Deposition Of Calcium Phosphate Crystals Research Articles

Pseudo Tumoral Calcinosis and Renal Ostedystrophy in Pathien under Hemodialysis: About a Case

Pseudo tumoral calcinosis is a rare affection, characterized by the deposition of calcium phosphate crystals in peri- articular soft tissues, forming giant mass, calcified and appearing as a compressive tumor. This affection can be hereditary of secondary to chronic renal failure undergoing hemodialysis. In this case, this affection is included in renal osteodystrophy. Radiology is the key for the diagnosis. We herein report the case of 68 years old, with chronic hemodialysis history, presenting with a latero-cervical mass, evolving since one year. CT showed diverse locations of pseudo tumoral calcinosis, associated to renal osteodystrophy and surgical resection was indicated for symptomatic locations.

Calcium Pyrophosphate and Basic Calcium Phosphate Deposition Diseases: The Year in Review 2022

Calcium-containing crystal deposition diseases are a common cause of pain and disability but remain relatively under-investigated. No drug has been identified that can prevent deposition or effect dissolution of either calcium pyrophosphate (CPP) or basic calcium phosphate (BCP) crystals. In comparison to the field of gout and urate biology, published research in relation to calcium crystal deposition diseases in 2022 was relatively modest in quantity. In CPP deposition (CPPD) disease, progress was made mainly in epidemiology, imaging, surgical management and Gitelman’s syndrome. In relation to BCP crystals, the effect on tenocytes in vitro was explored and results indicate that BCP crystals likely reduce tendon matrix integrity via their interaction with tenocytes. The involvement of calcification in the progression of osteoarthritis (OA) was elegantly demonstrated contributing to further discovery of the process of OA progression. There was a paucity of mechanistic and genetic studies in calcium crystal deposition diseases published in 2022, nor any breakthrough in therapy, showing that there is abundant scope for investigation under these themes in the future.

#4000 ROLE OF ENDOTHELIAL CELLS IN VASCULAR CALCIFICATION

Abstract Background and Aims Vascular calcification (VC) represents a common pathological feature of cardiovascular disease and VC caused by hyperphosphatemia is one of the hallmarks of chronic kidney disease (CKD). Therefore, elucidating the pathogenesis of VC may have significant clinical benefits for CKD patients. Vascular smooth muscle cells challenged with high-phosphate (Pi) actively participate to VC by trans-differentiating into simil-osteoblastic cells that acquire the capability to deposit hydroxyapatite crystals in the extracellular matrix of tunica media in arteries. The role of endothelial cells (ECs) in high-Pi calcification has been poorly investigated until now. Nonetheless, following particular stimuli ECs can lose their endothelial characteristics and acquire several different phenotypes transdifferentiating with a process known as endothelial-to-mesenchymal transition (EndMT). Given the central role of VC for CKD patients wellbeing we decided to investigate whether ECs may have a role in VC process. Method We set up an in vitro calcification model with human aortic ECs (HAECs) challenged with 2, 2.5, 3, 3.5 mM high-Pi for 7 days. Calcium deposition, cell viability and cell trans-differentiation were evaluated. Results In our model we observed deposition of calcium phosphate crystals induced by high-Pi that resulted significant from 3 up to 3.5 mM Pi (Ctr 0.43 ± 0.01; 3 mM Pi 6.72 ± 1.15; 3.5 mM Pi 17.31 ± 1.88; micromol Ca++/mg protein; P < .01; Fig 1A-B). We found that calcification is mediated by Pi-influx, as demonstrated by the inhibition of calcium deposition by Phosphonoformic acid (PFA), an inhibitor of Na/Pi cotransporter Pit-1 (3.5 mM Pi 15.52 ± 1.97; 1 mM PFA 0.6 ± 0.05; micromol Ca++/mg protein; P < .0001; Fig 1A, C). In addition, in our model high-Pi did not show any significant toxic effect at every concentration tested compared to control cells. In Pi-treated ECs we observed a clear change in cell-morphology from cobblestone to spindle shaped, a recognized sign of EndMT. Moreover, we observed a change in extracellular matrix composition more similar to osteoblastic matrix. In facts, ECs treated with Pi were Alcian Blue positive, a staining that binds glycosaminoglycans contained in bone matrix, with blue granules of calcium-phosphate in the cytoplasm of treated cells. Starting from day 5 up to day 7 the transition was confirmed by an up-regulation of SNAIL and N-cadherin mRNA expressions, one of the master genes and one the markers of EndMT process, respectively. A modification of endothelial phenotype was also supported by a progressive decrease in protein expression of Von Willebrand factor and VE-Cadherin observed at day 9 of calcification (−53.7% and −32.6% respectively). Moreover, from day 5 till day 7 we found some osteoblastic differentiation signs such as a progressive increase of RUNX2 and BMP2 mRNA expression and an increase of protective MGP mRNA levels. Conclusion Our data demonstrate that HAECs are able to calcify. High-Pi induces trans-differentiation in simil-osteoblastic cells and a progressive loss of endothelial characteristics through the EndMT process. More studies are needed to better elucidate the role for ECs in CKD VC process.

