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Calcif Tissue Int DOI 10.1007/s00223-017-0260-9

Histological and Ultrastructural Characterization of Alkaptonuric Tissues

Lia Millucci1 · Giulia Bernardini1 · Adriano Spreafico2 · Maurizio Orlandini1 · Daniela Braconi1 · Marcella Laschi1 · Michela Geminiani1 · Pietro Lupetti3 · Giovanna Giorgetti4 · Cecilia Viti4 · Bruno Frediani5 · Barbara Marzocchi1,6 · Annalisa Santucci1 

Received: 10 November 2016 / Accepted: 20 February 2017 © Springer Science+Business Media New York 2017

of ochronotic spondyloarthropathy is known, there are only few data regarding an exhaustive ultrastructural and histo-logic study of different tissues in AKU. Moreover, an in-depth analysis of tissues from patients of different ages, having varied symptoms, is currently lacking. A complete microscopic and ultrastructural analysis of different AKU tissues, coming from six differently aged patients, is here presented thus significantly contributing to a more compre-hensive knowledge of this ultra-rare pathology.

Keywords Alkaptonuria · Histology · Tissues · Amyloidosis

Introduction

AKU is the clinical result of loss of function, missense mutations of the gene that codes for homogentisate 1,2 dioxygenase [1, 2]. Raised blood levels of HGA lead to the deposition of its pigmented oxidation products in many tissues [3]. Virchow [4] noted the ochre-coloured pigmen-tation in histological sections of post-mortem tissues that lead to ochronosis. The main clinical manifestation is the premature degeneration of articular cartilage leading to osteoarthritis and to scoliosis and fusion of the vertebrae [3, 5]. Pigment is deposited in chondrocytes and in the matrix of articular cartilage, as well as in ligaments and elastic cartilages [6]. Pigment deposition around the aortic valve may give rise to aortic valve stenosis, and pigmen-tation of atheromatous plaques has been described [7, 8]. Currently, there is no licensed cure for this disorder [9].

The processes that govern pigment deposition and the mechanisms of pigment toxicity are not well understood, although possible factors include the co-presence of amy-loidosis and angiogenesis [10] in the tissues [10–12] and

Abstract Alkaptonuria (AKU) is a hereditary disor-der that results from altered structure and function of homogentisate 1,2 dioxygenase (HGD). This enzyme, pre-dominantly produced by liver and kidney, is responsible for the breakdown of homogentisic acid (HGA), an intermedi-ate in the tyrosine degradation pathway. A deficient HGD activity causes HGA levels to rise systemically. The disease is clinically characterized by homogentisic aciduria, bluish-black discoloration of connective tissues (ochronosis) and joint arthropathy. Additional manifestations are cardiovas-cular abnormalities, renal, urethral and prostate calculi and scleral and ear involvement. While the radiological aspect

Lia Millucci and Giulia Bernardini have contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s00223-017-0260-9) contains supplementary material, which is available to authorized users.

* Annalisa Santucci annalisa.santucci@unisi.it

1 Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, 53100 Siena, Italy

2 Immunoematologia Trasfusionale, Azienda Ospedaliera Universitaria Senese, Viale Bracci, Siena, Italy

3 Dipartimento di Scienze della Vita, Università degli Studi di Siena, via A. Moro 2, Siena, Italy

4 Dipartimento di Scienze Fisiche, della Terra e dell’Ambiente, Università degli Studi di Siena, Strada Laterina 8, Siena, Italy

5 Dipartimento di Scienze Mediche, Chirurgiche e Neuroscienze, Università degli Studi di Siena, Viale Bracci, Siena, Italy

6 U. O. C. Patologia Clinica, Azienda Ospedaliera Universitaria Senese, Viale Bracci, Siena, Italy

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the oxidative and inflammatory effects of HGA on tissues and cells [13–19]. Pigmentation of tissues in AKU is well documented and is the end point of a process that is not understood. Although HGA is excreted in gram quantities in the urine, its high affinity for collagen can allow it to deposit as a pigmented polymer, particularly in the articu-lar cartilages of the weight-bearing joints. But analysis of different body districts showed that this deposition vary at either the macroscopic or ultrastructural level [20, 21]. Research has shown the close relationship between colla-gen fibres and the ochronotic pigment but as yet there are no data to demonstrate the effects that the pigment has on the cells and functionality of tissues other than cartilage or synovia. Most of the clinical findings may be explained by inhibition of collagen crosslinks, but some require addi-tional interpretation.

Most pathological observations are based on case reports about surgical material that, of necessity, demonstrate end-stage changes at the time of surgery. There is a paucity of information regarding the physiopathological implications of AKU related to different body districts [22] and to pecu-liar specific alterations, especially in young patients.

