules can lead to severe diseases such as osteoarthritis or rheumatoid arthritis. To date, PG‐associated ECM alterations are routinely diagnosed by invasive analytical methods. Here, we employed Raman microspectroscopy, a laser‐based, marker‐free and non‐destructive technique that allows the generation of spectra with peaks originating from molecular vibrations within a sample, to identify specific Raman bands that can be assigned to PGs within human and porcine cartilage samples and chondrocytes. Based on the non‐invasively acquired Raman spectra, we further revealed that a prolonged in vitro culture leads to phenotypic alterations of chondrocytes, resulting in a decreased PG synthesis rate and loss of lipid contents. Our results are the first to demonstrate the applicability of Raman microspectroscopy as an analytical and potential diagnostic tool for non‐invasive cell and tissue state monitoring of cartilage in biomedical research. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects of ionizing radiations on human costal cartilage and exploration of the procedures to protect the tissue from radiation damage

Synoposis The chemical changes produced following⁶⁰Co--irradiation of human costal cartilage have been monitored using critical electrolyte concentration (CEC) measurements utilizing Alcian Blue-magnesium chloride and Toluidine Blue-sodium chloride systems. The decrease in the CEC with radiation can be related to the decrease in hexose and hexosamine contents of the cartilage. Such changes arise as a result of degradation of the glycosaminoglycan component of the tissue. The state of the collagen fibres after irradiation has been examined using the Van Giesson stain and by biochemical assay of the hydroxyproline moeity. Energy transfer methods have been utilized to protect the glycosaminoglycan component of the cartilage from radiation damage.     

Detection and imaging of lipids of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> based on confocal Raman microspectroscopy

In this study, confocal Raman microspectroscopy was used to detect lipids in microalgae rapidly and non-destructively. Microalgae cells were cultured under nitrogen deficiency. The accumulation of lipids in Scenedesmus obliquus was observed by Nile red staining, and the total amount of lipids accumulated in the cells was measured by gravimetric method. The signals from different microalgae cells were collected by confocal Raman microspectroscopy to establish a prediction model of intracellular lipid content, and surface scanning signals for drawing pseudo color images of lipids distribution. The images can show the location of pyrenoid and lipid accumulation in cells. Analyze Raman spectrum data and build PCA-LDA model using four different bands (full bands, pigments, lipids, and mixed features). Models of full bands or pigment characteristic bands were capable of identifying S. obliquus cells under different nitrogen stress culture time. The prediction accuracy of model of lipid characteristic bands is relatively low. The correlation between the fatty acid content measured by the gravimetric method and the integral Raman intensity of the oil characteristic peak (1445 cm^?1) measured by Raman spectroscopy was analyzed. There was significant correlation (R ² = 0.83), which means that Raman spectroscopy is applicable to semi-quantitative detection of microalgal lipid content.     

FT-IR Microspectroscopy of Rat Ear Cartilage

Rat ear cartilage was studied using Fourier transform-infrared (FT-IR) microspectroscopy to expand the current knowledge which has been established for relatively more complex cartilage types. Comparison of the FT-IR spectra of the ear cartilage extracellular matrix (ECM) with published data on articular cartilage, collagen II and 4-chondroitin-sulfate standards, as well as of collagen type I-containing dermal collagen bundles (CBs) with collagen type II, was performed. Ear cartilage ECM glycosaminoglycans (GAGs) were revealed histochemically and as a reduction in ECM FT-IR spectral band heights (1140–820 cm^-1) after testicular hyaluronidase digestion. Although ear cartilage is less complex than articular cartilage, it contains ECM components with a macromolecular orientation as revealed using polarization microscopy. Collagen type II and GAGs, which play a structural role in the stereo-arrangement of the ear cartilage, contribute to its FT-IR spectrum. Similar to articular cartilage, ear cartilage showed that proteoglycans add a contribution to the collagen amide I spectral region, a finding that does not recommend this region for collagen type II quantification purposes. In contrast to articular cartilage, the symmetric stretching vibration of –SO₃^- groups at 1064 cm^-1 appeared under-represented in the FT-IR spectral profile of ear cartilage. Because the band corresponding to the asymmetric stretching vibration of –SO₃^- groups (1236–1225 cm^-1) overlapped with that of amide III bands, it is not recommended for evaluation of the –SO₃^- contribution to the FT-IR spectrum of the ear cartilage ECM. Instead, a peak (or shoulder) at 1027–1016 cm^-1 could be better considered for this intent. Amide I/amide II ratios as calculated here and data from the literature suggest that protein complexes of the ear cartilage ECM are arranged with a lower helical conformation compared to pure collagen II. The present results could motivate further studies on this tissue under pathological or experimental states involving ear cartilage.     

