Beginnings of nephrolithiasis: insights into the past, present and future of Randall's plaque formation research.

PURPOSE OF REVIEW: Kidney stones form as a result of heterogeneous nucleation on a calcium phosphate lesion in the renal papilla known as Randall's plaque. Stone disease has plagued humans for millennia with relatively little progress made in the realm of prevention. An understanding of the historical aspects of research into Randall's plaque is necessary to interpret novel correlative imaging discoveries. Focus for the past several decades has been on the distal papillary tip, and the overlooked Anderson-Carr-Randall progression is revitalized with novel supporting evidence. RECENT FINDINGS: Novel correlative techniques of three-dimensional micro-XCT imaging combined with electron and light microscopy techniques have revealed that the earliest mineralization event in the papilla is a distinct event that occurs proximal to the region where Randall's plaque has traditionally been identified. SUMMARY: The history of Randall's plaque research and the Anderson-Carr-Randall progression is reviewed. Proximal intratubular mineral deposits in normal and Randall's plaque affected papillae may be a target for future therapeutic interventions for nephrolithiasis. Further collaboration between nephrologists and urologists is necessary to cure this debilitating disease.

Anatomic variations and stone formation.

PURPOSE OF REVIEW: The pathophysiological mechanisms in kidney stone formation are insufficiently understood. In order to achieve a better understanding of the complexity of stone formation, studies evaluating anatomical variations in the renal papillae have been performed. This review intends to illuminate recent findings. Moreover, new techniques to improve the understanding and interpretation of crystallization mechanisms are reviewed. RECENT FINDINGS: Due to improvements of digital ureteroscopes, detailed endoscopic mapping of renal papillae is now possible. Connections between papillary morphology and histopathological changes in different subsets of stone formers have been documented. The formation of kidney stones seems to take place in relation to Randall's plaques, Ducts of Bellini or by free formation. Additionally, theories of kidney stone formation because of vascular injury or inflammatory events in the papillae have been suggested. SUMMARY: Novel techniques including improved digital endoscopic visualization, microcomputed tomography (CT), electron microscopy and energy dispersive compositional analyses of kidney stones seem essential in the search for effective and reliable methods to understand stone forming processes, which ultimately should result in effective measures for more personalized stone prevention strategies in the future.

Renal papillary attenuation differences between primary and recurrent idiopathic calcium stone disease patients.

AIM: The aim of this paper was to investigate whether renal papillae of patients with nephrolithiasis are more radiodense than that of control patients and to evaluate the predictability of urolithiasis using papillary density differences between stone and non-stone formers. METHODS: Renal papillary Hounsfield Unit (HU) measurements were conducted at the level of upper pole, middle region and lower pole of both kidneys in a total of 126 primary (group 1), 133 recurrent (group 2) stone disease patients and 108 controls (group 3). RESULT: Mean patient age did not differ significantly between groups (P>0.05). Mean stone diameters (±SD) were 5.0±3.1 mm (3-9 mm) and 6.1±3.3 mm (3-15 mm) for primary and recurrent groups, respectively and group distributions and variances were similar (P>0.05). Mean papillary attenuation values (±SD) were 27.26±9.30 (4.00-56.00) in group 1, 30.42±9.88 (12.00-64.00) in group 2 and 25.83±2.72 (20.30-32.56) in the control group. The difference between the mean papillary attenuation value of the primary stone disease group and the control group was statistically insignificant (P=0.104). When the control group and the recurrent stone group was compared without variances, in terms of the mean renal papillary attenuation value, a statistical significance was achieved (P=0.000). CONCLUSION: With increasing renal papillary HU values, the risk of recurrent calcium stone disease is increased.

Renal papillary calcification and the development of calcium oxalate monohydrate papillary renal calculi: a case series study.

