Fourier-Transform Infrared Spectroscopy of Epiretinal Membranes and Internal Limiting Membranes after Pars Plana Vitrectomy with Membrane Peeling.

INTRODUCTION: Fourier-transform infrared imaging (FTIRI) enables examination of protein secondary structure in the analyzed tissues. The aim of our study was to examine the distribution of secondary structures in epiretinal membranes (ERMs) and internal limiting membranes (ILMs), and to explore possible associations to other diagnostic variables. METHODS: This prospective pilot study included patients scheduled for pars plana vitrectomy with membrane peeling. ERMs and ILMs were harvested during surgery and placed on a BaF2 window for postsurgical FTIRI analysis. Infrared hyperspectral images were subjected to second and fourth derivative analysis to obtain information of the protein secondary structures present in the tissues. RESULTS: Samples of 43 patients were analyzed, with the triple helical domain showing the highest prevalence in the examined tissues. The other secondary structures (beta-sheet, random coil, and beta-turn) showed a heterogenous distribution in the examined samples, without specific associations to indication of surgery, comorbidities, outcomes from optical coherence tomography, and intrasurgical findings. CONCLUSIONS: FTIRI enables analysis of the spatial distribution of protein secondary structures in the examined tissues; thus, it is a useful analytical technique for the analysis of ERMs and ILMs.

Alterations of bone material properties in adult patients with X-linked hypophosphatemia (XLH).

X-linked hypophosphatemia (XLH) caused by PHEX mutations results in elevated serum FGF23 levels, renal phosphate wasting and low 1,25-dihydroxyvitamin D. The glycophosphoprotein osteopontin, a potent inhibitor of mineralization normally degraded by PHEX, accumulates within the bone matrix. Conventional therapy consisting of supplementation with phosphate and vitamin D analogs is burdensome and the effects on bone material poorly characterized. We analyzed transiliac bone biopsies from four adult patients, two of them severely affected due to no diagnosis and no treatment until adulthood. We used light microscopy, qBEI and FTIRI to study histology, histomorphometry, bone mineralization density distribution, properties of the organic matrix and size of hypomineralized periosteocytic lesions. Non-treatment resulted in severe osteomalacia, twice the amount of mineralized trabecular volume, multiple osteon-like perforations, continuity of lamellae from mineralized to unmineralized areas and distinctive patches of woven bone. Periosteocytic lesions were larger than in treated patients. The latter had nearly normal osteoid thicknesses, although surface was still elevated. The median calcium content of the matrix was always within normal range, although the percentage of lowly mineralized bone areas was highly increased in non-treated patients, resulting in a marked heterogeneity in mineralization. Divalent collagen cross-links were evident independently of the mineral content of the matrix. Broad osteoid seams lacked measurable pyridinoline, a mature trivalent cross-link and exhibited considerable acidic lipid content, typically found in matrix vesicles. Based on our results, we propose a model that possibly integrates the relationship between the observed mineralization disturbances, FGF23 secretion and the known osteopontin accumulation in XLH.

Bone Matrix Mineralization and Response to Burosumab in Adult Patients With X-Linked Hypophosphatemia: Results From the Phase 3, Single-Arm International Trial.

X-linked hypophosphatemia (XLH) is characterized by excess fibroblast growth factor 23 (FGF23) secretion, renal phosphate wasting, and low 1,25(OH)2 D3 . Adult patients present with osteomalacia, hypomineralized periosteocytic lesions, bone fragility, and pain. Burosumab is a fully human monoclonal FGF23 antibody approved for XLH treatment. UX023-CL304 was an open-label, phase 3 study investigating the effects of burosumab on osteomalacia in adults with XLH, who remained untreated at least 2 years prior enrollment. Here, we present the effect of burosumab on bone material properties. We analyzed transiliac bone biopsy samples from 11 individuals before and after 48 weeks of subcutaneous burosumab treatment (1.0 mg/kg administered every 4 weeks). We used quantitative backscattered electron imaging (qBEI) and Fourier transform infrared imaging (FTIRI) to assess bone mineralization density distribution (BMDD), mineralized bone volume, properties of the organic matrix, and size of periosteocytic lesions. The outcomes were compared with reference values from healthy adults and with four XLH patients either untreated or treated by conventional therapy. Prior to burosumab, the average mineralization in cancellous bone was lower than in healthy reference. CaLow, the fraction of lowly mineralized matrix, and CaHigh, the fraction of highly mineralized matrix, were both elevated resulting in a broad heterogeneity in mineralization (CaWidth). Burosumab resulted in a decrease of CaHigh toward normal range, whereas CaLow and CaWidth remained elevated. The mineralized bone volume was notably increased (+35.9%). The size of the periosteocytic lesions was variable but lower than in untreated XLH patients. FTIRI indicated decreased enzymatic collagen crosslink ratio heterogeneity. In summary, matrix mineralization in XLH is very heterogeneous. Highly mineralized regions represent old bone packets, probably protected from osteoclastic resorption by osteoid seams. The concomitant decrease of highly mineralized matrix, persistence of lowly mineralized matrix, and increase in mineralized bone volume after burosumab suggest a boost in mineralization of preexisting unmineralized or very lowly mineralized matrix, providing a potential explanation for previously observed improved osteomalacia. © 2022 American Society for Bone and Mineral Research (ASBMR).