In this work, for the first time, we present a histological study of different tissues from patients of different ages and with different relevance of symptoms. Our findings provide the first detailed overview of AKU pathology, emphasiz-ing the heterogeneity of AKU phenotype and considering possible pathogenic mechanisms in the decades of life. The histopathological changes in AKU tissues are illustrated by the detailed study at microscopic and ultrastructural level, offering a substantial support to physicians who are often unaware of the causal relationship between AKU, the patients’ arthritic complaints and the histopathological changes.

Materials and Methods

The study was conducted following the approval of the local University Hospital Ethics Committee. All patients gave a written informed consent agreeing with the use of their samples and publication of data (in print and electron-ically), prior to inclusion in the study.

All reagents were from Sigma-Aldrich (St. Louis, MO), if not differently specified.

Patients

AKU patients were recruited from the Rheumatology Clinic of Siena University Hospital. Biopsies and resec-tions of six ochronosis cases were evaluated for histopatho-logical and ultrastructural observations. Patient features are summarized in Supplementary Table 1 and histological and

ultrastructural analyses performed in different tissues of all AKU patients are presented in Supplementary Table 2. In radiological examination, mild-to-severe degenerative changes as joint space narrowing, cartilage irregularities, subchondral sclerosis or peripheral osteophytes as well as linear intervertebral disc calcifications were observed in all cases (Fig. 1).

Congo Red (CR)

CR staining method was adopted. Sections of 3–5  μm thickness of fresh specimens were fixed in cooled 96% ethanol 10 min, rinsed in distilled water, incubated in 1% CR for 40 min, washed in water, incubated 10  s in 1 mL 1% sodium hydroxide in 100 mL of 50% ethanol, incubated 30 s in Mayer’s hematoxylin, sequentially washed in 50, 75, 95% ethanol, mounted and observed under a polarized light microscope (Zeiss Axio Lab.A1, Arese, Milano).

Safranin‑O

Sections were deparaffinized and rehydrated. Slides were stained with Weigert iron hematoxylin and rinsed in water, incubated in Fast Green, differentiated in acetic acid and stained with Safranin-O. Dehydration was performed in ethanol. Tissue-Clear was used, before mounting the sections.

Alizarin Red

Staining for tissue mineralization was performed using standard protocol for Alizarin Red.

Picrosirius Red

Sections were mounted on slides and stained with Picro-sirius Red (Polysciences, Inc), following the manufacturer instructions.

Transmission Electron Microscopy (TEM)

Samples were fixed in 2.5% glutaraldehyde in 0.1 M caco-dylate buffer (CB) pH 7.2 for 3 h at 4 °C. After rinsing in CB, samples were post-fixed in 1% osmium tetroxide in CB for 2 h at 4 °C, dehydrated in a graded series of ethanol and embedded in a mixture of epon–araldite resins. Thin sections, obtained with a Reichert ultramicrotome, were stained with uranyl acetate and lead citrate and observed with a TEM FeiTecnai G2 spirit at 80 Kv.

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Scansion Electron Microscopy (SEM)

Scanning electron microscopy (SEM) was performed using a Philips XL30, operating at 20  kV and equipped with an EDAX-DX4 energy dispersive spectrometer (EDS). The atomic percent of single elements has been calculated following the ZAF correction method. The spectrometer detects the characteristic X-rays of elements with atomic number >6 (i.e. C). Counting rate was kept close to 2200–2300 counts per second over the whole energy spectrum, with a counting time of 50 live-sec-onds. Analytical precision, checked by repeated analyses,

was better than 0.5% for major elements and better than 20% relative for minor elements (i.e. those ranging from 0.3 wt% up to 3–5 wt%). The volume of sample ana-lysed with EDAX, at the actual operating condition, was estimated to have a diameter of ca. 3  μm; hence, areas smaller than ca. 5 μm will have chemical analyses mixed with adjacent phases. As a first step, the fragment was analysed without carbon coating to check the presence of C in the sample itself. Once verified that C was a com-ponent of the ochronotic pigment, the fragment was car-bon coated in order to obtain good quality images under SEM.

Fig. 1 Clinical examination of the pigment deposition and the mac-roscopic aspect of the pathology. a, b The pigment deposition is most conspicuous where the stratum corneum is thickest as the palm of the hands (case 3). c Bilateral deposition of ochronotic pigment in the sclera (case 2). d Generalized black pigmentation of the articular car-tilages. The joint surfaces are shown intraoperatively (case 2). e Sam-ples of AKU cartilage, meniscus and tendons after total knee replace-

ment (case 3). f Right femur head. The cartilage (black) is abraded in the weight-bearing zone (arrow) leaving a bare, eburnated and flattened underlying femoral head. A marginal overgrowth on—pig-mented hyperplastic synovium represents an extrinsic element of this osteoarthrotic remodelling, and thus can be considered as an equiva-lent of osteophyte. Some islets of pigmented cartilage splits grafted in the synovium are visible as black areas (case 5)

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Results

The clinical presentation of AKU is heterogeneous and virtually every organ can be involved. Despite important advancements in research, many key questions are still open about AKU. Response to these questions would be a premise for developing novel therapies aimed at interfering with specific components of the pathogenic cascade. Mac-roscopical features of AKU patients are depicted in Fig. 1, whereas different tissues were presented in individual sec-tions with the description of performed analysis.

