The manuscript tests the hypothesis that posttranslational modification of the SIBLING family of proteins in general and osteopontin in particular modify the abilities of these proteins to regulate in vitro hydroxyapatite (HA) formation. Osteopontin has diverse effects on hydroxyapatite (HA) mineral crystallite formation and growth depending on the extent of phosphorylation. We hypothesized that different regions of full-length OPN would also have distinct effects on the mineralization process. Thrombin fragmentation of milk OPN (mOPN) was used to test this hypothesis. Three fragments were tested in a de novo HA formation assay; an N-terminal fragment (aa 1–147), a central fragment (aa 148–204) denoted SKK-fragment and a C-terminal fragment (aa 205–262). Compared to intact mOPN the C- and N-terminal fragments behaved comparably, promoting HA formation and growth, but the central SKK-fragment acted as a mineralization inhibitor. In a seeded growth experiment all fragments inhibited mineral proliferation, but the SKK-fragment was the most effective inhibitor. These effects, seen in HA-formation and seeded growth assays in a gelatin gel system and in a pH-stat experiment were lost when the protein or fragments were dephosphorylated. Effects of the fully phosphorylated protein and fragments were also altered in the presence of fibrillar collagen. The diverse effects can be explained in terms of the intrinsically disordered nature of OPN and its fragments which enable them to interact with their multiple partners.
Osteopontin; hydroxyapatite; SIBLING proteins; mineralization mechanisms
The interface between bone and connective tissues such as the Anterior Cruciate Ligament (ACL) constitutes a complex transition traversing multiple tissue regions, including non-calcified and calcified fibrocartilage, which integrates and enables load transfer between otherwise structurally and functionally distinct tissue types. The objective of this study was to investigate region-dependent changes in collagen, proteoglycan and mineral distribution, as well as collagen orientation, across the ligament-to-bone insertion site using Fourier transform infrared spectroscopic imaging (FTIR-I). Insertion site-related differences in matrix content were also evaluated by comparing tibial and femoral entheses. Both region- and site-related changes were observed. Collagen content was higher in the ligament and bone regions, while decreasing across the fibrocartilage interface. Moreover, interfacial collagen fibrils were aligned parallel to the ligament-bone interface near the ligament region, assuming a more random orientation through the bulk of the interface. Proteoglycan content was uniform on average across the insertion, while its distribution was relatively less variable at the tibial compared to the femoral insertion. Mineral was only detected in the calcified interface region, and its content increased exponentially across the mineralized fibrocartilage region toward bone. In addition to new insights into matrix composition and organization across the complex multi-tissue junction, findings from this study provide critical benchmarks for the regeneration of soft tissue-to-bone interfaces and integrative soft tissue repair.
Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4mM inorganic phosphate and no organic-phosphate esters; control cultures had 1mM inorganic phosphate. Mineralization was monitored based on 45Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis.
Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating the phosphatases were, in part, providing a source of inorganic phosphate.
To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin’s previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.
micromass culture; avian; mineralization; casein kinase II; phosphoprotein phosphatase
The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co-event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21-23 chick limb buds were plated as micro-mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un-treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z-Val-Ala-Asp (O-Me)-fluoromethylketone (Z-VAD-fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT-IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo-deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40-120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte apoptosis is not a pre-requisite for calcification in this culture system.
micro-mass; apoptosis; calcification; staurosporine; caspase-inhibition; hydroxyapatite
The role of DMP1 in mineralization was analyzed by comparing bone mineral and matrix properties in dmp1-null female mice to heterozygous and wildtype controls by FTIR imaging spectroscopy. The observed decreased mineral content in dmp1 null mice indicates a key role for dmp1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the KO phenotype.
Dentin matrix protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, is highly expressed in mineralized tissues. In vitro, DMP1 peptides can promote or inhibit mineralization depending on the extent of phosphorylation, the peptide size, and concentration. To clarify the biological function of DMP1 protein on in vivo mineralization, this study analyzed bone properties of dmp1 knockout (KO) mice compared with heterozygous (HET) and wildtype (WT) controls.