Mineral and organic matrix composition at bone forming surfaces in postmenopausal women with osteoporosis treated with either teriparatide or zoledronic acid.

The ability of bone to resist fracture is dependent on the composite nature of its mineral and organic matrix content. Teriparatide (TPTD) and zoledronic acid (ZOL) are approved anabolic and antiresorptive therapies, respectively, to reduce fracture risk in women with postmenopausal osteoporosis. In the SHOTZ study, postmenopausal women with osteoporosis were treated with TPTD (20 µg daily, subcutaneous) or ZOL (5 mg/year, intravenous infusion) for 24 months. Iliac crest biopsies were obtained at 6 months and again at 24 months from approximately one third of the original study cohort. To investigate the early effects of these two drugs on the quality of newly formed bone, we used vibrational spectroscopic techniques to analyze tetracycline-labelled transiliac biopsies obtained from participants at the 6-month time point. Raman spectra were acquired at forming trabecular and intra-cortical surfaces (identified by fluorescent double labels), to determine mineral, organic matrix, glycosaminoglycan, and tissue water content, as well as mineral maturity/crystallinity at three specific tissue ages (1-5, 15, and ≥25 days). Fourier transformed infrared microspectroscopy was used to determine pyridinoline/divalent collagen cross-link ratios. At 6 months, treatment with TPTD versus ZOL resulted in lower mineral and higher organic matrix content, increased tissue water content, and lower mineral/matrix, mineral maturity/crystallinity, glycosaminoglycan content, and pyridinoline/divalent enzymatic collagen cross-link ratio. Our results suggest that TPTD and ZOL have differential effects on material properties of newly formed bone at individual remodeling sites, highlighting their different mechanisms of action.

Impact microindentation assesses subperiosteal bone material properties in humans.

Impact microindentation (IMI) is a Reference Point Indentation technique measuring tissue-level properties of cortical bone in humans in vivo. The nature, however, of the properties that can affect bone strength is incompletely understood. In the present study we examined bone material properties in transiliac bone biopsies obtained concurrently with measurements of Bone Material Strength index (BMSi) by IMI in 12 patients with different skeletal disorders and a wide range of BMD, with or without fractures (8 males, 4 females, mean age 48±12.2 (SD) years, range 15-60 years). IMI was performed in the mid-shaft of the right tibia with a hand-held microindenter (OsteoProbe). Cancellous and cortical bone mineralization density distributions (BMDD) were measured in the entire biopsy bone area by quantitative backscattered electron imaging. Raman measurements were obtained right at the outer edge of the cortex, and 5, 50, 100, 500µm inwards. The calculated parameters were: i) Mineral and organic matrix content as well as the mineral / matrix ratio. ii) Nanoporosity. iii) Glycosaminoglycan content. iv) Pyridinoline content. v) Maturity/crystallinity of the apatite crystallites. There was no relationship between BMSi values with any measurement of mineral content of whole bone tissue (BMD, BMDD) or maturity/crystallinity of bone mineral. On the other hand, a positive correlation between BMSi and local mineral content, and an inverse correlation between BMSi and nanoporosity at the mineralized subperiosteal edge of the sample and at 5µm inwards was found. A positive correlation was also observed between BMSi and pyridinoline content at the same locations. These results indicate that local mineral content, nanoporosity and pyridinoline content at the subperiosteal site in the transiliac bone biopsy are linked to the BMSi values measured in the tibia. As both high porosity at the nano level and low pyridinoline content of the bone matrix can negatively impact bone strength, our findings suggest that BMSi most likely assesses subperiosteal bone material properties.

Bone tissue material composition is compromised in premenopausal women with Type 2 diabetes.