Ultrasound Guided Arthroscopic Removal of Calcific Tendonitis: A Minimum of 2-Year Followup.

Background: We have developed a novel technique for managing rotator cuff calcific tendonitis, involving arthroscopic debridement of calcific tendonitis with localization assistance from a breast biopsy needle under ultrasound guidance. While we have demonstrated encouraging results at six-month follow-up, the medium-term outcomes and the long-term outcomes of this technique at 2 years or beyond are unknown. The aim of this paper was to determine if this technique was successful in resolving symptoms after two years and beyond. Study Design: Retrospective Cohort Study. Methods: Patients who underwent arthroscopic debridement of calcific tendonitis with localization assistance from a breast biopsy needle under ultrasound guidance by a senior surgeon were evaluated using patient-rated pain scores and functional status with the use of the Likert scales and via examiner-rated shoulder range-of-motion and strength at the pre-operative visit, at 1, 6, 12, and 24 weeks post-operatively, and long-term at a mean of 249 weeks after surgery. Results: At a mean follow-up period of 4.8 years (range, 2-10 years), 31 patients (33 shoulders) experienced significant improvement in the severity of pain at rest, with overhead activities, and during sleep compared to their pre-operative presentation (p < 0.001). The patient experienced less frequent pain during activities and sleep, and a decreased frequency of extreme pain (p < 0.001). Passive range of abduction (p = 0.003), forward flexion (p < 0.001), and supraspinatus strength (p = 0.018) improved compared to the presurgical presentation. Out of 27 patients, 24 patients (89%) had complete resolution of calcific tendonitis, and 26 patients (96%) had an intact rotator cuff. Conclusion: Arthroscopic debridement of calcific tendonitis with localization assistance from a breast biopsy needle under ultrasound guidance was very effective. Patients had significant pain relief, improved range of motion, and a reduction in stiffness at a mean post-operative period of 4.8 years. Patients had a significant reduction in residual calcification, and rotator cuff integrity was largely preserved by long-term follow-up. What is known about this subject: Calcific tendonitis of the rotator cuff is one of the most painful and debilitating disorders of the shoulder. This condition is characterized by the deposition of calcium-phosphate crystals within the rotator cuff tendons. Arthroscopic debridement and excision of rotator cuff calcifications have proven to be efficacious treatments with regards to clinical and functional outcomes in the short and medium term. Identifying the calcific lesion intra-operatively, however, can prove to be challenging. Furthermore, inadequate excision of the calcific deposit has been shown to have poorer clinical outcomes. We designed a technique that utilizes the assistance of ultrasound to guide a localization-biopsy wire to the calcific lesion. This technique aids in precisely identifying the location of the lesion intra-operatively to optimize accuracy in removing the maximum amount of calcific deposit possible. A short-term follow-up study by us has demonstrated successful outcomes with regards to the return of function and relief of pain. However, there have been no studies evaluating the effectiveness of this particular technique beyond six months. What this study adds to current knowledge: At a mean of 4.8 years, arthroscopic debridement of calcific tendonitis, using our technique, was successful in relieving the severity and frequency of pain with overhead activities, pain at rest, and pain during sleep, as well as improving range of motion.