The effect of mechanical load on integrin subunits α5 and β1 in chondrocytes from mature and immature cartilage explants

Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits 5 and 1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits 5 and 1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit 5 in immature and mature cartilage but not in the integrin subunit 1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin 51 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.This work was supported by NIH grant AR45748 to PAT and the HSS MacArthur Cartilage Fund.     

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.     

Epidermal growth factor and transforming growth factor alpha regulate extracellular matrix production by embryonic mouse palatal mesenchymal cells cultured on a variety of substrata

Mouse embryonic palatal mesenchymal (MEPM) cells were cultured either on plastic tissue culture dishes or on the surface of three-dimensional collagen gels or within collagen gel matrices in DMEM/F12 medium containing 2.5% donor calf serum. MEPM cells proliferated exponentially when cultured on collagen or on plastic. Cells cultured within collagen gels did not proliferate but remained viable. Addition of 10 ng/ml epidermal growth factor (EGF) or transforming growth factor alpha (TGF) stimulated the proliferation of those cells cultured on plastic or on collagen but not those cultured within collagen gels. Immunocytochemical analysis revealed that MEPM cells synthesise collagen types I, III, IV, V, VI and IX; fibronectin, heparan sulphate proteoglycan, laminin and tenascin in vitro. These molecules are all present in the developing palate in vivo. EGF and TGF produced a generalised stimulation of extracellular matrix (ECM) synthesis by MEPM cells in vitro. Biochemical analysis indicated that cells cultured within collagen gels had the highest intrinsic rate of protein synthesis. On all substrata neither EGF nor TGF markedly altered the types of ECM molecules synthesised but rather caused a general increase in the total amount produced. This stimulation was most marked where the cells were cultured within collagen gels. The lack of stimulation of proliferation of MEPM cells cultured within collagen gels (i.e. in a physiologically-relevant environment) by EGF or TGF together with the marked stimulation of ECM synthesis suggests that these factors may act as differentiation signals via their effects on ECM production. Correspondence to: M.J. Dixon     

The effect of collagen degradation on chondrocyte volume and morphology in bovine articular cartilage following a hypotonic challenge

Collagen degradation is one of the early signs of osteoarthritis. It is not known how collagen degradation affects chondrocyte volume and morphology. Thus, the aim of this study was to investigate the effect of enzymatically induced collagen degradation on cell volume and shape changes in articular cartilage after a hypotonic challenge. Confocal laser scanning microscopy was used for imaging superficial zone chondrocytes in intact and degraded cartilage exposed to a hypotonic challenge. Fourier transform infrared microspectroscopy, polarized light microscopy, and mechanical testing were used to quantify differences in proteoglycan and collagen content, collagen orientation, and biomechanical properties, respectively, between the intact and degraded cartilage. Collagen content decreased and collagen orientation angle increased significantly (p < 0.05) in the superficial zone cartilage after collagenase treatment, and the instantaneous modulus of the samples was reduced significantly (p < 0.05). Normalized cell volume and height 20 min after the osmotic challenge (with respect to the original volume and height) were significantly (p < 0.001 and p < 0.01, respectively) larger in the intact compared to the degraded cartilage. These findings suggest that the mechanical environment of chondrocytes, specifically collagen content and orientation, affects cell volume and shape changes in the superficial zone articular cartilage when exposed to osmotic loading. This emphasizes the role of collagen in modulating cartilage mechanobiology in diseased tissue.     