Type 2 diabetes mellitus (T2DM) patients are at an increased risk of fracture despite normal to high bone mineral density (BMD) values. In this cross-sectional study we establish bone compositional properties in tetracycline labeled iliac crest biopsies from premenopausal women diagnosed with T2DM (N = 26). Within group comparisons were made as a function of tissue age (TA), presence of chronic complications (CC), glycosylated haemoglobin (HbA1c) levels, and morphometric fracture (MFx). We also compared these data at actively trabecular bone forming surfaces against sex- and age-matched healthy controls (N = 32). The bone quality indices determined by Raman microspectroscopic analysis were: mineral/matrix (MM), tissue water content (nanoporosity; NanoP), mineral maturity/crystallinity (MMC), and glycosaminoglycan (GAG), pyridinoline (Pyd), N-(carboxymethyl)lysine (CML), and pentosidine (PEN) content. Within the T2DM group, at the oldest tissue, CML and PEN contents were significantly elevated in the cancellous compared to cortical compartment. The outcomes were not dependent on MFx. On the other hand, both were significantly elevated in patients with CC, as well as those with HbA1c levels > 7%. At actively forming surfaces, the cortical compartment had higher NanoP compared to cancellous. Still within the T2DM group, patients with MFx had significantly elevated MM and GAGs compared to the ones that did not. At actively forming trabecular surfaces, compared to healthy women, T2DM patients had elevated GAGs content and MMC. The results of this study indicate increased AGEs in those with poor glycation control and chronic complications. Additionally, T2DM patients had elevated MMC and decreased GAGs content compared to healthy controls. These alterations may be contributing to the T2DM inherent elevated fracture risk and suggest a role for hyperglycemia on bone quality.

Impact microindentation measurements correlate with cortical bone material properties measured by Fourier transform infrared imaging in humans.

Bone Material Strength index (BMSi) measured by Impact Microindentation is generally lower in subjects with fragility fractures independently of BMD values. We recently reported that in humans, BMSi values are strongly associated with material properties of subperiosteal mineralized bone surface (local mineral content, nanoporosity, pyridinoline content). In the present study we investigated the relationship of BMSi with material properties of the whole bone cortex, by analyzing thin sections of iliac crest biopsies (N = 12) from patients with different skeletal disorders and a wide range of BMD with or without fractures, by Fourier transform infrared imaging (FTIRI). The calculated parameters were: i) mineral and organic matrix content and their ratio (MM), ii) mineral maturity/crystallinity (MMC) and iii) the ratio of pyridinoline (Pyd) and divalent collagen cross-links (XLR). Results were expressed as images, which were converted to histogram distributions. For each histogram the characteristics recorded were: mean value, mode (most often occurring value), skewness, and kurtosis and their association with BMSi values was examined by correlation analysis. BMSi values were significantly correlated only with MM mean and mode values (r = 0.736, p = 0.0063, and r = 0.855, p = 0.0004, respectively), and with XLR mode values (r = -0.632, p = 0.0274). The results of the present study demonstrate that BMSi values are strongly associated with MM, a metric that corrects the mineral content for the organic matrix content, and may also depend on organic matrix quality. These and our previous observations strongly suggest that BMSi assesses material properties of cortical bone.

Bone matrix hypermineralization associated with low bone turnover in a case of Nasu-Hakola disease.

Analysis of tissue from a 34-years-old male patient from Austrian origin with a history of multiple fractures associated with painful episodes over the carpal, tarsal and at the end of the long bones respectively is presented. Radiographic images and axial 3DCT scans showed widespread defects in trabecular bone architecture and ill-defined cortices over these skeletal sites in the form of discrete cystic-like lesions. Family history indicated two sisters (one half and one full biological sisters) also with a history of fractures. Whole exome sequencing revealed two heterozygous missense mutations in TYROBP (MIM 604142; NM_003332.3) gene encoding for a cell-surface adaptor protein, which is part of a signaling complex triggering activation of immune responses. It is expressed in cells of the ectoderm cell linage such as NK and dendritic cells, macrophages, monocytes, myeloid cells, microglia cells and osteoclasts. The phenotype and genotype of the patient were consistent with the diagnosis of Nasu-Hakola disease (NHD) (OMIM 221770). Investigations at the bone material level of a transiliac bone biopsy sample from the patient using polarized light microscopy and backscatter electron imaging revealed disordered lamellar collagen fibril arrangement and extensively increased matrix mineralization. These findings are the first bone material data in a patient with NHD and point toward an osteoclast defect involvement in this genetic condition.

Influence of artificially-induced porosity on the compressive strength of calcium phosphate bone cements.

The biological and mechanical nature of calcium phosphate cements (CPC's) matches well with that of bone tissues, thus they can be considered as an appropriate environment for bone repair as bone defect fillers. The current study focuses on the experimental characterization of the mechanical properties of CPCs that are favorably used in clinical applications. Aiming on evaluation of their mechanical performance, tests in compression loading were conducted in order to determine the mechanical properties of the material under study. In this context, experimental results occurring from the above mechanical tests on porous specimens that were fabricated from three different porous additives, namely albumin, gelatin and sodium alginate, are provided, while assessment of their mechanical properties in respect to the used porous media is performed. Additionally, samples reinforced with hydroxyapatite crystals were also tested in compression and the results are compared with those of the above tested porous CPCs. The knowledge obtained allows the improvement of their biomechanical properties by controlling their structure in a micro level, and finds a way to compromise between mechanical and biological response.