Cartilage

Macroscopically, all cases showed brown to black col-ouring in articular cartilages (Fig.  1d, e). The fragility of AKU cartilage leads to fragmentation of upper layer, caus-ing the detachment of small fragments (shards), included in the synovium, resulting in inflammatory and degenera-tive aspects and exposure of the subchondral bone [23]. Fibrillation and eburnation were present in samples of AKU elderly patients, but not in the 2 young patients exam-ined. Histologic evaluation of not stained samples (Fig. 2) showed thickened and fibrotic areas of cartilage with dark pigmentation in AKU cartilage of elderly patients, while none of similar features were present in specimens from the young patients, where chondrocytes were regular in shape and no tears or fissures were visible. Nevertheless, also cartilage from patients 4 and 6 showed the presence of extracellular perilacunar pigmentation (Supplemen-tary Fig. 1). Cartilage from elderly patients appeared more encrusted of pigment, scattered in the tissue and isogenous groups located in pigmented areas appear distrophic and irregular in shape (Supplementary Fig.  1). We also per-formed comparative analysis with a healthy young control and an osteoarthritic (OA) control, age-matched with aged AKU patients (Fig. 2), and the difference was evident for the absence of pigmentation. OA cartilage showed some degeneration of the tissue and the presence of distrophic lacunae, confirming the parallelism with this condition and AKU. To assess the presence of systemic amyloidosis, we performed Congo Red staining and in all AKU cases, and a strong birefringence was observed, especially evident in more fibrillated and degraded areas of the tissue. Pigmented areas are particularly birefringent, suggesting a strong cor-relation between ochronotic pigment and amyloid fibrils deposition (Fig. 2). No sign of amyloid presence was found in control samples. In elderly AKU patients, the integrity of the superficial zone of cartilage was lost, changes extended into deeper zones and separation, and disorganization of the superficial collagen fibrils was evident as evidenced by Picrosirius staining (Fig.  2). In normal cartilage, collagen fibres were cross-linked covalently to other type collagen

molecules, in particular, to homotypic polymers of type II collagen (yellow stained by Picrosirius), the physiologi-cally more abundant form of collagen in human adult artic-ular cartilage. On the contrary, significant amounts of type III collagen were present in all AKU cartilage samples, evidenced by green colour (Fig. 2). In healthy controls, the main collagen was the normal type II collagen, while in OA a little green colour can be observed, suggesting that also in this case repair process of matrix damage also started but to a lesser extent than in AKU. The present findings empha-size the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage. The Safranin-O staining of cartilage coming from patients 4 and 6 (at initial stage of pathology) showed a normal structure, with superficial horizontally aligned chondrocytes, while in the deep layers vertically orientated rows of chondrocytes were disturbed. Proteoglycans (PGs) were reduced (Fig.  2), although intensified staining was observed around the chondrocytes. These chondrocytes are supposed to produce more Glycosaminoglycans (GAGs) as a reaction to the loss of PGs from the matrix in the super-ficial zone. A pale discoloration characterized the superfi-cial articular cartilage, which appeared smooth with only slight erosions, but with very low presence of PGs, similar to OA cartilage. On the contrary, cartilage from patient 2 was abnormally damaged, completely disrupted and totally lacking PGs as evidenced by Safranin absence of staining. Mineralization was observed in degraded and fibrillated areas of cartilage of elderly patients, mainly in proximity of articular surface (Fig. 2 Alizarin Red). No calcification nor foci of mineralization were observed in articular cartilage of young patients as well as in healthy control. OA carti-lage showed a more intense discoloration only in peripheral area of the sample, probably indicating a marginal zone of calcification. At an ultrastructural observation (Fig.  3), pigment seemed to be present in large amounts filling up the whole chondrocyte (asterisk) and, in some cases, cov-ering the nucleus. Numerous amyloid fibrils were diffused around the rest of an apoptotic chondrocyte releasing cel-lular debris (Fig.  2a, arrows). Cells overfilled with dense vacuoles were observable (Fig.  2b–d), from which pig-ment granules were released to gradually form deposited granules and lumps between the fibres (Fig. 2b big arrows, e, f). Collagen fibres appeared completely disorganized (Fig.  2e, f; Supplementary Fig.  2a) and intertwined with drop-like pigment granules (big arrows). Amyloid bundles of protofibrils and fibrils were scattered in the whole tissue and in some area superimposed to collagen fibres (Fig. 2e arrows). Irregularity in the plasma membrane are frequent and nuclei are often pycnotic (Fig.  2b–d; Supplementary Fig.  2b). OA cartilage was adopted as positive control.