BACKGROUND: The objective of this study is to determine in a case series (four patients) how calcified deposits in renal papillae are associated with the development of calcium oxalate monohydrate (COM) papillary calculi. METHODS: From the recently collected papillary calculi, we evaluated retrospectively patients, subjected to retrograde ureteroscopy, with COM papillary lithiasis. RESULTS: The COM papillary calculi were found to result from subepithelial injury. Many of these lesions underwent calcification by hydroxyapatite (HAP), with calculus morphology and the amount of HAP in the concave zone dependent on the location of the calcified injury. Most of these HAP deposits grew, eroding the epithelium covering the renal papillae, coming into contact with urine and starting the development of COM calculi. Subepithelial HAP plaques may alter the epithelium covering the papillae, resulting in the deposit of COM crystals directly onto the epithelium. Tissue calcification depends on a pre-existing injury, the continuation of this process is due to modulators and/or crystallization inhibitors deficiency. CONCLUSIONS: Since calculus morphology and the amount of detected HAP are dependent on the location and widespread of calcified injury, all types of papillary COM calculi can be found in the same patient. All patients had subepithelial calcifications, with fewer papillary calculi, demonstrating that some subepithelial calcifications did not further evolve and were reabsorbed. A high number of subepithelial calcifications increases the likelihood that some will be transformed into COM papillary calculi.

Role of osteopontin in early phase of renal crystal formation: immunohistochemical and microstructural comparisons with osteopontin knock-out mice.

Osteopontin (OPN) is an important matrix protein of renal calcium stone. However, the function of OPN in the early phase of renal crystal formation is not well defined. In this study, we examined OPN expression in the early phase of renal crystal formation with ultra-microstructural observations and immuno-TEM (transmission electron microscopy) in control and OPN knock-out (OPN-KO) mice. Glyoxylate (100 mg/kg) was intra-abdominally administered to male wild-type mice (C57BL/6, 8 weeks of age) and OPN-KO mice (C57BL/6, 8 weeks of age). Kidney was collected before and 6, 12, and 24 h after administration. We examined the relation between renal crystal formation and microstructural OPN location using TEM and immunohistochemical staining of OPN as well as western blotting and quantitative RT-PCR for OPN. OPN protein expression gradually increased in the renal cortex-medulla junction after glyoxylate administration, and OPN mRNA was increased until 12 h, but decreased at 24 h. In ultra-microstructural observation, OPN began to appear on the luminal side of renal distal tubular cells at 6 h and was gradually detected in the tubular lumen at 12 h. OPN was present in the crystal nuclei and collapsed mitochondria in the tubular lumen. In the OPN-KO mice, collapsed mitochondria were present, but no crystal nuclei formation were detected at 24 h. Based on the results this study proposed that the appearance of organelles, such as mitochondria and microvilli, in the tubular lumen after cell injury may be the starting point of crystal nucleus formation due to the aggregation ability of OPN.

[Infectious genesis of nephroliths (electron-microscopic study)].

Scanning electron microscopy and x-ray microstructural analysis were employed in the study of nephroliths from patients suffering from nephrolithiasis. Bacterial biofilms, urease producing microorganisms, alkaline reaction of the urine are basic factors for local urine crystallization, formation of the base of the nephroliths and its rigid fixation to the pelvic mucosa. Mechanic trauma of the pelvic tissues by the concrement results in destruction of the pelvic mucosa epithelium at the site of the nephrolith. Subsequent inflammation in the underlying connective tissue contributes to formation of connective tissue commissures fixing the conrement in the kidney. It is shown that bacteria as a part of a biofilm are capable to persist in nephroliths for a long time. Destruction of the stones during operation or lithotripsy can trigger activation of growth of bacteria integrated in the biofilm and cause septic complications. Preservation of commissures with elements of the destroyed stone after lithotripsy or surgical removal is one of the leading causes of recurrent nephrolithiasis.

Non-infectious phosphate renal calculi: fine structure, chemical and phase composition.

BACKGROUND: Chemical composition of internally non-homogeneous phosphate stones should be related to the conditions prevailing during the formation of each individual part. OBJECTIVE: The object of this paper was to provide a detailed study of phosphate stone composition on the micro- and macro-scales. METHODS: Fine inner structure, chemical and phase composition of 10 phosphate calculi from different patients were determined by chemical (wet) analysis, observation by scanning microscope, semi-quantitative determination of Ca, Mg, P and C by energy dispersive X-ray and by X-ray diffraction. Results. Eight calculi are formed by amorphous calcium phosphate and two by hydroxyapatite. Magnesium was inversely related to Ca/P ratio. Point chemical composition of solid phase varies in wide limits, i.e. composition of calculus interior is highly inhomogeneous on the microscale. All studied calculi contained an abundance of organic matter incorporated in their volume; the content of carbon was double the calcium content in molar quantities. CONCLUSIONS: Phosphate renal calculi with the low Ca/P molar ratio predominantly consist of amorphous calcium phosphate whereas those with a high Ca/P molar ratio are composed of poorly crystalline hydroxyapatite which can be partially carbonated. Magnesium may be an inhibitor of HAP formation from urine. Abundant organic matter incorporated into the calculus volume indicates its decisive role at stone formation. Variable point composition of stones implies widely varying conditions during their development.