Regression of a Primary Pseudo-Tumoral Calcinosis Progressing During 16 Years under Combined Treatment with Acetazolamide and Aluminum Hydroxide: A Case Report

Primary pseudo-tumoral calcinosis is a rare genetic disease due to a disorder of phosphorus metabolism characterized by the formation and deposition of calcium phosphate crystals in the peri-articular regions. The treatment is based on the reduction of hyperphosphatemia by hygienic and nutritional measures, hypophosphatemic drugs and surgery. We report the case of a 19-year-old patient followed since the age of 3 years for a primary pseudo-tumoral calcinosis that was resistant to surgery and that had well progressed with aluminum hydroxide and acetazolamide. Treatment combining aluminum hydroxide and acetazolamide with hygienic and nutritional measures seems to be an interesting therapeutic option for this pathology.

A Proteomic Screen to Unravel the Molecular Pathways Associated with Warfarin-Induced or TNAP-Inhibited Arterial Calcification in Rats

Arterial media calcification refers to the pathological deposition of calcium phosphate crystals in the arterial wall. This pathology is a common and life-threatening complication in chronic kidney disease, diabetes and osteoporosis patients. Recently, we reported that the use of a TNAP inhibitor, SBI-425, attenuated arterial media calcification in a warfarin rat model. Employing a high-dimensionality unbiased proteomic approach, we also investigated the molecular signaling events associated with blocking arterial calcification through SBI-425 dosing. The remedial actions of SBI-425 were strongly associated with (i) a significant downregulation of inflammatory (acute phase response signaling) and steroid/glucose nuclear receptor signaling (LXR/RXR signaling) pathways and (ii) an upregulation of mitochondrial metabolic pathways (TCA cycle II and Fatty Acid β-oxidation I). Interestingly, we previously demonstrated that uremic toxin-induced arterial calcification contributes to the activation of the acute phase response signaling pathway. Therefore, both studies suggest a strong link between acute phase response signaling and arterial calcification across different conditions. The identification of therapeutic targets in these molecular signaling pathways may pave the way to novel therapies against the development of arterial media calcification.

Mitochondrial Dysfunction and Oxidative Stress in Hereditary Ectopic Calcification Diseases

Ectopic calcification (EC) is characterized by an abnormal deposition of calcium phosphate crystals in soft tissues such as blood vessels, skin, and brain parenchyma. EC contributes to significant morbidity and mortality and is considered a major health problem for which no effective treatments currently exist. In recent years, growing emphasis has been placed on the role of mitochondrial dysfunction and oxidative stress in the pathogenesis of EC. Impaired mitochondrial respiration and increased levels of reactive oxygen species can be directly linked to key molecular pathways involved in EC such as adenosine triphosphate homeostasis, DNA damage signaling, and apoptosis. While EC is mainly encountered in common diseases such as diabetes mellitus and chronic kidney disease, studies in rare hereditary EC disorders such as pseudoxanthoma elasticum or Hutchinson-Gilford progeria syndrome have been instrumental in identifying the precise etiopathogenetic mechanisms leading to EC. In this narrative review, we describe the current state of the art regarding the role of mitochondrial dysfunction and oxidative stress in hereditary EC diseases. In-depth knowledge of aberrant mitochondrial metabolism and its local and systemic consequences will benefit the research into novel therapies for both rare and common EC disorders.

Navigation-assisted resection of tumoral calcinosis of the lumbosacral spine: illustrative case

BACKGROUNDTumoral calcinosis is an uncommon disease resulting from dystrophic calcium phosphate crystal deposition, with only 7% of cases involving the spine, and it may diagnostically mimic neoplasms.OBSERVATIONSIn this case, a 54-year-old woman with history of systemic scleroderma presented with 10 months of progressive left lumbosacral pain. Imaging revealed an expansile, 4 × 7-cm, well-circumscribed mass in the lumbosacral spine with L5–S1 neuroforaminal compression. Because intractable pain and computed tomography (CT)-guided needle biopsy did not entirely rule out malignancy, operative management was pursued. The patient underwent L4–S2 laminectomies, left L5–S1 facetectomy, L5 and S1 pediculectomies, and en bloc resection, performed under stereotactic CT-guided intraoperative navigation. Subsequently, instrumented fusion was performed with L4 and L5 pedicle screws and S2 alar-iliac screws. Pathological examination was consistent with tumoral calcinosis, with multiple nodules of amorphous basophilic granular calcified material lined by histiocytes. There was no evidence of recurrence or neurological deficits at 5-month follow-up.LESSONSBecause spinal tumoral calcinosis may mimic neoplasms on imaging or gross intraoperative appearance, awareness of this clinical entity is essential for any spine surgeon. A review of all case reports of lumbosacral tumoral calcinosis (n = 14 from 1952 to 2016) was additionally performed. The case featured in this report presents the first known case of navigation-assisted resection of lumbosacral tumoral calcinosis.