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Fig. 2 Images of AKU cartilage from young and elderly patients, healthy controls and OA cartilage. Not stained samples of articular cartilage from young AKU patient showing extracellular perilacunar pigmentation (arrow) evident also in non-pigmented areas. The sam-ple is not fissurated or fibrotic and the typical columnar orientation of chondrocytes is maintained. Sample from elderly patient showed more intense pigmentation, more prominent in upper middle parts of the articular cartilage, and the integrity of the superficial zone of car-tilage was lost. Congo Red staining of articular cartilage of young and elderly AKU patients showed green birefringence demonstrating the massive presence of amyloid deposits in AKU cartilage specimens, especially in correspondence to pigmented areas. In the articular car-tilage from elderly AKU patient, the green staining of Picrosirius Red under polarized light indicates the presence of huge amount of col-

lagen III, sign of cartilage repair process in a seriously damaged zone. The yellow colour of healthy cartilage confirms the presence of type II collagen as expected in normal cartilage, while in OA sample some green fibril is visible indicating a partial repair process of ECM also in this pathology. Safranin-O/fast green staining for GAG showed that hyaline cartilage from young AKU patient, even though less degraded and fissured in appearance, did not exhibit GAG staining suggesting an early proteoglycans depauperation in AKU, while cartilage from elderly AKU patient is massively disrupted and no Safranin-O stain-ing was observed indicating complete absence of PGs. Alizarin Red showed foci of mineralization in AKU cartilage of elderly patient, principally in the more fibrillated and damaged zone. On the contrary, no foci of mineralization were observed in cartilage of young patient also in widely pigmented area

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Images G and H show regular collagen fibrils arrangement and size distribution, with normal axial periodicity.

Bone

The femoral head showed full-depth ochronotic pigmen-tation across all regions of AKU femoral head and loss of articular cartilage from bone surface (Fig.  1f), a sharp distinction between the weight-bearing and non-weight-bearing zones (Fig.  4a). In the latter, the cartilaginous pigmented surface remained and the underlying bone was osteoporotic. In the overloaded part (Fig.  1f), ochro-notic pigment was found in the bone matrix, resulting in degeneration and fragility of the tissue (Fig.  4a). In sam-ples not completely pigmented, the focal ochronotic depos-its were in regions subjected to the greatest load bearing during locomotion. This is in accordance with reports in literature that confirmed ochronotic pigmentation to be accentuated in more loaded zone of the bone [24, 25]. SEM images (Fig.  4b) showed that the pigmentation was consistently associated with the territorial matrix and a boundary line between black and white bones was clearly visible (Fig. 4b arrow). The SEM low-magnification image (Fig. 4c) showed that most of the bone surface was covered by a dark-grey groundmass, disseminated of micrometre

white granules and clumps with an idiomorphic habit. EDS analyses revealed that the dark material was composed of C, O, N, S, Na and variable amount of Ca, while the white areas were composed of P and Ca, hence showing an apa-tite composition (Fig. 4d).

Synovia

Macroscopically (Fig.  5a), synovia appeared as a dull grey and mild villous hypertrophy is present. Oedematous aspect was common in all samples and increased vascu-larity was observed (Fig. 5b). The brittle cartilage formed “spicule-like” pieces, grouped or isolated in the synovia (Fig.  5c–h), occasionally embedded in the marrow of

Fig. 3 TEM imaging of AKU. Representative images are reported. a AKU chondrocyte are characterized by an abundant rough endoplas-mic reticulum (RER) and by prominent Golgi complexes. Nucleus show irregular outlines (arrow) and contains a very condensed chro-matin (asterisk). Multiple protrusions of the cell membrane are evi-dent spaced out with zones of disruption of the cellular membrane. b Disintegration of cell resulting from a combination of digestion in autophagic vacuoles and of the release of resultant cell fragment into the extracellular space is here observable. In this terminal stage of cell death, cytoplasm organelles were not recognizable and only some nuclear remnant is often visible. Arrows indicate amyloid showing the fibrillar nature of the deposits. c AKU chondrocytes showed char-acteristic signs of degeneration and organelle destruction: increased amounts of condensed heterochromatin in the nuclei, anular chroma-tin condensation at the nuclear envelope, budding of the nuclear enve-lope and clusters of swollen mitochondria. d Prominent RER showing hardly visible ribosomes, enhanced, fragmented and expanded Golgi membranes, dense cytosol, dense chromatin, cell fragmentation and disintegration. Arrow indicates amyloid scattered fibrils in the ECM. e, f Ultrastructural appearance of AKU ECM is shown: patchy pig-ment deposition is visible (light arrows) and disruption of collagen fibres is evident. The remarkable sparse dotted pigmentation, distrib-uted within the tissue, appears to be responsible of the collagen fibres fragmentation and of the lack of their periodicity. Trace of amyloid fibrils thinly interconnected to collagen broken fibrils (big arrows) is randomly diffused and interposed with pigment droplets in the dis-organized net of collagen fibres. g, h Healthy and OA controls show a more regular collagen network, with appreciable periodicity. The fibrils of the AKU pigmented radial zone were irregular in diam-eter, being narrower than those of healthy cartilage in some tract but presenting some fragment generally wider than in control samples. Moreover, in AKU, many fibrils appeared to be swollen into an amor-phous structure with loss of striations