Raman spectroscopy of blood serum for Alzheimer's disease diagnostics: specificity relative to other types of dementia

The key moment for efficiently and accurately diagnosing dementia occurs during the early stages. This is particularly true for Alzheimer's disease (AD). In this proof‐of‐concept study, we applied near infrared (NIR) Raman microspectroscopy of blood serum together with advanced multivariate statistics for the selective identification of AD. We analyzed data from 20 AD patients, 18 patients with other neurodegenerative dementias (OD) and 10 healthy control (HC) subjects. NIR Raman microspectroscopy differentiated patients with more than 95% sensitivity and specificity. We demonstrated the high discriminative power of artificial neural network (ANN) classification models, thus revealing the high potential of this developed methodology for the differential diagnosis of AD. Raman spectroscopic, blood‐based tests may aid clinical assessments for the effective and accurate differential diagnosis of AD, decrease the labor, time and cost of diagnosis, and be useful for screening patient populations for AD development and progression.

Multivariate data analysis of blood serum Raman spectra allows for the differentiation between patients with Alzheimer's disease, other types of dementia and healthy individuals.     

Cover Picture: J. Biophotonics 2/2013

Employing Raman microspectroscopy, biochemical fingerprint patterns of human and porcine cartilage were obtained in this study. Using this non‐contact screening tool, it was also shown that prolonged in vitro culture can lead to phenotypic changes in chondrocytes. Picture: M. Pudlas et al., pp. 206–212 in this issue)     

Raman spectroscopic identification of Mycobacterium tuberculosis

In this study, Raman microspectroscopy has been utilized to identify mycobacteria to the species level. Because of the slow growth of mycobacteria, the per se cultivation‐independent Raman microspectroscopy emerges as a perfect tool for a rapid on‐the‐spot mycobacterial diagnostic test. Special focus was laid upon the identification of Mycobacterium tuberculosis complex (MTC) strains, as the main causative agent of pulmonary tuberculosis worldwide, and the differentiation between pathogenic and commensal nontuberculous mycobacteria (NTM). Overall the proposed model considers 26 different mycobacteria species as well as antibiotic susceptible and resistant strains. More than 8800 Raman spectra of single bacterial cells constituted a spectral library, which was the foundation for a two‐level classification system including three support vector machines. Our model allowed the discrimination of MTC samples in an independent validation dataset with an accuracy of 94% and could serve as a basis to further improve Raman microscopy as a first‐line diagnostic point‐of‐care tool for the confirmation of tuberculosis disease.

     

A Multiscale Vibrational Spectroscopic Approach for Identification and Biochemical Characterization of Pollen

Background

Analysis of pollen grains reveals valuable information on biology, ecology, forensics, climate change, insect migration, food sources and aeroallergens. Vibrational (infrared and Raman) spectroscopies offer chemical characterization of pollen via identifiable spectral features without any sample pretreatment. We have compared the level of chemical information that can be obtained by different multiscale vibrational spectroscopic techniques.

Methodology

Pollen from 15 different species of Pinales (conifers) were measured by seven infrared and Raman methodologies. In order to obtain infrared spectra, both reflectance and transmission measurements were performed on ground and intact pollen grains (bulk measurements), in addition, infrared spectra were obtained by microspectroscopy of multigrain and single pollen grain measurements. For Raman microspectroscopy measurements, spectra were obtained from the same pollen grains by focusing two different substructures of pollen grain. The spectral data from the seven methodologies were integrated into one data model by the Consensus Principal Component Analysis, in order to obtain the relations between the molecular signatures traced by different techniques.