Endothelial dysfunction aggravates arterial media calcification in warfarin administered rats.

Arterial media calcification is an active cell process. This encompasses osteochondrogenic transdifferentiation of vascular smooth muscle cells followed by the deposition of calcium-phosphate crystals. Increasing evidence suggests a significant role for endothelial cells (ECs) in the development of arterial media calcification. This manuscript explores a role for endothelial dysfunction in the disease progression of arterial media calcification. Male rats were randomly assigned to four different groups. The first group received standard chow. The second group was given L-NAME (≈50mg kg-1 · d-1 ), to induce endothelial dysfunction, in addition to standard chow. The third group and fourth group received a warfarin-supplemented diet to induce mild calcification and the latter group was co-administered L-NAME. Prior to sacrifice, non-invasive measurement of aortic distensibility was performed. Animals were sacrificed after 6weeks. Arterial media calcification was quantified by measuring aortic calcium and visualized on paraffin-embedded slices by the Von Kossa method. Arterial stiffness and aortic reactivity was assessed on isolated carotid segments using specialized organ chamber setups. Warfarin administration induced mineralization. Simultaneous administration of warfarin and L-NAME aggravated the arterial media calcification process. Through organ chamber experiments an increased vessel tonus was found, which could be linked to reduced basal NO availability, in arteries of warfarin-treated animals. Furthermore, increased calcification because of L-NAME administration was related to a further compromised endothelial function (next to deteriorated basal NO release also deteriorated stimulated NO release). Our findings suggest early EC changes to impact the disease progression of arterial media calcification.

Abstract 149: Hyperphosphatemic familial tumoral calcinosis due to homozygous mutation at Leu366Arg: First case report

Background: Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disabling autosomal recessive metabolic disorder characterized by hyperphosphatemia and manifestations ranging from progressive deposition of calcium phosphate crystals in the skin, soft tissues and bone, and hyperostosis. The most common cause of HFTC is related to loss-of-function mutations in GALNT3 (Polypeptide N-Acetylgalactosaminyl transferase 3) gene causing defective glycosylation of FGF23.Material and Methods: A 16 year boy presented with lump in the left gluteal area and hyperphosphatemia. Serum ionized calcium, alkaline phosphate, intact PTH, 25-OH vitamin D, serum creatinine and C terminal FGF 23, magnetic resonance imaging of hip, dental CBCT , bone mineral density at lumbar spine and hip by dual x-ray absorptiometry (DEXA) BMD, and 99Tc MDP bone scan, followed by next generation sequencing (NGS) and Sanger's sequencing.Results: A 16 year otherwise healthy boy presented with hard painless lump in the left gluteal area and hyperphosphatemia for last one year. His parents had third degree consanguinity with ancestral roots in Middle East. Father has small calcinosis in the right hand and mother was asymptomatic. Child had no associated bone pain, proximal muscle weakness, fracture or constitutional symptoms. S. phosphorus was 6.98mg/dl (normal: 3.5–5.5) with inappropriately increased TRP (95%) and TMP-GFR (7.2). Serum ionized calcium, alkaline phosphate, intact PTH, 25-OH vitamin D and serum creatinine were normal. MRI showed 5.7x3.7x3 cm lobulated heterogenous hypointensity near left greater trochanter. This mass was excised and showed tissue calcification. 99Tc-MDP bone scan and bone density parameters were normal. CBCT scan maxilla and mandible showed generalized hypercementosis with irregular root morphology. Serum C-terminal FGF 23 was 2401 RU/ml (normal: <150). NGS revealed normal FGF23 and Alpha Klotho genes. Exon 5 of GALNT3 gene showed homozygous mutation at Leu 366 Arg. GALNT3 Leu 366 Arg mutation causing HFTC has been described as compound heterozygous.Conclusion: This is the first case report of homozygous mutation at Leu366Arg causing HFTC.