Materials and Methods
Tibias from dmp1 KO and age-, sex-, and background-matched HET and WT mice at 4 and 16 weeks (Ntotal = 60) were examined by Fourier transform infrared imaging (FTIRI), histology (n = 6 per genotype and age; N = 36), and geometry by μCT (n = 4 per genotype and age; N = 24). Serum ionic calcium and phosphate concentrations were also determined.
The mineral-to-matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in dmp1 KO mice tibias compared with WT and HET at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in dmp1 KO and HET mice relative to WT. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of nonreducible/reducible collagen cross-links) in dmp1 KO were not significantly different from WT and HET. Based on μCT, cortical bone cross-sectional areas at 16 but not 4 weeks were significantly reduced in the KO compared with controls. Maximum, minimum, and polar cross-sectional moments of inertia were significantly lower in dmp1 KO than in HET at 16 weeks but not at 4 weeks. Histological analysis and μCT 3-D images suggested that dmp1 KO mice had osteomalacia. Dmp1 KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values.
The findings of decreased mineral-to-matrix ratio and increased crystal size in bones of dmp1 KO mice suggest that DMP1 has multiple roles (both direct and indirect) in the regulation of postnatal mineralization. We suggest that direct effects on mineral formation, crystal growth, and indirect effects on regulation of Ca × P concentrations and matrix turnover all contribute to the dominant phenotype in the dmp1 KO mouse.
dentin matrix protein-1; Fourier transform infrared imaging; mineralization; osteomalacia model; mouse; bone geometry
Bone mineral composition, crystallinity, and bone mineral content of osteoporotic patients are different from those of normal subjects. We review the evidence that these mineralization parameters contribute to the strength (fracture resistance) of bone and the methods that have been used to examine them. A specific example is provided from analysis of biopsies from the Multiple Outcomes in Raloxifene Evaluation trial. For the analyses, randomly selected biopsies from placebo, low-dose, and high-dose groups (n = 5 per group) obtained at time zero and 2 years after treatment were examined by infrared imaging spectroscopy. In all cases, comparable increases in mineral content were found, but there were no significant variations in mineral crystallinity.
The Brtl/+ mouse is a knock-in model for osteogenesis imperfecta type IV in which a Gly349Cys substitution was introduced into one COL1A1 allele. To gain insight into the changes in dentin structure and mineral composition in these transgenic mice, the objective of this study was to use microcomputed tomography (micro-CT), scanning electron microscopy (SEM), and Fourier transform infrared imaging (FTIRI) to analyze these structures at 2 and 6 months of age. Results, consistent with the dental phenotype in humans with type IV OI, showed decreased molar volume and reduced mineralized tissue volume in the teeth without changes in enamel properties. Increased acid phosphate content was noted at 2 and 6 months by FTIRI, and a trend towards altered collagen structure was noted at 2 but not 6 months in the Brtl/+ teeth. The increase in acid phosphate content suggests a delay in the mineralization process, most likely associated with the defect in the collagen structure. It appears that in the Brtl/+ teeth slow maturation of the mineralized structures allows correction of altered mineral content and acid phosphate distribution.
The extent to which bone tissue composition varies across anatomic sites in normal or pathologic tissue is largely unknown, although pathologic changes in bone tissue composition are typically assumed to occur throughout the skeleton. Our objective was to compare the composition of normal cortical and trabecular bone tissue across multiple anatomic sites. The composition of cadaveric bone tissue from three anatomic sites was analyzed using Fourier transform infrared imaging: iliac crest (IC), greater trochanter (GT), and subtrochanteric femur (ST). The mean mineral:matrix ratio was 20% greater in the subtrochanteric cortex than in the cortices of the iliac crest (p=0.004) and the greater trochanter (p=0.02). There were also trends toward 30% narrower crystallinity distributions in the subtrochanteric cortex than in the greater trochanter (p=0.10) and 30% wider crystallinity distributions in the subtrochanteric trabeculae than in the greater trochanter (p=0.054) and the iliac crest (p=0.11). Thus the average cortical tissue mineral content and the widths of the distributions of cortical crystal size/perfection differ at the subtrochanteric femur relative to the greater trochanter and the iliac crest. In particular, the cortex of the iliac crest has lower mineral content relative to that of the subtrochanteric femur and may have limited utility as a surrogate for subtrochanteric bone.
