Adam Michael Taylor

The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria

OBJECTIVE Alkaptonuria is a genetic disorder of tyrosine metabolism, resulting in elevated circulating concentrations of homogentisic acid. Homogentisic acid is deposited as a polymer, termed ochronotic pigment, in collagenous tissues, especially cartilages of weight-bearing joints, leading to a severe osteoarthropathy. We undertook this study to investigate the initiation and progression of ochronosis from the earliest detection of pigment through complete joint failure. METHODS Nine joint samples with varying severities of ochronosis were obtained from alkaptonuria patients undergoing surgery and compared to joint samples obtained from osteoarthritis (OA) patients. Samples were analyzed by light and fluorescence microscopy, 3-dimensional scanning electron microscopy (SEM), and the quantitative backscattered electron mode of SEM. Cartilage samples were mechanically tested by compression to determine Youngs modulus of pigmented, nonpigmented, and OA cartilage samples. RESULTS In alkaptonuria samples with the least advanced ochronosis, pigment was observed intracellularly and in the territorial matrix of individual chondrocytes at the boundary of the subchondral bone and calcified cartilage. In more advanced ochronosis, pigmentation was widespread throughout the hyaline cartilage in either granular composition or as blanket pigmentation in which there is complete and homogenous pigmentation of cartilage matrix. Once hyaline cartilage was extensively pigmented, there was aggressive osteoclastic resorption of the subchondral plate. Pigmented cartilage became impacted on less highly mineralized trabeculae and embedded in the marrow space. Pigmented cartilage samples were much stiffer than nonpigmented or OA cartilage as revealed by a significant difference in Youngs modulus. CONCLUSION Using alkaptonuria cartilage specimens with a wide spectrum of pigmentation, we have characterized the progression of ochronosis. Intact cartilage appears to be resistant to pigmentation but becomes susceptible following focal changes in calcified cartilage. Ochronosis spreads throughout the cartilage, altering the mechanical properties. In advanced ochronosis, there is aggressive resorption of the underlying calcified cartilage leading to an extraordinary phenotype in which there is complete loss of the subchondral plate. These findings should contribute to better understanding of cartilage-subchondral interactions in arthropathies.

Development of an in vitro model to investigate joint ochronosis in alkaptonuria

OBJECTIVES Alkaptonuria (AKU) is a genetic disorder caused by lack of the enzyme responsible for breaking down homogentisic acid (HGA), an intermediate in tyrosine metabolism. HGA is deposited as a polymer, termed ochronotic pigment, in collagenous tissues. Pigmentation is progressive over many years, leading to CTDs including severe arthropathies. To investigate the mechanism of pigmentation and to determine how it leads to arthropathy, we aimed to develop an in vitro model of ochronosis. METHODS Osteosarcoma cell lines MG63, SaOS-2 and TE85 were cultured in medium containing HGA from 0.1 μM to 1 mM. Cultures were examined by light microscopy and transmission electron microscopy, and Schmorls stain was used to detect pigment deposits in vitro, following the observation that this stain identifies ochronotic pigment in AKU tissues. The effects of HGA on cell growth and collagen synthesis were also determined. RESULTS There was a dose-related deposition of pigment in cells and associated matrix from 33 μM to 0.33 mM HGA. Pigmentation in vitro was much more rapid than in vivo, indicating that protective mechanisms exist in tissues in situ. Pigment deposition was dependent on the presence of cells and was observed at HGA concentrations that were not toxic. There was an inhibition of cell growth and a stimulation of type I collagen synthesis up to 0.33 mM HGA, but severe cell toxicity at 1 mM HGA. CONCLUSION We have developed an in vitro model of ochronosis that should contribute to understanding joint destruction in AKU and to the aetiology of OA.

Ultrastructural examination of tissue in a patient with alkaptonuric arthropathy reveals a distinct pattern of binding of ochronotic pigment

SIR, we report a female with known alkaptonuria (AKU) undergoing routine hip replacement surgery due to alkaptonuric arthropathy. AKU is caused by a deficiency in the enzyme that breaks down homogentisic acid (HGA), resulting in elevated circulation of HGA levels in the body. HGA is deposited as a polymerized pigment in collagenous connective tissues, predominantly in the weight-bearing joints [1]. It has also been shown to affect other non-joint tissues [2, 3]. Ligamentous capsule was processed routinely for histology and electron microscopy. Macroscopically, ochronotic pigment was clearly visible alongside non-pigmented regions. Haematoxylin and eosin (H&E) staining showed the presence of extracellular pigmentation associated with the collagen fibres and also intracellular pigmentation within fibroblasts. This novel intracellular pigmentation appeared as numerous individual granules located within the cytoplasm of single fibroblasts (Fig. 1A). Extracellular pigmentation appeared as two distinct types: a granular deposition, similar to that within the cells, but also more homogeneous pigmentation that completely encrusted the collagen fibres. Ultrastructural analysis revealed that collagen fibres in transverse section had numerous electron-dense granules of ochronotic pigment associated with them. These granules varied in position and distribution among the fibres. Some had single granules of pigment located within the cross-section of …

Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone

Background Alkaptonuria (AKU) is a rare metabolic disease caused by deficiency of homogentisate 1,2 dioxygenase, an enzyme involved in tyrosine catabolism, resulting in increased circulating homogentisic acid (HGA). Over time HGA is progressively deposited as a polymer (termed ochronotic pigment) in collagenous tissues, especially the cartilages of weight bearing joints, leading to severe joint disease. Objectives To characterise blood biochemistry and arthropathy in the AKU mouse model (Hgd−/−). To examine the therapeutic effect of long-term treatment with nitisinone, a potent inhibitor of the enzyme that produces HGA. Methods Lifetime levels of plasma HGA from AKU mice were measured by high-performance liquid chromatography (HPLC). Histological sections of the knee joint were examined for pigmentation. The effect of nitisinone treatment in both tissues was examined. Results Mean (±SE) plasma HGA levels were 3- to 4-fold higher (0.148±0.019 mM) than those recorded in human AKU. Chondrocyte pigmentation within the articular cartilage was first observed at 15 weeks, and found to increase steadily with mouse age. Nitisinone treatment reduced plasma HGA in AKU mice throughout their lifetime, and completely prevented pigment deposition. Conclusions The AKU mouse was established as a model of both the plasma biochemistry of AKU and its associated arthropathy. Early-stage treatment of AKU patients with nitisinone could prevent the development of associated joint arthropathies. The cellular pathology of ochronosis in AKU mice is identical to that observed in early human ochronosis and thus is a model in which the early stages of joint pathology can be studied and novel interventions evaluated.

Proteomic and redox‐proteomic evaluation of homogentisic acid and ascorbic acid effects on human articular chondrocytes

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues up to the deposition of melanin‐like pigments (ochronosis). Since little is known on the effects of HGA and its metabolites on articular cells, we carried out a proteomic and redox‐proteomic analysis to investigate how HGA and ascorbic acid (ASC) affect the human chondrocytic protein repertoire. We settled up an in vitro model using a human chondrocytic cell line to evaluate the effects of 0.33 mM HGA, alone or combined with ASC. We found that HGA and ASC significantly affect the levels of proteins with specific functions in protein folding, cell organization and, notably, stress response and cell defense. Increased protein carbonyls levels were found either in HGA or ASC treated cells, and evidences produced in this paper support the hypothesis that HGA‐induced stress might be mediated by protein oxidation. Our finding can lay the basis towards the settling up of more sophisticated models to study AKU and ochronosis. J. Cell. Biochem. 111: 922–932, 2010.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition

OBJECTIVE Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorls reagent. RESULTS Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

Identification of alkaptonuria in the general population : a United Kingdom experience describing the challenges, possible solutions and persistent barriers.

Progress in research into rare diseases is challenging. This paper discusses strategies to identify individuals with the rare genetic disease alkaptonuria (AKU) within the general population. Strategies used included a questionnaire survey of general practitioners, a dedicated website and patient network contact, targeted family screening and medical conference targeting. Primary care physicians of the UK were targeted by a postal survey that involved mailing 11,151 UK GPs; the response rate was 18.2%. We have identified 75 patients in the UK with AKU by the following means: postal survey (23), targeted family screening (11), patient networks and the website (41). Targeting medical conferences (AKU, rare diseases, rheumatology, clinical biochemistry, orthopaedics, general practitioners) did not lead to new identification in the UK but helped identify overseas cases. We are now aware of 626 patients worldwide including newly identified non-UK people with AKU in the following areas: Slovakia (208), the rest of Europe (including Turkey) (79), North America (including USA and Canada) (110), and the rest of the world (154). A mechanism for identifying individuals with AKU in the general population—not just in the UK but worldwide—has been established. Knowledge of patients with AKU, both in the UK and outside, is often confined to establishing their location in a particular GP practice or association with a particular medical professional. Mere identification, however, does not always lead to full engagement for epidemiological research purposes or targeting treatment since further barriers exist.

Primary Human Osteoblast Cultures

Osteoblast cultures can be used to investigate the mechanisms of bone formation, to probe the cellular and molecular basis of bone disease, and to screen for potential therapeutic agents that affect bone formation. Here, we describe the methods for establishing and characterising primary human osteoblast cultures.

Calculi and intracellular ochronosis in the submandibular tissues from a patient with alkaptonuria.

Alkaptonuria (AKU) is a rare autosomal recessive condition caused by deficiency of the enzyme homogentisate 1,2 dioxygenase, resulting in widespread deposition of oxidised homogentisic acid (HGA) polymer, primarily in joint tissues but also in other connective tissues. Macroscopic pigmentation of connective tissues in AKU is well documented and is the end point of a process that is not understood. Deposition in less common regions may provide clues to the pigment formation process. This is the first report of detection of ochronotic pigment in acinar cells and lumina in the submandibular gland of a patient with AKU. Deposition was noted in the apical region of the cells. A lobar duct presented a large calculus with unusual deposits possibly associated with calcium salts. This report highlights the effect that local and intracellular factors may have on converting HGA into polymeric derivatives in the absence of an extracellular matrix.

Collagen atomic scale molecular disorder in ochronotic cartilage from an alkaptonuria patient, observed by solid state NMR

In pilot studies of the usefulness of solid state nuclear magnetic resonance spectroscopy in characterizing chemical and molecular structural effects of alkaptonuria on connective tissue, we have obtained 13 C spectra from articular cartilage from an AKU patient. An apparently normal anatomical location yielded a cross polarization magic angle spinning spectrum resembling literature spectra and dominated by collagen and glycosaminoglycan signals. All spectral linewidths from strongly pigmented ochronotic cartilage however were considerably increased relative to the control indicating a marked increase in collagen molecular disorder. This disordering of cartilage structural protein parallels, at the atomic level, the disordering revealed at higher length scales by microscopy. We also demonstrate that the abnormal spectra from ochronotic cartilage fit with the abnormality in the structure of collagen fibres at the ultrastructural level, whereby large ochronotic deposits appear to alter the structure of the collagen fibre by invasion and cross linking. Summary: Increased signal linewidths in solid state NMR spectra of ochronotic articular cartilage from an AKU patient relative to linewidths in normal, control, cartilage reveals a marked decrease in collagen molecular order in the diseased tissue. This atomic level disordering parallels higher length scale disorder revealed by microscopic techniques.