Кристаліндукований остеоартрит: роль кристалів основного фосфату кальцію

Кристали основних фосфатів кальцію вже давно асоціюються з патогенезом остеоартриту. Основні фосфати кальцію з великою частотою визначаються в синовіальній рідині і суглобовому хрящі при цьому захворюванні. У той час як осадження основних фосфатів кальцію розглядається багатьма вченими як наслідок прогресуючого перебігу остеоартриту, з’явилися суттєві докази того, що ці кристали можуть виступати активними патогенними медіаторами остеоартриту. «Кристали основних фосфатів кальцію» — загальний термін для опису декількох видів фосфатів кальцію, у тому числі карбонатапатиту, гідроксиапатиту, три- і октакальцієвих фосфатів, кристалів вітлокіта магнію. Кристали основних фосфатів кальцію патогенетично пов’язані з остеоартритом, кальцинуючим тендинітом, гострим кристалічним артритом та атеросклерозом. Кристали кальцію визначаються в синовіальній рідині в 65 % випадків остеоартриту колінного суглоба, вони з високою частотою виявляються в гіаліновому хрящі при остеоартриті, а також у 100 % пацієнтів у суглобовому хрящі при проведенні операції тотального ендопротезування. Кристали основних фосфатів кальцію також були виявлені в синовіальній рідині та синовіальній мембрані пацієнтів із легким, помірним і тяжким ураженням колінного суглоба при артроскопії. Основні фосфати кальцію проявляють множинні біологічні ефекти in vitro, у тому числi здатність стимулювати мітогенез, продукцію простагландинів, цитокінів і матриксних металопротеїназ у різних типах клітин, включаючи синовіоцити, хондроцити і макрофаги. Кристали основних фосфатів кальцію також обумовлюють запалення при остеоартриті шляхом прямої взаємодії з вродженою імунною системою. У цьому огляді обговорюються останні досягнення в цій галузі і підбиваються підсумки сучасного розуміння ролі кристалів основних фосфатів кальцію в патогенезі остеоартриту. Погляди на роль кристалів основних фосфатів кальцію в патогенезі остеоартриту продовжують розширюватися, проте вже сьогодні існує достатньо експериментальних і клінічних доказів найважливішої ролі кальційумісних кристалів у розвитку цього захворювання і прогресуючій дегенерації суглобового хряща.

The Interplay of WNT and PPARγ Signaling in Vascular Calcification.

Vascular calcification (VC), the ectopic deposition of calcium phosphate crystals in the vessel wall, is one of the primary contributors to cardiovascular death. The pathology of VC is determined by vascular topography, pre-existing diseases, and our genetic heritage. VC evolves from inflammation, mediated by macrophages, and from the osteochondrogenic transition of vascular smooth muscle cells (VSMC) in the atherosclerotic plaque. This pathologic transition partly resembles endochondral ossification, involving the chronologically ordered activation of the β-catenin-independent and -dependent Wingless and Int-1 (WNT) pathways and the termination of peroxisome proliferator-activated receptor γ (PPARγ) signal transduction. Several atherosclerotic plaque studies confirmed the differential activity of PPARγ and the WNT signaling pathways in VC. Notably, the actively regulated β-catenin-dependent and -independent WNT signals increase the osteochondrogenic transformation of VSMC through the up-regulation of the osteochondrogenic transcription factors SRY-box transcription factor 9 (SOX9) and runt-related transcription factor 2 (RUNX2). In addition, we have reported studies showing that WNT signaling pathways may be antagonized by PPARγ activation via the expression of different families of WNT inhibitors and through its direct interaction with β-catenin. In this review, we summarize the existing knowledge on WNT and PPARγ signaling and their interplay during the osteochondrogenic differentiation of VSMC in VC. Finally, we discuss knowledge gaps on this interplay and its possible clinical impact.

Idiopathic Auricular Calcinosis Cutis.

Calcinosis cutis is a rare disease characterized by the deposition of insoluble calcium phosphate crystals in the epidermis. Etiologically it is divided into 4 types: dystrophic, metastatic, iatrogenic and idiopathic. The most common type is the dystrophic type and the rarest idiopathic type. The pathophysiology of calcinosis cutis is not clearly known. Its definitive diagnosis is made histopathologically by showing calcium accumulation in the skin and clinically excluding other types. A pediatric patient was admitted to our clinic with a left auricular mass, and histopathological findings were diagnosed as idiopathic calcinosis cutis. Our case is presented in the light of literature because it is seen in auricula, an atypical localization of idiopathic calcinosis cutis, which is a rare type.