iliac crest; subtrochanteric femur; Fourier transform infrared imaging; cortical bone; material properties
Bisphosphonates are highly effective agents for reducing osteoporotic fractures in women and men, decreasing fracture incidence at the hip and spine up to 50%. In a small subset of patients, however, these agents have recently been associated with 'atypical femoral fractures' (AFFs) in the subtrochanteric region or the diaphysis. These fractures have several atypical characteristics, including occurrence with minimal trauma; younger age than typical osteoporotic fractures; occurrence at cortical, rather than cancellous sites; early radiographic appearance similar to that of a stress fracture; transverse fracture pattern rather than the familiar spiral or transverse-oblique morphologies; initiation on the lateral cortex; and high risk of fracture on the contralateral side, at the same location as the initial fracture. Fracture is a mechanical phenomenon that occurs when the loads applied to a structure such as a long bone exceed its load-bearing capacity, either due to a single catastrophic overload (traumatic failure) or as a result of accumulated damage and crack propagation at sub-failure loads (fatigue failure). The association of AFFs with no or minimal trauma suggests a fatigue-based mechanism that depends on cortical cross-sectional geometry and tissue material properties. In the case of AFFs, bisphosphonate treatment may alter cortical tissue properties, as these agents are known to alter bone remodeling. This review discusses the use of bisphosphonates, their effects on bone remodeling, mechanics and tissue composition, their significance as an effective therapy for osteoporosis, and why these agents may increase fracture risk in a small population of patients.
For those seeking to model biomineralization in vitro, hydrogels can serve as excellent models of the extracellular matrix (ECM) microenvironment. A major challenge posed in implementing such systems is the logistics involved, from fundamental engineering to experimental design. For the study of calcium phosphate (e.g., hydroxyapatite) formation, many researchers use hydrogel-based double-diffusion systems (DDSs). The various designs of these DDSs are seemingly as unique as their applications. In this Highlight, we present a survey of four distinct types of double-diffusion systems and evaluate them in the context of fundamental diffusion theory. Based upon this analysis, we present the design and evaluation of an optimized system. The techniques and framework for the evaluation and construction of a DDS presented here can be applied to any DDS that a researcher may want to implement for their particular studies of biomineralization.
Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1−/− mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1−/− mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1−/− callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair.
Fracture repair; endochondral bone formation; transcription factor; early growth response gene
Bone strength depends on both bone quantity and quality. The former is routinely estimated in clinical settings through bone mineral density measurements but not the latter. Bone quality encompasses the structural and material properties of bone. Although its importance is appreciated, its contribution in determining bone strength has been difficult to precisely quantify partly because it is multifactorial and requires investigation of all bone hierarchical levels. Fourier transform infrared spectroscopy provides one way to explore these levels.
The purposes of our review were to (1) provide a brief overview of Fourier transform infrared spectroscopy as a way to establish bone quality, (2) review the major bone material parameters determined from Fourier transform infrared spectroscopy, and (3) review the role of Fourier transform infrared microspectroscopic analysis in establishing bone quality.
We used the ISI Web of Knowledge database initially to identify articles containing the Boolean term “infrared” AND “bone.” We then focused on articles on infrared spectroscopy in bone-related journals.
Infrared spectroscopy provides information on bone material properties. Their microspectroscopic versions allow one to establish these properties as a function of anatomic location, mineralization extent, and bone metabolic activity. It provides answers pertaining to the contribution of mineral to matrix ratio, mineral maturity, mineral carbonate substitution, and collagen crosslinks to bone strength. Alterations of bone material properties have been identified in disease (especially osteoporosis) not attainable by other techniques.
Infrared spectroscopic analysis is a powerful tool for establishing the important material properties contributing to bone strength and thus has helped better understand changes in fragile bone.