Results

The vibrational spectroscopy enabled biochemical characterization of pollen and detection of phylogenetic variation. The spectral differences were clearly connected to specific chemical constituents, such as lipids, carbohydrates, carotenoids and sporopollenins. The extensive differences between pollen of Cedrus and the rest of Pinaceae family were unambiguously connected with molecular composition of sporopollenins in pollen grain wall, while pollen of Picea has apparently higher concentration of carotenoids than the rest of the family. It is shown that vibrational methodologies have great potential for systematic collection of data on ecosystems and that the obtained phylogenetic variation can be well explained by the biochemical composition of pollen. Out of the seven tested methodologies, the best taxonomical differentiation of pollen was obtained by infrared measurements on bulk samples, as well as by Raman microspectroscopy measurements of the corpus region of the pollen grain. Raman microspectroscopy measurements indicate that measurement area, as well as the depth of focus, can have crucial influence on the obtained data.     

Localization of collagen X in human fetal and juvenile articular cartilage and bone

The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilagebone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often spotty distribution of collagen X with increasing cartilage degeneration associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and spotty collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.     

Subcellular measurements of mechanical and chemical properties using dual Raman‐Brillouin microspectroscopy

Brillouin microspectroscopy is a powerful technique for noninvasive optical imaging. In particular, Brillouin microspectroscopy uniquely allows assessing a sample's mechanical properties with microscopic spatial resolution. Recent advances in background‐free Brillouin microspectroscopy make it possible to image scattering samples without substantial degradation of the data quality. However, measurements at the cellular‐ and subcellular‐level have never been performed to date due to the limited signal strength. In this report, by adopting our recently optimized VIPA‐based Brillouin spectrometer, we probed the microscopic viscoelasticity of individual red blood cells. These measurements were supplemented by chemically specific measurements using Raman microspectroscopy.

     

Interrelationship of cartilage composition and chondrocyte mechanics after a partial meniscectomy in the rabbit knee joint – Experimental and numerical analysis

Site-specific and depth-dependent properties of cartilage were implemented within a finite element (FE) model to determine if compositional or structural changes in the tissue could explain site-specific alterations of chondrocyte deformations due to cartilage loading in rabbit knee joints 3 days after a partial meniscectomy (PM). Depth-dependent proteoglycan (PG) content, collagen content and collagen orientation in the cartilage extracellular matrix (ECM), and PG content in the pericellular matrix (PCM) were assessed with microscopic and spectroscopic methods. Patellar, femoral groove and samples from both the lateral and medial compartments of the femoral condyle and tibial plateau were extracted from healthy controls and from the partial meniscectomy group. For both groups and each knee joint site, axisymmetric FE models with measured properties were generated. Experimental cartilage loading was applied in the simulations and chondrocyte volumes were compared to the experimental values. ECM and PCM PG loss occurred within the superficial cartilage layer in the PM group at all locations, except in the lateral tibial plateau. Collagen content and orientation were not significantly altered due to the PM. The FE simulations predicted similar chondrocyte volume changes and group differences as obtained experimentally. Loss of PCM fixed charge density (FCD) decreased cell volume loss, as observed in the medial femur and medial tibia, whereas loss of ECM FCD increased cell volume loss, as seen in the patella, femoral groove and lateral femur. The model outcome, cell volume change, was also sensitive to applied tissue geometry, collagen fibril orientation and loading conditions.     

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster–specific decrease in mitochondrial DNA–encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.     

Raman spectroscopy for the non‐contact and non‐destructive monitoring of collagen damage within tissues