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the remodelled bone. Sometimes, cartilage pieces were phagocytosed by histiocytes and giant cells and contain-ing pigment granules (Fig.  5c, d). They induced a more

or less pronounced synovial hyperplasia, as proved by intensive inflammatory cellular reaction. Synoviocytic hyperplastic layer was visible, where shards could be also

Fig. 4 AKU bone. a Sagit-tal and transverse section of preceding femur head specimen. Macroscopic view. Pre-existing pigmented cartilage remains also in the non-weight-bearing zones in this case, i.e. supero-lateral and infero-medial; some pigmented tissue (cartilaginous splits or ligamentous bundles) remains in the fovea capi-tis (arrow) as well as in the periosteum. b SEM observa-tion of AKU bone. White bone areas (circles) correspond to mineral component (Apatite), dark areas are incrusted of ochronotic pigment. Boundary line between black and white areas is not well defined, as the pigment seems scattered in granular manner to invade white zones (arrow). c SEM observations on the ribbon-like fragments and detritus of femur head, showing a homogeneous area with small white clumps, having an idiomorphic, platy habit. Square indicates the mag-nificated area of panel A and the zone analysed by EDS. d EDS spectra of pigmented area of AKU bone and ochronotic pigment element composition. Microanalyses of the dark sur-face are quite constant revealing that the area is composed of C, O, N, S, Na and Ca. The white areas are composed of Apatite

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found (Fig.  5e). The sub-lining showed fibrotic aspects with focal or diffuse lymphomonocitar infiltrates (Fig. 5b, c). Most of the ochronotic shards were surrounded by inflammatory cells and multinucleated cells typical of

a foreign body inclusion (Fig.  5e, f). Massive amyloid deposits were also present in synovial tissue, confirm-ing the co-localization of ochronosis and amyloid fibrils (Fig. 5g, h).

Fig. 5 AKU Synovia. a AKU synovial hyperplasia with black pigmentation. b H&E staining of synovial capsular tissue with signs of lymphocyte infiltration and a high grade of vasculariza-tion, which indicates inflamma-tion but not an acute reaction. c, d Big cartilage fragments detached and embedded in syn-ovial tissue (shards), surrounded by macrophages and giant cells, swallowed by pigment granules. e, f Severely degraded synovial with shards and numerous lym-phocytic infiltrates. g, h Congo Red staining of AKU synovial showing diffuse amyloid depos-its specially in correspondence of ochronotic shards

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Tendon

In fibrous tissues as tendons, ochronotic pigment deposited homogeneously. The ligamentum patellae were ochronotic and the tendon resulted partially necrotic, due to the pig-ment imbibition. Morphological observations indicated an initiation of calcification occurring on the surface of col-lagen fibrils associated with changes in the collagen fibril structure that appeared straighter and pack into narrower bundles (Fig.  6a, b). In areas far from the mineralization site, tendon cells were spindle-shaped and extended into the ECM, eventually connecting with processes of neighbour-ing cells (Fig. 6c). The tendon cells had increased amounts of endoplasmic reticulum, Golgi apparatus and thin cellu-lar processes that weave between tightly packed collagen fibrils (Fig.  6d; Supplementary Fig.  3). Vesicles contain-ing pigment were observable within and outside cells. In the dense network of pigmented collagen fibrils, bundles of amyloid fibrils finelly interdispersed and surrounding cellu-lar debris were visible (Fig. 6d). These features were com-pared to those reported in the literature for normal tendons [26, 27].

Infrapatellar Fat Pad (IFP)

IFP can be regarded as a peculiar form of fibro-adipose tissue localized close to the synovial membrane and articular cartilage. The precise function of the IFP is unknown, although studies showed that it may play a role in the biomechanics of the knee or act as a store for repar-ative cells after injury. In AKU patients, inflammation and fibrosis within the IFP were evident because it was vascularized by a rich anastomotic network and presented

a rich innervation. Ochronotic deposition was observed in the form of granular material, uniformly strewn in the tis-sue and also in proximity of numerous Pacinian corpus-cles (Fig. 6a, b). Amyloid was present clumped in regular bundles and frequently associated to scattered pigment granules (Fig. 6c, d, f). Elastic fibres were absent in the