Differential bound proteins and adhesive capabilities of calcium oxalate monohydrate crystals with various sizes.

Adhesion of calcium oxalate (CaOx) crystals onto renal tubular epithelial cells is one of the critical steps in kidney stone formation. However, effects of crystal size on the crystal adhesive capability remained unclear. This study compared the adhesive capabilities of CaOx monohydrate (COM) crystals with various sizes (<10 µm, 20-30 µm, 50-60 µm, and > 80 µm). Crystal-cell adhesion assay showed size-dependent increase of COM crystal adhesion onto epithelial cell surface using the larger crystals. Identification of apical membrane proteins that could bind to COM crystals by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) demonstrated size-specific sets of the COM crystal-binding proteins. Among these, numbers of known oxalate-binding proteins and COM crystal receptors were greatest in the set of the largest size (>80 µm). Atomic force microscopy (AFM) revealed that adhesive forces between carboxylic-immobilized AFM tip and COM crystal surface and between COM-mounted AFM tip and renal epithelial cell surface were size-dependent (greater for the larger crystals). In summary, the adhesive capability of COM crystals is size-dependent - the larger the greater adhesive capability. These data may help better understanding of the pathogenic mechanisms of kidney stone formation at an initial stage when renal tubular cells are exposed to various sizes of COM crystals.

Antibacterial effect of fosfomycin tromethamine on the bacteria inside urinary infection stones.

OBJECTIVE: This study sought to evaluate the antibacterial effect of fosfomycin tromethamine (FT) on the bacteria inside urinary infection stones. METHODS: The internal structures of urinary stones were observed via scanning electron microscopy to verify the presence of internal bacteria. We randomly assigned equal numbers of patients with kidney stones who met the inclusion criteria into two groups in a prospective study and treated them with different perioperative antibiotics. One group (experimental group) was treated with FT, and the other (control group) was treated with cefuroxime sodium. All stone specimens were collected via percutaneous nephrolithotomy (PCNL). The primary infection stones were screened via a stone component analysis, 30 cases in the experimental group and 31 cases in the control group. High-performance liquid chromatography (HPLC)-mass spectrometry was used to measure the drug concentration inside the stones, the bacterial count was calculated via stone culture, and the clinical infection index were monitored for between-group comparisons. RESULTS: Compared with the control group, the experimental group had a higher internal drug concentration, a higher drug sensitivity against various pathogenic bacteria, a lower bacterial colony count in the stone culture, and a lower incidence of postoperative clinical infection. CONCLUSIONS: FT is more effective than cefuroxime, which is commonly used during the perioperative period of urinary stones, and exerts a high antibacterial effect on these internal bacteria, and effectively reduces the probability of infection and sepsis after urinary stone surgery. FT can be used as an antibiotic during the perioperative period of urinary stones.

Proteomic analysis of a matrix stone: a case report.

Matrix stones are radiolucent bodies that present as soft muco-proteinaceous material within the renal collecting system. Following wide-angle X-ray diffraction (XRD) and scanning electron microscopy (SEM), we homogenized a surgically removed matrix stone, extracted and purified protein, and analyzed samples using tandem mass spectrometry for proteomic composition. Resulting spectra were searched using ProteinPilot 2.0, and identified proteins were reported with >95% confidence. Primary XRD mineral analysis was a biological apatite, and SEM revealed fibrous, net-like laminations containing bacterial, cellular, and crystalline material. Of the 33 unique proteins identified, 90% have not been previously reported within matrix stones and over 70% may be considered inflammatory or defensive in nature. Characterization of other matrix stone proteomes, in particular from non-infectious populations, may yield insights into the pathogenesis of this rare stone as well as the mineralogical process that occurs within crystalline calculi.