The non‐destructive and label‐free monitoring of extracellular matrix (ECM) remodeling and degradation processes is a great challenge. Raman spectroscopy is a non‐contact method that offers the possibility to analyze ECM in situ without the need for tissue processing. Here, we employed Raman spectroscopy for the detection of heart valve ECM, focusing on collagen fibers. We screened the leaflets of porcine aortic valves either directly after dissection or after treatment with collagenase. By comparing the fingerprint region of the Raman spectra of control and treated tissues (400–1800 cm^–1), we detected no significant differences based on Raman shifts; however, we found that increasing collagen degradation translated into decreasing Raman signal intensities. After these proof‐of‐principal experiments, we compared Raman spectra of native and cryopreserved valve tissues and revealed that the signal intensities of the frozen samples were significantly lower compared to those of native tissues, similar to the data seen in the enzymatically‐degraded tissues. In conclusion, our data demonstrate that Raman microscopy is a promising, non‐destructive and non‐contact tool to probe ECM state in situ. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple‐helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ～60‐80°C for hydrated samples. We report high temperature Raman spectra for freeze‐dried cartilage samples that show an increase in laser‐excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H₂O is retained within the freeze‐dried tissue. This is confirmed by thermogravimetric analysis that show 5‐7 wt% H₂O remaining within freeze‐dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re‐hydration following recovery indicate that the capacity of the denatured collagen to re‐absorb water is reduced. Our results are important for revealing the presence of bound H₂O within the collagen component of connective tissue even after freeze‐drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.     

Carotenoids Co-Localize with Hydroxyapatite,Cholesterol, and Other Lipids in Calcified Stenotic Aortic Valves. Ex Vivo Raman Maps Compared to Histological Patterns

Unlike its application for atherosclerotic plaque analysis, Raman microspectroscopy was sporadically used to check the sole nature of bioapatite deposits in stenotic aortic valves, neglecting the involvement of accumulated lipids/lipoproteins in the calcific process. Here, Raman microspectroscopy was employed for examination of stenotic aortic valve leaflets to add information on nature and distribution of accumulated lipids and their correlation with mineralization in the light of its potential precocious diagnostic use. Cryosections from surgically explanted stenotic aortic valves (n=4) were studied matching Raman maps against specific histological patterns. Raman maps revealed the presence of phospholipids/triglycerides and cholesterol, which showed spatial overlapping with one another and Raman-identified hydroxyapatite. Moreover, the Raman patterns correlated with those displayed by both von-Kossa-calcium- and Nile-blue-stained serial cryosections. Raman analysis also provided the first identification of carotenoids, which co-localized with the identified lipid moieties. Additional fit concerned the distribution of collagen and elastin. The good correlation of Raman maps with high-affinity staining patterns proved that Raman microspectroscopy is a reliable tool in evaluating calcification degree, alteration/displacement of extracellular matrix components, and accumulation rate of different lipid forms in calcified heart valves. In addition, the novel identification of carotenoids supports the concept that valve stenosis is an atherosclerosis-like valve lesion, consistently with their previous Raman microspectroscopical identification inside atherosclerotic plaques.Key words: Valve calcification, stenosis, carotenoids, lipids, Raman microspectroscopy     

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

Recent work by a number of investigators has demonstrated that the process of bone matrix formation and mineralization is under the influence of growth factors and cytokines present in the local environment. Utilizing primary and established osteoblast cell culture systems, these studies have examined the regulation of bone matrix protein synthesis and deposition into the extracellular matrix (ECM) and subsequent mineralization. In previous studies, we have utilized the human osteoblastic cell line, HOS TE85, to study the effects of Tumor Necrosis Factor - alpha (TNF-) on the regulation of matrix proteins and proteolytic function in monolayer cultures as well as during the development and calcification of ECM formed by HOS TE85 cells during extended culture. Our studies demonstrate that TNF- inhibited formation and mineralization of nodules. In the study reported here, we evaluated the ultrastructural morphology of the cell-matrix complex formed by HOS TE85 cells in the presence and absence of TNF- at selected time points during the matrix development process utilizing both transmission electron microscopy and light microscopy. In the presence of TNF-, the cell-matrix complex does not develop normally, with a lack of organization and mineralization, when compared to untreated cells. The lack of mineralization appears to result from the lack of normal collagen fibril deposition and formation of an appropriate ECM essential for the mineralization process. These results support our previous observations that TNF- inhibits HOS TE85 cells from forming a mineralizing ECM by inhibiting incorporation of collagen into the ECM and inducing the synthesis of proteolytic enzymes capable of degrading collagen in the ECM.