Fig. 6 TEM images of AKU Tendon and IFP. Upper panel repre-sentative fascicle structure found in an AKU tendon observed by TEM. a Bundles of collagen fibrils encrusted by pigment deposits. b Axial section of collagen fibrils in AKU tendon. Drop-like pig-ment granules are present and diffusely scattered in the inner part of the fibres. c Tenocytes with abnormal Golgi apparatus, dysmorphic nuclei and numerous vesicles containing pigment. d Amyloid fibrils (circles) are present in the tendons arranged in disorganized clusters. In this photo, amyloid fibrils are near cellular debris and interspersed with granules of pigment. Lower panel TEM images of AKU IFP. a The subcutaneous adipose tissue of the knee showing nerves sur-rounded by thick multilaminated connective tissue capsules both in the adipose lobules and in the interlobular septa. Pacinian corpuscles were recognizable along the interlobular septa and in the adipose lob-ules (arrow). b Free nerve fibres (arrows) were recognizable within adipose lobules (arrowheads). c A cell with degenerative changes, such as an irregular cytoplasmatic membrane (arrow) and disaggre-gated nucleus, near a big fat vesicle (arrowhead). d Amyloid fibrils (arrow) passing near big granular pigment mass. e At the level of the IFP, the intercellular space is larger and thick fibrils of collagen are evident. Near the adipocytes of the knee, the collagen fibres are organized in bundles. f Circle highlights massive deposition of amy-loid disposed in parallel bundles of fibrils

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IFP where, on the contrary, we found a prevalence of col-lagen fibres (Fig. 6e).

Cardiovascular Tissue

Analysing the aortic valve of case 1, severe annular cal-cification extending into the left ventricular outflow tract

transitioning towards the aorta and dark blue discoloration of the valve cusps were noted (Fig.  7a). The aortic valve showed mild cusp fusion and calcification of the cusps with prominent pigment deposition, particularly the left coro-nary cusp (Fig. 7a).

Histological examination showed pigmented cells in the ECM (Fig.  7b, c). Pigment appeared as fine granules or

Fig. 7 AKU Aortic valve. a Brown pigmentation is present in aortic valve leaflets. b, c Light microscopy (H&E) examinations of AKU aortic valve with intense inflammatory cell infiltrate pigment appeared as fine granules (asterisk) or crystals, mixed with areas of calcification (arrows). d, e Congo Red staining of AKU aortic valve amyloid deposits. The pigmented areas were also birefringent (arrows), indicat-ing overlapping of ochronosis and amyloid. f Myelin figures in the aortic valve confirmed the presence of advanced lipid peroxidation. g ECM organiza-tion is disrupted and contains fragmented collagens and elas-tin fibres strictly interconnected to pigment deposits. In this photo, amyloid fibrils are in the proximity of a pigment granule

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crystals, mixed with areas of calcification. Diffuse amyloid deposits were found (Fig.  7d, e) in correspondence with pigmented areas.

Amyloid was evident at ultrastructural examination. Numerous myelin figures were visible (Fig. 7g), confirming that lipid peroxidation was extensively present.

Salivary Gland

The presence of amyloid was diffuse in the tissue, not being confined around the acini and excretory ducts, suggesting

a peculiar pathway of amyloid deposition in AKU. TEM confirmed the massive presence of amyloid fibrils located in the secretory stroma and adjacent to the basement mem-brane (Fig. 8c, d), as well as in the interstitial connective tissue, interspersed with collagen fibrils (Fig.  8e, f) and around interlobular ducts. The glandular stroma contained numerous broken collagen fibrils interconnected with amyloid fibrils, arranged in a scattered manner. Evident pigment traces finely sprinkled over the tissue (Fig. 8e, f) as individual “drop-like” deposits between the collagen fibres and scattered over amyloid fibrils. This confirmed

Fig. 8 AKU LSG. a, b Congo Red staining under polarized light detected the presence of massive amyloid deposits within AKU salivary gland. c, d TEM images showing fine amyloid fibrils located in the interstitial connective tissue stroma of AKU LSG. Pigment granules are visible amongst collagen fibrils in transverse and longi-tudinal section (black arrows) and near secretory acini. e, f Pigment deposits are present on broken collagen fibres and scattered between amyloid fibrils. Collagen fibres present-ing electron dense ochronotic deposits located amongst fibres are shown. Dark ochronotic pigment granules are indicated (arrows) scattered amongst the collagen fibre as well as amongst amyloid fibrils

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the presence of pigment also in LSG, not pigmented when observed at naked eye.

Discussion

The recognition of the distinctive features of AKU, includ-ing its histological and ultrastructural appearance, is neces-sary to catalogue AKU clinical behaviour and to define the most appropriate treatment.

The examination of six AKU cases with the pathological study of different tissues, demonstrates the following gen-eral aspects of ochronosis:

1. Cartilage deterioration was observed not only after long exposure and impregnation with HGA and its derivatives, but also in young asymptomatic people.

2. Alteration in collagen composition and PGs depletion were present also in young patients, whereas calcifica-tion and fibrillation were observed in elderly patients.