Fourier transform infrared imaging spectroscopy (FTIRI)-assessed bone composition parameters (mineral content, collagen maturity, crystal size and perfection, and carbonate content) describe bone quality and correlate to bone fracture risk. The challenge with studying bone quality in patients treated with antiresorptive drugs such as bisphosphonates (e.g., alendronate) and selective estrogen receptor modulators (SERMs) (e.g. raloxifene) is being able to test bone mechanical performance and material properties pre- and posttreatment. The purpose of this study was to evaluate the FTIRI changes in a large animal model of osteoporosis (female sheep with dietary induced metabolic acidosis; MA). Previous studies have investigated the relationship between bone material properties and bone strength in humans and smaller animals and have shown that changes in compositional properties influence fracture risk. Here we characterize the MA model at 6 and 12 months, demonstrate the loss of bone and changes in compositional properties, and show that 6 months of treatment with both antiresorptives ameliorate the bone loss as assessed by bone mineral density and FTIRI. This preliminary data suggest that the MA sheep model allows investigation of whether drug treatments preserve bone properties that exist at the time of treatment or if they induce further beneficial changes.
Osteoporosis; Antiresorptive treatment; Fourier transform infrared spectroscopy; Sheep
Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.
Osteogenesis imperfecta; C-propeptide; collagen; C-proteinase; mineralization; high bone mass
Alginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calcium-crosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1–6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mM bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcification.
Calcification; Alginate; Microencapsulation; Adipose stem cell microbeads; Bone tissue engineering
Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed.
CHICK LIMB-BUD; MICROMASS CULTURE; MINERALIZATION; MICROARRAY; GENE EXPRESSION
Material property changes in bone tissue with ageing are a crucial missing component in our ability to understand and predict age-related fracture. Cortical bone osteons contain a natural gradient in tissue age, providing an ideal location to examine these effects. This study utilized osteons from baboons aged 0 to 32 years (n=12 females), representing the baboon lifespan, to examine effects of tissue and animal age on mechanical properties and composition of the material. Tissue mechanical properties (indentation modulus and hardness), composition (mineral-to-matrix ratio, carbonate substitution, and crystallinity), and aligned collagen content (aligned collagen peak height ratio) were sampled along three radial lines in three osteons per sample by nanoindentation, Raman spectroscopy, and second harmonic generation microscopy, respectively. Indentation modulus, hardness, mineral-to-matrix ratio, carbonate substitution, and aligned collagen peak height ratio followed biphasic relationships with animal age, increasing sharply during rapid growth before leveling off at sexual maturity. Mineral-to-matrix ratio and carbonate substitution increased 12% and 6.7%, respectively, per year across young animals during growth, corresponding with a nearly 7% increase in stiffness and hardness. Carbonate substitution and aligned collagen peak height ratio both increased with tissue age, increasing 6 to 12% across the osteon radii. Indentation modulus most strongly correlated with mineral-to-matrix ratio, which explained 78% of the variation in indentation modulus. Overall, the measured compositional and mechanical parameters were the lowest in tissue of the youngest animals. These results demonstrate that composition and mechanical function are closely related and influenced by tissue and animal age.
ageing; primate; osteon; nanoindentation; tissue properties
Bones provide mechanical and protective function, while also serving as housing for marrow and a site for regulation of calcium ion homeostasis. The properties of bones do not remain constant with age; rather they change throughout life, in some cases improving in function, but in others, function deteriorates. Here we review the modifications in the mechanical function and shape of bones, the bone cells, the matrix they produce, and the mineral that is deposited on this matrix while presenting recent theories about the factors leading to these changes.