Urates in uric acid renal calculi.

OBJECTIVES: To investigate the presence of calcium urate, sodium, potassium and calcium in 'pure' uric acid calculi. METHODS: Ten spontaneously passed uric acid urinary calculi have been examined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and energy dispersive X-Ray analysis. RESULTS: The analysis of selected uric acid calculi revealed the presence of a near-pure calcium urate in two cases and of calcium enriched urate zones in all of the samples. Furthermore, in some cases complex urates containing sodium, potassium and calcium in different proportions appeared on the surface of the uric acid calculi studied, potassium being generally predominant. Ammonium urate was not detected. CONCLUSIONS: Most urinary uric acid calculi are not pure in composition. 'Complex urates', sodium, potassium and calcium were found together in various proportions in many areas of uric acid stones.

The Agreement Between Current Stone Analysis Techniques and SEM-EDAX in Urolithiasis.

PURPOSE: Nowadays, there are many physical and chemical methods available for urinary stone analysis. According to the latest guidelines, infrared spectroscopy (IR) or x-ray diffraction (XRD) are the two preferred methods in this issue. Therefore, we decided to do a practical comparison between the two above-mentioned techniques with a reference method in order to set up a proper analysis method in our clinical laboratories. MATERIALS AND METHODS: A total of 60 kidney stones were obtained at Labbafinejad hospital through open surgery or percutaneous nephrolithotomy. Then stone analysis techniques included both a morphological examination by SEM (Scanning Electron Microscopy) and internal structure analysis by EDAX (Elemental distribution analysis X-ray), XRD, IR and wet chemical analysis. SEM together with EDAX (SEM-EDAX) was considered as reference methods. RESULTS: The results of XRD had the highest agreement with SEM-EDAX analysis (93%), while the total agreement of FTIR and wet chemical analysis was 81% and 71% respectively. The agreement of FTIR for calcium oxalate stones was acceptable (90%), but for uric acid and cystine stones was challenging (65% and 76% respectively). CONCLUSION: Our results revealed that XRD is more reliable than FTIR; but considering cost issues, FTIR is more suitable for routine clinical laboratory. Moreover, wet chemical analysis, which is routinely used in our laboratories is insufficient for stone analysis and it is mandatory to be replaced by techniques that are more accurate.

Delving into the Antiurolithiatic Potential of Tribulus terrestris Extract Through -In Vivo Efficacy and Preclinical Safety Investigations in Wistar Rats.

Modern treatment interventions for kidney stones are wrought with side-effects, hence the need for alternative therapies such as plant-based medicines. We have previously documented through in vitro studies that statistically optimized aqueous extract of Tribulus terrestris (Zygophyllaceae family) possesses antiurolithic and antioxidant potential. This provides strong scientific foundation to conduct in vivo efficacy and preclinical safety studies to corroborate and lend further proof to its ability to prevent and cure kidney stones. The preventive and curative urolithiatic efficacy in experimentally induced nephrolithiatic Wistar rats, along with preclinical toxicity was evaluated following oral administration of statistically optimized aqueous extract of T. terrestris. Treatment showed augmented renal function, restoration of normal renal architecture and increase in body weight. Microscopic analysis of urine revealed excretion of small sized urinary crystals, demonstrating that treatment potentially modulated the morphology of renal stones. Tissue enzymatic estimation affirmed the antioxidant efficacy of treatment with reduced free radical generation. Significant upregulation of p38MAPK at both the gene and protein level was noted in hyperoxaluric group and interestingly treatment reversed it. Acute oral toxicity study established the Median Lethal Dose (LD50) to be greater than 2000 mg/kg body weight (b.wt.) No observed adverse effect level (NOAEL) by repeated oral toxicity for 28 days at 750 mg/kg b.wt. was noted. This study lends scientific evidence to the safe, preventive and curative potential of statistically optimized aqueous extract of T. terrestris at a dose of 750 mg/kg b.wt. and suggests that the extract shows promise as a therapeutic antiurolithic agent.

Kidney stone disease.