Depletion of GAGs was present also in young patients. In a recent work of Taylor et al. [28], the study of tissue turnover in AKU cartilage showed significant reduc-tion in GAG and demonstrated the ochronotic pigment effect on other matrix components. This supports our finding that the biological ageing process had surely influence on joint tissue turnover of these patients [23], but the degradation of ECM is not related only to in the last decades of life. The GAG depletion, especially in superficial and intermediate zones, exposes the superficial collagen fibrils and, in elderly, leads either to disorganization of the superficial zone collagen network, water influx and thickening and softening of cartilage. Simultaneously, calcified cartilage and sub-chondral bone plate grow thicker with remodelling of subchondral trabecular bone as a consequence of both increased resorption and formation of the subchondral bone matrix. These functional and biophysical changes may likely contribute to the matrix changes described by us and Taylor and others in a number of elegant and rigorous works [29–33]. Moreover, the observed changes in cartilage matrix composition described here may induce the observed alterations in articular carti-lage and bone and may contribute to their progression by facilitating HGA to polymerize substituting GAG, or alternatively but not exclusively, to prey GAGs as a nucleation seed. There is a possibility that the greater loss of GAG in AKU allows for greater extracellular amyloid deposition, and this is potentially combined with an increased amount of collagen of type III found in the matrix, binding larger amounts of protein as reported by Taylor et  al. [27]. Due to the early onset

of AKU, rapidly progressing to osteoarthropathy, our finding that GAG are lost very early in AKU patients may play a key role in the accelerated disease progres-sion typical of this disease.

3. Most information in the literature is representative of the end stage of the disease, when the disruptive and degenerative changes are almost complete. Here we showed that the deterioration of the cartilage was char-acterized by brittleness and fragmentation and was observed only in tissues from elderly patients. This is in agreement with a previous report from Taylor et al. [34] that specifically measures Young’s modulus. Nev-ertheless, we found molecular changes also in young patients. An important AKU feature was the presence of collagen III in young and elderly AKU patients. The collagen framework of hyaline cartilages, includ-ing articular cartilage, normally consists largely of type II collagen. In AKU articular cartilages, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues [35]. These results suggest a phenotypic alteration in AKU chon-drocytes [36, 37] in conditions of diseased cartilage [38, 39], expressing and synthesizing collagen type III. The lack of collagen II, at least at the detection levels, besides high levels of type III collagen in AKU carti-lage was unexpected. It is possible that chondrocytes can remodel micro-anatomical domains of collagen-ous matrix (e.g. replacing fibril-surface molecules, damaged fibrils or pericellular collagen) more rapidly than bulk collagen of the inter-territorial matrix. The collagen type III expression by articular chondrocytes appeared to be relatively early in the AKU pathogen-esis, at least before overt destruction of all articular surfaces in AKU joint has occurred. It has been shown that pigmentation starts pericellularly [32], but due to the complex relationship existing between chondro-cytes and their pericellular matrices, this likely exac-erbates processes that drive alteration in tissue com-position. In virtue of this, our finding may indicate a metabolic response of AKU chondrocytes depositing type III collagen in regions of articular cartilage, pre-sumably as a response to inflammatory injury or other matrix damage. The effect may be akin to the wound-healing role of type III collagen and can open new per-spective to explore the potential for a novel biomarker of AKU process.

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These data represent an important advance in the under-standing of AKU cartilage modifications and turnover that could help to find new therapeutical approaches.

In all synovia samples, severely inflamed AKU tissues containing lymphoid aggregates were observed, considered diagnostic of inflammatory joint disease. The vascular bed is particularly well represented in the subsynoviocytic layer. The overall appearance shows both degenerative charac-ter and clear inflammatory aspects that may be related to a foreign body reaction induced by cartilage shards and to the pro-oxidative action of HGA. Therefore, AKU angio-genesis and inflammation [9] seem to be both essential pro-moters of the damage of the AKU joint, through cartilage degradation and osteophyte formation.

Tendon disorders are a major problem for AKU patients and rupture of Achilles tendon is an important clinical man-ifestation of AKU [Mistry 2013]. Deposits of HGA pig-ment in the collagenous tissues affect the structural integ-rity of collagen [Mistry JB 2013]. This probably increases the possibility of tendon rupture due to their high collagen content, since HGA may inhibits collagen cross-linking. However, there are limited data on ultrastructural appear-ance of tendons in ochronosis. Ultrasound examination of Achilles tendon showed loss of fibrillary pattern, increased thickness, small focus of calcification and increase in size of retrocalcaneal bursa [40, 41]. In our case, ultrastruc-tural analysis of AKU Achille-tendon biopsies showed the degenerated and altered structure of collagen network and of tenocytes in AKU patients. AKU tendons appear grey and amorphous to the naked eye as reported in similar AKU patients [42] and microscopy revealed discontinuous and disorganized collagen fibres. These changes accom-panied by the increasingly conspicuous presence of pig-mented fibroblasts within the tendon tissue. A discontinuity of collagen fibres, all encrusted of drop-like pigment was evident. The most significant feature was the absence of inflammatory cells.