Extracellular Nucleotides Regulate Arterial Calcification by Activating Both Independent and Dependent Purinergic Receptor Signaling Pathways.

Arterial calcification, the deposition of calcium-phosphate crystals in the extracellular matrix, resembles physiological bone mineralization. It is well-known that extracellular nucleotides regulate bone homeostasis raising an emerging interest in the role of these molecules on arterial calcification. The purinergic independent pathway involves the enzymes ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), 5′-nucleotidase and alkaline phosphatase. These regulate the production and breakdown of the calcification inhibitor—pyrophosphate and the calcification stimulator—inorganic phosphate, from extracellular nucleotides. Maintaining ecto-nucleotidase activities in a well-defined range is indispensable as enzymatic hyper- and hypo-expression has been linked to arterial calcification. The purinergic signaling dependent pathway focusses on the activation of purinergic receptors (P1, P2X and P2Y) by extracellular nucleotides. These receptors influence arterial calcification by interfering with the key molecular mechanisms underlying this pathology, including the osteogenic switch and apoptosis of vascular cells and possibly, by favoring the phenotypic switch of vascular cells towards an adipogenic phenotype, a recent, novel hypothesis explaining the systemic prevention of arterial calcification. Selective compounds influencing the activity of ecto-nucleotidases and purinergic receptors, have recently been developed to treat arterial calcification. However, adverse side-effects on bone mineralization are possible as these compounds reasonably could interfere with physiological bone mineralization.

Imaging of crystalline arthropathy in 2020.

Crystal-related arthropathies are the result of crystal deposition in joint and periarticular soft tissues. Identification of urate crystals is mandatory to distinguish gout from other crystalline arthropathies, including calcium pyrophosphate dihydrate and basic calcium phosphate crystal deposition diseases. ACR/EULAR classification criteria for gout included dual-energy computed tomography and ultrasound with equal impact to the final score. Different diagnostic strengths of these imaging modalities depend on disease duration and scanned anatomic site. While ultrasound has been indicated as the first-choice imaging technique, especially in the early stages of the disease, dual-energy computed tomography has shown to be highly specific, allowing the detection of crystal deposits in anatomic sites not accessible by ultrasound, such as the spine. At the spinal level, MRI findings are usually nonspecific. Finally, there is preliminary evidence that at the knee, dual-energy computed tomography may discriminate calcium pyrophosphate dihydrate from basic calcium phosphate crystal deposits.

In-situ polymerized polypyrrole nanoparticles immobilized poly(ε-caprolactone) electrospun conductive scaffolds for bone tissue engineering.

Despite intensive attempts to fabricate polypyrrole nanoparticles (PPy-NPs) incorporated nanofibrous scaffolds, a low-cost facile strategy is still demanded. Herein, we developed a novel strategy- in-situ polymerization of PPy-NPs and immobilized them into the PCL polymeric matrix in a single step. For the in-situ polymerization of PPy-NPs, ferric chloride hexahydrate (FeCl3.6H2O) was introduced as an oxidant into the blended solution of PCL and pyrrole monomers. Due to the chemical oxidative polymerization process, the clear solution changed into a black PCL/PPy solution. After electrospinning the solution, PCL/PPy composite nanofibers were fabricated. The immobilization of PPy-NPs into PCL matrix was clearly revealed by Bio-TEM images. The Field emission scanning electron microscopy (FESEM) results exhibited that the PCL/PPy scaffolds showed significantly decreased fiber diameter. The atomic force microscopy (AFM) study showed increased surface roughness in the PCL/PPy scaffolds. The mechanical strength test of PCL/PPy scaffolds showed improved Young's Modulus (YM=2 to 4-folds) and tensile strength (TS=3 to 4-folds). As well as the YM and TS were gradually increased with increased concentration of PPy-NPs in composite scaffolds. The conductivity measurement conducted on polymeric solution and electrospun scaffolds showed an increasing trend of conductive property in the PCL/PPy solution and scaffolds too. The surface wettability test exhibited decreased water contact angle measurement from 126° for pure PCL to 93° for the PCL/PPy-200 composite scaffold. The biomineralization test conducted by simulated body fluid (SBF) incubation showed enhanced calcium-phosphate crystal deposition on the PCL/PPy scaffolds. The CCK-8 assay and confocal laser scanning microscopic (CLSM) imaging conducted without and with electrical stimulation (ES) displayed enhanced cell adhesion, growth, and proliferation of MC3T3-E1 cells on the PCL/PPy conductive scaffolds. Furthermore, ALP and ARS staining assays showed significant enhancement of the calcium-phosphate deposition on the PCL/PPy scaffolds after ES treatment. Hence, the current study provides a novel strategy for the fabrication of PCL/PPy conductive scaffolds with enhanced bioactivity, biocompatibility, and osteogenic differentiation under electrical stimulation confirmed its promising application towards bone tissue engineering.