bone properties; aging; senescence; bone cells; bone mineral; review
Bone geometry and tissue material properties jointly govern whole-bone structural behavior. While the role of geometry in structural behavior is well characterized, the contribution of the tissue material properties is less clear, partially due to the multiple tissue constituents and hierarchical levels at which these properties can be characterized. Our objective was to elucidate the contribution of the mineral phase to bone mechanical properties across multiple length scales, from the tissue material level to the structural level. Vitamin D and calcium deficiency in 6-week-old male rats was employed as a model of reduced mineral content with minimal collagen changes. The structural properties of the humeri were measured in three-point bending and related to the mineral content and geometry from microcomputed tomography. Whole-cortex and local bone tissue properties were examined with infrared (IR) spectroscopy, Raman spectroscopy, and nanoindentation, to understand the role of altered mineral content on the constituent material behavior. Structural stiffness (-47%) and strength (-50%) were reduced in vitamin D-deficient (-D) humeri relative to controls. Moment of inertia (-38%), tissue mineral density (TMD, -9%), periosteal mineralization (-28%), and IR mineral:matrix ratio (-19%) were reduced in -D cortices. Thus, both decreased tissue mineral content and changes in cortical geometry contributed to impaired skeletal load bearing function. In fact, 97% of the variability in humeral strength was explained by moment of inertia, TMD, and IR mineral:matrix ratio. The strong relationships between structural properties and cortical material composition demonstrate a critical role of the microscale material behavior in skeletal load-bearing performance.
bone strength; material properties; mineral; rat; Fourier transform infrared spectroscopy
Chondrocyte apoptosis is thought to be an important step in the calcification of cartilage in vivo; however there are conflicting reports as to whether or not this apoptosis is a necessary precursor to mineralization. The goal of this study was to determine whether or not apoptosis is necessary for mineralization in an in vitro murine micromass model of endochondral ossification. C3H10T1/2 murine mesenchymal stem cells were plated in micromass culture in the presence of 4mM inorganic phosphate with the addition of the apoptogens, camptothecin or staurosporine, to induce apoptosis. The rate and total accumulation of mineralization was measured with 45Ca uptake. In these studies, both apoptogens increased the rate of mineralization, with staurosporine increasing 45Ca accumulation by about 2.5 times that of controls and camptothecin increasing total amounts of mineralization about 1.5 times that of controls. Inhibiting cell apoptosis with the caspase inhibitor, ZVAD-fmk, to prevent apoptosis, caused slower rates of 45Ca uptake, however total amounts of 45Ca accumulation reached the same values by Day 30 of culture. FTIR data showed mineralization in all samples treated with 4mM inorganic phosphate, with the highest mineral to matrix ratios in the camptothecin treated samples.
apoptosis; mineralization; micromass cultures
The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with 19Flourine magnetic resonance spectroscopic imaging (19FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using 19Fluorine magnetic resonance spectroscopy (19FMRS) and 19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. 19FMRS and 19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. 19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized 19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of 19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, 19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications.
Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues.
ribosomal protein knockout; bone mineral content; bone quality; Fourier transform infrared microspectroscopy; teeth
Osteoporosis and fractures occur frequently in patients with beta thalassemias, a group of congenital hemolytic anemias characterized by decreased synthesis of the beta chain of hemoglobin. In this study, we determined the bone abnormalities of the th3 thalassemia mouse, generated by deletion of the mouse beta chain genes. The heterozygote th3/+ mouse has moderate anemia, and serves as a model of beta thalassemia intermedia (TI), which represents the mild thalassemia phenotype. The th3/th3 mouse has lethal anemia and is a model of beta thalassemia major (TM), which is characterized by life-threatening anemia requiring regular transfusions to sustain life.
Compared to controls: i) Micro-CT of trabecular bone showed decreased bone volume fraction, number of trabeculae and trabecular thickness in both th3/+ and th3/th3 (p<0.05). ii) Cortical bone analysis showed thinner cortices and increased marrow area in th3/+ animals (p<0.05). iii) Micro-CT abnormalities in th3/+ mice were present by 2 months and did not worsen with age. iv) Histomorphometry was significant for decreased bone formation and resorption in both th3/+ and th3/th3. Similarly, cathepsin K and osteocalcin expression from bone of both th3/+and th3/th3 animals was reduced (p<0.05). vi) Biomechanics showed reduced maximum load, maximum moment and structural stiffness in both th3/+and th3/th3 (p<0.01).
In conclusion, the th3 mouse model of thalassemia manifests bone changes reminiscent of those in humans, and can be used for further bone studies in thalassemia. Bone changes are associated with decreased bone turnover, and develop early on during the period of bone accrual.