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.
Tissue engineering with adult stem cells is a promising approach for the restoration of focal defects in articular cartilage. For this, progenitor cells would ideally be delivered to (and maintained within) the defect site via a biocompatible material and in combination with soluble factors to promote initial cell differentiation and subsequent tissue maturation in vivo. While growth factor delivery methods are continually being optimized, most offer only a short (days to weeks) delivery profile at high doses. To address this issue, we investigated mesenchymal stem cell (MSC) differentiation and maturation in photocrosslinkable hyaluronic acid (HA) hydrogels with transient exposure to the pro-chondrogenic molecule transforming growth factor-beta3 (TGF-β3), at varying doses (10, 50 and 100 ng/mL) and durations (3, 7, 21 and 63 days). Mechanical, biochemical, and histological outcomes were evaluated through 9 weeks of culture. Results showed that a brief exposure (7 days) to a very high level (100 ng/mL) of TGF-β 3 was sufficient to both induce and maintain cartilage formation in these 3D constructs. Indeed, this short delivery resulted in constructs with mechanical and biochemical properties that exceeded that of continuous exposure to a lower level (10 ng/mL) of TGF-β 3 over the entire 9-week time course. Of important note, the total TGF delivery in these two scenarios was roughly equivalent (200 vs. 180 ng), but the timing of delivery differed markedly. These data support the idea that acute exposure to a high dose of TGF will induce functional and long-term differentiation of stem cell populations, and furthers our efforts to improve cartilage repair in vivo.
Hyaluronic Acid; Mesenchymal Stem Cells; Cartilage Tissue Engineering
Little is known about how the biochemical properties of collagen change during tissue regeneration following cartilage damage. In the current study, temporal changes in cartilage repair tissue biochemistry were assessed in a rabbit osteochondral defect.
Bilateral full thickness 3mm osteochondral trochlear groove defects were created in 54 adult male skeletally mature New Zealand white rabbits and tissue repair monitored over 16 weeks. Collagen content, cross-links, lysyl hydroxylation, gene expression, histological grading, and FTIR analyses were performed at 2, 4, 6, 8, 12, and 16 weeks.
Defect fill occurred at ~4 weeks post-injury, however, histological grading showed that the repair tissue never became normal, primarily due to the presence of fibrocartilage. Gene expression levels of Col1a1 and Col1a2 were higher in the defect compared to adjacent regions. Collagen content in the repair tissue reached the level of normal cartilage at 6 weeks, but it took 12 weeks for the extent of lysine hydroxylation to return to normal. Divalent immature cross-links markedly increased in the early stages of repair. Though the levels gradually diminished thereafter, they never returned to the normal levels. The mature cross-link, pyridinoline, gradually increased with time and nearly reached normal levels by week 16. Infrared imaging data of protein content paralleled the biochemical data. However, collagen maturity, a parameter previously shown to reflect collagen cross-link ratios in bone, did not correlate with the biochemical determination of cross-links in the repair tissue..
Collagen biochemical data could provide markers for clinical monitoring in a healing defect.
Collagen cross-links; cartilage repair; pyridinoline; FTIR
Hyaline cartilage and mechanically inferior fibrocartilage consisting of mixed collagen types are frequently found together in repairing articular cartilage. The present study seeks to develop methodology to identify collagen type and other tissue components using Fourier transform infrared (FTIR) spectral evaluation of matrix composition in combination with multivariate analyses. FTIR spectra of the primary molecular components of repair cartilage, types I and II collagen, and aggrecan, were used to develop multivariate spectral models for discrimination of the matrix components of the tissues of interest. Infrared imaging data were collected from bovine bone, tendon, normal cartilage, meniscus and human repair cartilage tissues, and composition predicted using partial least squares analyses. Histology and immunohistochemistry results were used as standards for validation. Infrared fiber optic probe spectral data were also obtained from meniscus (a tissue with mixed collagen types) to evaluate the potential of this method for identification of collagen type in a minimally-invasive clinical application. Concentration profiles of the tissue components obtained from multivariate analysis were in excellent agreement with histology and immunohistochemistry results. Bone and tendon showed a uniform distribution of predominantly type I collagen through the tissue. Normal cartilage showed a distribution of type II collagen and proteoglycan similar to the known composition, while in repair cartilage, the spectral distribution of both types I and II collagen were similar to that observed via immunohistochemistry. Using the probe, the outer and inner regions of the meniscus were shown to be primarily composed of type I and II collagen, respectively, in accordance with immunohistochemistry data. In summary, multivariate analysis of infrared spectra can indeed be used to differentiate collagen type I and type II, even in the presence of proteoglycan, in connective tissues, using both imaging and fiber optic methodology. This has great potential for clinical in situ applications for monitoring tissue repair.
Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by ectopic mineralization. However, the structure of the mineral deposits, their interactions with the connective tissue matrix, and the details of the progressive maturation of the mineral crystals are currently unknown. In this study, we examined the mineralization processes in Abcc6−/− mice, a model system for PXE, by energy dispersive X-ray, and Fourier transform infrared imaging spectroscopy (FT-IRIS). The results indicated that the principal components of the mineral deposits were calcium and phosphate which co-localized within the histologically demonstrable lesions determined by topographic mapping. The Ca/P ratio increased in samples with progressive mineralization reaching the value comparable to that in endochondral bone. A progressive increase in mineralization was also reflected by increased mineral-to-matrix ratio determined by FT-IRIS. Determination of the mineral phases by FT-IRIS suggested progressive maturation of the mineral deposits from amorphous calcium phosphate to hydroxyapatite. These results provide critical information of the mechanisms of mineralization in PXE, with potential pharmacologic implications.
Pseudoxanthoma elasticum; ectopic connective tissue mineralization; mineral composition; hydroxyapatite; FT-IRIS; treatment of mineralization disorders
Noninvasive early detection of specific matrix alterations in degenerative cartilage disease would be of substantial use in basic science studies and clinically, but remains an elusive goal. Recently-developed MRI methods exhibit some specificity, but require contrast agents or nonstandard pulse sequences and hardware. We present a multiexponential approach which does not require contrast agents or specialized hardware, and uses a standard multiple-echo spin-echo sequence. Experiments were performed on tissue models of degenerative cartilage using enzymes with distinct actions. MR results were validated with histologic, biochemical, and infrared spectroscopic analyses. The sulfated glycosaminoglycan (sGAG) per dry weight (dw) in bovine nasal cartilage (BNC) was 0.72±0.06 mg/mg dw and was reduced through chondroitinase AC (ChAC) and collagenase digestion to 0.56±0.12 and 0.58±0.13 mg/mg dw, respectively. Multiexponential analysis of data obtained at 9.4T permitted identification of tissue compartments assigned to the proteoglycan (PG) component of the matrix and to bulk water. Enzymatic treatment resulted in a significant reduction in the ratio of PG-bound to free water from 0.13±0.02 in control cartilage to 0.03±0.02 and 0.05±0.06 under ChAC and collagenase treatment, respectively. As expected, monoexponential T2 increased with both degradation protocols, but without further specificity to the nature of the degradation. An important eventual extension of this approach may be to map articular cartilage degeneration in the clinical setting. As an initial step towards this, localized multiexponential T2 analysis was performed on excised bovine patella. Results obtained on this articular cartilage sample were readily interpretable in terms of PG-associated and relatively free water compartments. In potential clinical applications, SNR constraints will define the threshold for detection of macromolecular compartment changes at a given spatial scale. The multiexponential approach has potential application to the early detection of cartilage degradation with use of appropriate pulse parameters under high SNR conditions.
cartilage MRI; transverse relaxation; multiexponential T2; proteoglycan; cartilage degradation
Fetal cartilage anlage provides a framework for endochondral ossification and organization into articular cartilage. We previously reported differences between mechanical properties of talar cartilage anlagen and adult articular cartilage. However, the underlying development-associated changes remain to be established. Delineation of the normal evolvement of mechanical properties and its associated compositional basis provides insight into the natural mechanisms of cartilage maturation. Our goal was to address this issue.
Materials and methods
Human fetal cartilage anlagen were harvested from the tali of normal stillborn fetuses from 20 to 36 weeks of gestational age. Data obtained from stress relaxation experiments conducted under confined and unconfined compression configurations were processed to derive the compressive mechanical properties. The compressive mechanical properties were extracted from a linear fit to the equilibrium response in unconfined compression, and by using the nonlinear biphasic theory to fit to the experimental data from the confined compression experiment, both in stress-relaxation. The molecular composition was obtained using FTIR, and spatial maps of tissue contents per dry weight were created using FTIR imaging. Correlative and regression analyses were performed to identify relationships between the mechanical properties and age, compositional properties and age, and mechanical versus compositional parameters.
All of the compositional quantities and the mechanical properties excluding the Poisson’s ratio changed with maturation. Stiffness increased by a factor of ~2.5 and permeability decreased by 20% over the period studied. Collagen content and degree of collagen integrity increased with age by ~3-fold, while the proteoglycan content decreased by 18%. Significant relations were found between the mechanical and compositional properties.