Molecular and Cellular Mechanisms that Induce Arterial Calcification by Indoxyl Sulfate and P-Cresyl Sulfate.

The protein-bound uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are considered to be harmful vascular toxins. Arterial media calcification, or the deposition of calcium phosphate crystals in the arteries, contributes significantly to cardiovascular complications, including left ventricular hypertrophy, hypertension, and impaired coronary perfusion in the elderly and patients with chronic kidney disease (CKD) and diabetes. Recently, we reported that both IS and PCS trigger moderate to severe calcification in the aorta and peripheral vessels of CKD rats. This review describes the molecular and cellular mechanisms by which these uremic toxins induce arterial media calcification. A complex interplay between inflammation, coagulation, and lipid metabolism pathways, influenced by epigenetic factors, is crucial in IS/PCS-induced arterial media calcification. High levels of glucose are linked to these events, suggesting that a good balance between glucose and lipid levels might be important. On the cellular level, effects on endothelial cells, which act as the primary sensors of circulating pathological triggers, might be as important as those on vascular smooth muscle cells. Endothelial dysfunction, provoked by IS and PCS triggered oxidative stress, may be considered a key event in the onset and development of arterial media calcification. In this review a number of important outstanding questions such as the role of miRNA’s, phenotypic switching of both endothelial and vascular smooth muscle cells and new types of programmed cell death in arterial media calcification related to protein-bound uremic toxins are put forward and discussed.

Dual-energy computed-tomography-based discrimination between basic calcium phosphate and calcium pyrophosphate crystal deposition in vivo.

Background:Dual-energy computed tomography (DECT) is being considered as a non-invasive diagnostic and characterization tool in calcium crystal-associated arthropathies. Our objective was to assess the potential of DECT in distinguishing between basic calcium phosphate (BCP) and calcium pyrophosphate (CPP) crystal deposition in and around joints in vivo.Methods:A total of 13 patients with calcific periarthritis and 11 patients with crystal-proven CPPD were recruited prospectively to undergo DECT scans. Samples harvested from BCP and CPP calcification types were analyzed using Raman spectroscopy and validated against synthetic crystals. Regions of interest were placed in BCP and CPP calcifications, and the following DECT attenuation parameters were obtained: CT numbers (HU) at 80 and 140 kV, dual-energy index (DEI), electron density (Rho), and effective atomic number (Zeff). These DECT attenuation parameters were compared and validated against crystal calibration phantoms at two known equal concentrations. Receiver operating characteristic (ROC) curves were plotted to determine the highest accuracy thresholds for DEI and Zeff.Results:Raman spectroscopy enabled chemical fingerprinting of BCP and CPP crystals both in vitro and in vivo. DECT was able to distinguish between HA and CPP in crystal calibration phantoms at two known equal concentrations, most notably by DEI (200 mg/cm3: 0.037 ± 0 versus 0.034 ± 0, p = 0.008) and Zeff (200 mg /cm3: 9.4 ± 0 versus 9.3 ± 0, p = 0.01) analysis. Likewise, BCP calcifications had significantly higher DEI (0.041 ± 0.005 versus 0.034 ± 0.005, p = 0.008) and Zeff (9.5 ± 0.2 versus 9.3 ± 0.2, p = 0.03) than CPP crystal deposits with comparable CT numbers in patients. With an area under the ROC curve of 0.83 [best threshold value = 0.0 39, sensitivity = 90. 9% (81.8, 97. 7%), specificity = 64.6% (50.0, 64. 6%)], DEI was the best parameter in distinguishing between BCP and CPP crystal depositions.Conclusion:DECT can help distinguish between crystal-proven BCP and CPP calcification types in vivo and, thus, aid in the diagnosis of challenging clinical cases, and in the characterization of CPP and BCP crystal deposition occurring in osteoarthritis.