The microscopic characteristics of the IFP in the general population are poorly described. In a previous work [12], we described the presence of numerous long, straight and parallel bundles of amyloid fibrils in the IFP of an AKU patient. Here we present a more exhaustive ultrastructural analysis of IFP in AKU to give an in-depth view also of this tissue, anatomically crucial for the knee biomechan-ics. The fibrous adipose tissue of AKU patients presented a very thick collagenic peri-adipocyte net which was prob-ably related to the peculiar mechanical tissue performances that can be found in such fat pad. In fact, the IFP is located in the spaces between the patella, the patellar tendon, the femoral condyles and the tibial plateau, and it can absorb forces at the level of the knee. TEM analysis confirmed that intercellular space was rich in collagen fibres, due to the mechanical role of the subcutaneous tissues. But

the disruptive presence of pigment and the massive inva-sion of amyloid fibrils in AKU contributed to the loss of protective role of knee subcutaneous tissue, with respect to deep structures, such as tendons, articular capsules and ligaments. Therefore, knowledge of the IFP lesions in AKU may be important for the appropriate management of these cases.

The study of aortic valve from an AKU patient showed the presence of deposits of amyloid in AKU valve. This may represent an evident example of degenerative tissue change due to the action of ochronotic pigment. Therefore, it is strongly likely that, in AKU, this condition is a sequel of local complication of progressive destruction of the val-vular connective tissue and may be worsened by the local HGD expression in heart [7, 8]. This may explain why, although valve is poorly vascularized, ochronosis is rel-evant in such type of tissue and why aortic valve stenosis and/or regurgitation is the most significant AKU clinical cardiovascular manifestation.

Finally, serum amyloid A (SAA) amyloidosis was reported as a complication in AKU [7], thus detection of amyloid deposits at an early phase in patients may be important for treatment, as in the majority of other kinds of systemic amyloidosis [43, 44] With this aim, we performed labial salivary gland (LSG) biopsies in all patients. Sys-temic amyloidosis was diagnosed in all patients (Fig.  7a, b). The analysis of LSG of all AKU patients confirmed the massive presence of amyloid fibrils. The fibrils were also present in the interstitial connective tissue, interspersed with collagen and around interlobular ducts of salivary glands. Moreover, thanks to TEM analysis, it was possi-ble to see evident pigment traces finely sprinkled over the whole tissue. This was a very interesting finding, since even though macroscopic pigmentation of connective tis-sues in AKU is well documented, deposition in less com-mon regions is not deeply studied as well. The fact that also tissues not apparently pigmented reveal a sparse dotted pig-mentation at ultrastructural observation may provide clues to the pigment formation process. An interesting work of Taylor et al. [45] showed for the first time the presence of ochronotic pigment in acinar cells and lumina in the sub-mandibular gland of a patient with AKU. In this work, authors showed that the ochronotic pigment deposited in the acinar cells and duct lumina of the submandibular gland can be intracellular.

This is perfectly in accordance with our analysis of AKU LSG. In the LSG of all analysed patients, we found exten-sive dotted pigmentation, finely sprinkled over the whole tissue as individual “drop-like” deposits between the colla-gen fibres and also scattered over amyloid fibrils. Moreover, pigment was seen intracellularly, confined into numerous vacuoles in acinar cells and also scattered in the cytoplasm of fibroblasts, near the lamina propria (Supplementary

L. Millucci et al.

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Fig.  4). This supports the thesis that pigment may not require a collagenous scaffold to deposit and that local and intracellular factors may play a role in his deposition. The fact that also in younger patients this pigmentation was observable and abundant, intra- and extracellularly, sug-gests that, analogously to other AKU tissues, accumulation of oxidized HGA (BQA) may be also due to local produc-tion of ochronotic pigment and that HGA aggregation and polymerization can be an autonomous process, not neces-sarily requiring the presence of collagen or amyloid. Being now ascertained that this deposition undoubtedly starts in an early phase in AKU, this may provide insights into the mechanisms of pigment deposition and may alert clini-cians to monitor AKU patients even in the absence of other symptoms.

Our findings depict a novel biological framework in which AKU may be viewed either as a systemic disease or as a metastatic disorder leading to multi-organ failure, rep-resenting a challenge requiring a multi-organ approach.

Overall, our data underscore the importance of recog-nizing the pathological involvement of multiple tissues in AKU, not only cartilage. The clinical burden of AKU may notably increase due to the development of major attention that physicians can pay to histological features of AKU. Not least, the co-presence of amyloidosis and cartilage and tendons degeneration also in young patients is worrying.

Acknowledgements This work has been supported by Tel-ethon Italy Grant GGP10058 and FP7 Research & Innovation Grant 304985-2–DevelopAKUre. The authors also thank Toscana Life Sciences Orphan_1 project, Fondazione Monte dei Paschi di Siena and aimAKU–Associazione Italiana Malati di Alcaptonu-ria (ORPHA263402). Prof. P. P. Mariani and Dr. D. Gambera are acknowledged for providing cartilage and Dr. E. Vannuccini and Dr. E. Paccagnini for their technical assistance.

Compliance with Ethical Standards

Conflict of interest None.

Informed Consent All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethi-cal standards. Informed consent was obtained from all patients being included in the study.

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