The mechanics of fetal talar cartilage is related to its composition, where the collagen and proteoglycan network play a prominent role. An understanding of the mechanisms of early cartilage maturation could provide a framework to guide tissue-engineering strategies.
cartilage anlage; fetal development; Fourier transform infrared spectroscopy; mechanical properties; collagen; proteoglycan
Association of MR parameters with cartilage matrix components remains an area of ongoing investigation. Multiexponential analysis of non-localized transverse relaxation data has previously been used to quantify water compartments associated with matrix macromolecules in cartilage. We extend this to mapping the proteoglycan-bound water fraction (wPG) in cartilage, using mature and young bovine nasal cartilage model systems, towards the goal of matrix component-specific imaging. wPG from mature and young bovine nasal cartilage was 0.3±0.04 and 0.22±0.06, respectively, in agreement with biochemically-derived proteoglycan content and proteoglycan-to-water weight ratios. Fourier transform infrared imaging spectroscopic-derived proteoglycan maps normalized by water content (IR-PGww) showed spatial correspondence with wPG maps. Extensive simulation analysis demonstrated that the accuracy and precision of our determination of wPG was within 2%, which is substantially smaller than the observed tissue differences. Our results demonstrate the feasibility of performing imaging-based multiexponential analysis of transverse relaxation data to map proteoglycan in cartilage.
transverse relaxation; proteoglycan mapping; multiexponential relaxation; cartilage
The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of fibrillin-1 or fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in fibrillin-1 or fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that fibrillin-1 plays a predominantly greater role than fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by fibrillin proteins. Our study therefore advance our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in fibrillin-1 or -2.
bone material and mechanical properties; congenital contractural arachnodactyly; fibrillin; Marfan syndrome; TGFβ
Noninvasive monitoring of matrix development in tissue-engineered cartilage constructs would permit ongoing assessment with the ability to modify culture conditions during development to optimize tissue characteristics. In this study, chondrocytes seeded in a collagen hydrogel were exposed for 20 min/day to pulsed low-intensity ultrasound (PLIUS) at 30 mWcm−2 and cultured for up to 5 weeks. Biochemical assays, histology, immunohistochemistry, Fourier transform infrared spectroscopy, and magnetic resonance imaging (MRI) were performed at weeks 3 and 5 after initiation of growth. The noninvasive MRI measurements were correlated with those from the invasive studies. In particular, MRI transverse relaxation time (T2) and magnetization transfer rate (km) correlated with macromolecular content, which was increased by application of PLIUS. This indicates the sensitivity of MR techniques to PLIUS-induced changes in matrix development, and highlights the potential for noninvasive assessment of the efficacy of anabolic interventions for engineered tissue.
Noninvasive assessment of engineered cartilage properties would enable better control of the developing tissue towards the desired structural and compositional endpoints through optimization of the biochemical environment in real time. The objective of this study is to assess the matrix constituents of cartilage using near-infrared spectroscopy (NIRS), a technique that permits full-depth assessment of developing engineered tissue constructs. Mid-infrared (mid-IR) and NIR data were acquired from full-thickness cartilage constructs that were grown up to 4 weeks with and without mechanical stimulation. Correlations were assessed between established mid-IR peak areas that reflect the relative amount of collagen (amide I, amide II, and 1338 cm−1) and proteoglycan (PG), (850 cm−1), and the integrated area of the NIR water absorbance at 5190 cm−1. This analysis was performed to evaluate whether simple assessment of the NIR water absorbance could yield information about matrix development. It was found that an increase in the mid-IR PG absorbance at 850 cm−1 correlated with the area of the NIR water peak (Spearman’s rho = 0.95, p < 0.0001). In the second analysis, a partial least squares method (PLS1) was used to assess whether an extended NIR spectral range (5400–3800 cm−1) could be utilized to predict collagen and proteoglycan content of the constructs based on mid-IR absorbances. A subset of spectra was randomly selected as an independent prediction set in this analysis. Average of the normalized root mean square errors of prediction of first-derivative NIR spectral models were 7% for 850 cm−1 (PG), 11% for 1338 cm−1 (collagen), 8% for amide II (collagen), and 8% for amide I (collagen). These results demonstrate the ability of NIRS to monitor macromolecular content of cartilage constructs and is the first step towards employing NIR to assess engineered cartilage in situ.
Near-infrared spectroscopy; NIR spectroscopy; Fourier transform infrared spectroscopy; FT-IR spectroscopy; Partial least squares; PLS; Multivariate analysis; Cartilage; Tissue engineering
Evaluation of the morphology and matrix composition of repair cartilage is a critical step toward understanding the natural history of cartilage repair and efficacy of potential therapeutics. In the current study, short-term articular cartilage repair (3 and 6 weeks) was evaluated in a rabbit osteochondral defect model treated with thrombin peptide (TP-508) using magnetic resonance imaging (MRI), quantitative T2 mapping, and Fourier transform–infrared imaging spectroscopy (FT-IRIS).