Enhanced bone tissue regeneration of a biomimetic cellular scaffold with co-cultured MSCs-derived osteogenic and angiogenic cells.

ObjectivesThe bone tissue engineering primarily focuses on three‐dimensional co‐culture systems, which physical and biological properties resemble the cell matrix of actual tissues. The complex dialogue between bone‐forming and endothelial cells (ECs) in a tissue‐engineered construct will directly regulate angiogenesis and bone regeneration. The purpose of this study was to investigate whether co‐culture between osteogenic and angiogenic cells derived by bone mesenchymal stem cells (MSCs) could affect cell activities and new bone formation.Materials and methodsMesenchymal stem cells were dually induced to differentiate into osteogenic cells (OMSCs) and ECs; both cell types were co‐cultured at different ratios to investigate their effects and underlying mechanisms through ELISA, RT‐qPCR and MTT assays. The selected cell mixture was transplanted onto a nano‐hydroxyapatite/polyurethane (n‐HA/PU) scaffold to form a cell‐scaffold construct that was implanted in the rat femoral condyles. Histology and micro‐CT were examined for further verification.ResultsELISA and gene expression studies revealed that co‐cultured OMSCs/ECs (0.5/1.5) significantly elevated the transcription levels of osteogenic genes such as ALP, Col‐I and OCN, as well as transcription factors Msx2, Runx2 and Osterix; it also upregulated angiogenic factors of vascular endothelial growth factor (VEGF) and CD31 when compared with cells cultured alone or in other ratios. The optimized OMSCs/ECs group had more abundant calcium phosphate crystal deposition, further facilitated their bone formation in vivo.ConclusionsThe OMSCs/ECs‐scaffold constructs at an optimal cell ratio (0.5/1.5) achieved enhanced osteogenic and angiogenic factor expression and biomineralization, which resulted in more effective bone formation.

Human proximal tubular cells can form calcium phosphate deposits in osteogenic culture: role of cell death and osteoblast-like transdifferentiation

Nephrocalcinosis is a clinicopathological entity characterized by microscopic calcium crystals in the renal parenchyma, within the tubular lumen or in the interstitium. Crystal binding to tubular cells may be the cause underlying nephrocalcinosis and nephrolithiasis. Pathological circumstances, such as acute cortical necrosis, may induce healthy cells to acquire a crystal-binding phenotype. The present study aimed to investigate whether human renal proximal tubular cells (HK-2 cells) can form calcium phosphate deposits under osteogenic conditions, and whether apoptosis and/or osteogenic-like processes are involved in cell calcification. HK-2 cells were cultured in standard or osteogenic medium for 1, 5, and 15 days. Von Kossa staining and ESEM were used to analyze crystal deposition. Apoptosis was investigated, analyzing caspase activation by in-cell Western assay, membrane translocation of phosphotidylserine by annexin V-FITC/propidium iodide staining, and DNA fragmentation by TUNEL assay. qRT/PCR, immunolabeling and cytochemistry were performed to assess osteogenic activation (Runx2, Osteonectin, Osteopontin and ALP), and early genes of apoptosis (BAX, Bcl-2). HK-2 cell mineralization was successfully induced on adding osteogenic medium. Calcium phosphate deposition increased in a time-dependent manner, and calcified cell aggregates exhibited characteristic signs of apoptosis. At 15 days, calcifying HK-2 cells revealed osteogenic markers, such as Runx2, ALP, osteonectin and osteopontin. Monitoring the processes at 1, 5, and 15 days showed apoptosis starting already after 5 days of osteogenic induction, when the first small calcium phosphate crystals began to appear on areas where cell aggregates were in apoptotic conditions. The cell death process proved caspase-dependent. The importance of apoptosis was reinforced by the time-dependent increase in BAX expression, starting from day 1. These findings strongly support the hypothesis that apoptosis triggered HK-2 calcification even before any calcium phosphate crystal deposition or acquisition of an osteogenic phenotype.