Three-mm-diameter osteochondral defects were made in the rabbit trochlear groove and filled with either TP-508 plus poly-lactoglycolidic acid microspheres or poly-lactoglycolidic acid microspheres alone (placebo). Repair tissue and adjacent normal cartilage were evaluated at 3 and 6 weeks postdefect creation. Intact knees were evaluated by magnetic resonance imaging for repair morphology, and with quantitative T2 mapping to assess collagen orientation. Histological sections were evaluated by FT-IRIS for parameters that reflect collagen quantity and quality, as well as proteoglycan (PG) content.
Results and Conclusion
There was no significant difference in volume of repair tissue at either time point. At 6 weeks, placebo repair tissue demonstrated longer T2 values (p < 0.01) than TP-508 did. Although both placebo and TP-508 repair tissue demonstrated longer T2 values than adjacent normal cartilage did, the 6-week T2 values of the TP-508 specimens were closer to those of the adjacent normal cartilage than were the placebo values. FT-IRIS analysis demonstrated a significant increase in collagen content, integrity, and PG content of the TP-508 repair tissue from 3 to 6 weeks (p ≤ 0.05). In addition, the collagen and PG content of the TP-508 samples were closer to normal cartilage at 3 weeks than were the placebo samples. Further, there was a significant inverse correlation between the T2 relaxation values and collagen orientation in the normal cartilage. However, there were no significant correlations between T2 relaxation values and any FT-IRIS parameter in the repair tissue. Together, the data demonstrate that MRI and FT-IRIS assessment of cartilage repair tissue provide molecular information that furthers understanding of the cartilage repair process.
The Hartley guinea pig develops articular cartilage degeneration similar to that seen in idiopathic human osteoarthritis. We investigated whether the application of pulsed low-intensity ultrasound (PLIUS) to the Hartley guinea pig joint would prevent or attenuate the progression of this degenerative process.
Treatment of male Hartley guinea pigs was initiated at the onset of degeneration (8 weeks of age) to assess the ability of PLIUS to prevent osteoarthritis, or at a later age (12 months) to assess the degree to which PLIUS acted to attenuate the progression of established disease. PLIUS (30 mW/cm2) was applied to stifle joints for 20 minutes per day over periods ranging from three to ten months, with contralateral limbs serving as controls. Joint cartilage histology was graded according to a modified Mankin scale to evaluate treatment effect. Immunohistochemical staining for IL-1 receptor antagonist (IL-1ra), MMP-3, MMP-13, and TGF-β1 was performed on the cartilage to evaluate patterns of expression of these proteins.
PLIUS did not fully prevent cartilage degeneration in the prevention groups, but diminished the severity of the disease, with the treated joints showing markedly decreased surface irregularities and a much smaller degree of loss of matrix staining as compared to controls. PLIUS also attenuated disease progression in the groups with established disease, although to a somewhat lesser extent as compared to the prevention groups. Immunohistochemical staining demonstrated a markedly decreased degree of TGF-β1 production in the PLIUS-treated joints. This indicates less active endogenous repair, consistent with the marked reduction in cartilage degradation.
PLIUS exhibits the ability to attenuate the progression of cartilage degeneration in an animal model of idiopathic human OA. The effect was greater in the treatment of early, rather than established, degeneration.
Recently, a new class of agents targeting the receptor activator of nuclear factor-κB ligand (RANKL) pathway has been developed for the treatment of osteoporosis and other bone diseases. In the current study, inhibition of the RANKL pathway was evaluated to assess effects on “bone quality” and fracture incidence in an animal model of osteogenesis imperfect (OI), the oim/oim mouse. Juvenile oim/oim (~6 weeks old) and wildtype (+/+) mice were treated with either a RANKL inhibitor (RANK-Fc) or saline. After treatment, bone density increased significantly in the femurs of both genotypes. Femoral length decreased with RANK-Fc in +/+ mice. Geometric measurements at mid-diaphysis in the oim/oim groups showed increases in the ML periosteal and endosteal diameters and AP cortical thickness in the treated groups. Within +/+ groups, ML cortical thickness and ML femoral periosteal diameter were significantly increased with RANK-Fc. Biomechanical testing revealed increased stiffness in oim/oim and +/+ mice. Total strain was increased with treatment in the +/+ mice. Histologically, RANKL inhibition resulted in retained growth plate cartilage in both genotypes. The average number of fractures sustained by RANK-Fc-treated oim/oim mice was not significantly decreased compared to saline treated oim/oim mice. This preclinical study demonstrated that RANKL inhibition at the current dose improved density and some geometric and biomechanical properties of oim/oim bone, but it did not decrease fracture incidence. Further studies that address commencement of therapy at earlier time points are needed to determine whether this mode of therapy will be clinically useful in OI.
Bone; Osteogenesis Imperfecta; Oim/oim; Mouse; RANK/RANKL
A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging including reduced albuminuria, decreased inflammation and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started mid-life.