About 5% of American women and 12% of men will develop a kidney stone at some time in their life, and prevalence has been rising in both sexes. Approximately 80% of stones are composed of calcium oxalate (CaOx) and calcium phosphate (CaP); 10% of struvite (magnesium ammonium phosphate produced during infection with bacteria that possess the enzyme urease), 9% of uric acid (UA); and the remaining 1% are composed of cystine or ammonium acid urate or are diagnosed as drug-related stones. Stones ultimately arise because of an unwanted phase change of these substances from liquid to solid state. Here we focus on the mechanisms of pathogenesis involved in CaOx, CaP, UA, and cystine stone formation, including recent developments in our understanding of related changes in human kidney tissue and of underlying genetic causes, in addition to current therapeutics.

Identifying small pelvic inclusions through SEM technology.

Tiny calcified structures may be occasionally recovered during excavation of human skeletal remains. Since taphonomic processes may displace these structures from their topographical relation with neighbouring organs or bones, differential diagnoses may pose a major challenge to the archeologist and/or anthropologist. Enteroliths, kidney stones or gallstones, phleboliths, calcified ganglia, or sesamoid bones account for most of such calcified tiny structures. In addition to their pure medical/paleopathological interest, some remains may be related to diet, to chronic haemolytic conditions, and/or to infections or chronic intestinal diseases. We here describe the technical procedures carried out to confirm or refute the identification of a sesamoid bone. The object in question was a small (5 × 3 × 2 mm) calcified structure that appeared over the right coxal bone of an 18th century individual buried in the church Nuestra Señora de La Concepción, in Santa Cruz de Tenerife (Canary Islands). For comparative purposes we also analyzed kidney stones and gallstones from modern individuals. As shown in this study, scanning electron microscope (SEM) analysis is the preferred method to establish a precise differential diagnosis in these cases.

Phase composition and morphological characterization of human kidney stones using IR spectroscopy, scanning electron microscopy and X-ray Rietveld analysis.

Pathological calcification in human urinary tract (kidney stones) is a common problem affecting an increasing number of people around the world. Analysis of such minerals or compounds is of fundamental importance for understanding their etiology and for the development of prophylactic measures. In the present study, structural characterization, phase quantification and morphological behaviour of thirty three (33) human kidney stones from eastern India have been carried out using IR spectroscopy (FT-IR), powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM). Quantitative phase composition of kidney stones has been analyzed following the Rietveld method. Based on the quantitative estimates of constituent phases, the calculi samples have been classified into oxalate (OX), uric acid (UA), phosphate (PH) and mixed (MX) groups. Rietveld analysis of PXRD patterns showed that twelve (36%) of the renal calculi were composed exclusively of whewellite (calcium oxalate monohydrate, COM). The remaining twenty one (64%) stones were mixture of phases with oxalate as the major constituent in fourteen (67%) of these stones. The average crystallite size of whewellite in oxalate stones, as determined from the PXRD analysis, varies between 93 (1) nm and 202 (3) nm, whereas the corresponding sizes for the uric acid and struvite crystallites in UA and PH stones are 79 (1)-155 (4) nm and 69 (1)-123(1) nm, respectively. The size of hydroxyapatite crystallites, 10 (1)-21 (1) nm, is smaller by about one order of magnitude compared to other minerals in the kidney stones. A statistical analysis using fifty (50) kidney stones (33 calculi from the present study and 17 calculi reported earlier from our laboratory) revealed that the oxalate group (whewellite, weddellite or mixture of whewellite and weddellite as the major constituent) is the most prevalent (82%) kidney stone type in eastern India.

Geobiology reveals how human kidney stones dissolve in vivo.

More than 10% of the global human population is now afflicted with kidney stones, which are commonly associated with other significant health problems including diabetes, hypertension and obesity. Nearly 70% of these stones are primarily composed of calcium oxalate, a mineral previously assumed to be effectively insoluble within the kidney. This has limited currently available treatment options to painful passage and/or invasive surgical procedures. We analyze kidney stone thin sections with a combination of optical techniques, which include bright field, polarization, confocal and super-resolution nanometer-scale auto-fluorescence microscopy. Here we demonstrate using interdisciplinary geology and biology (geobiology) approaches that calcium oxalate stones undergo multiple events of dissolution as they crystallize and grow within the kidney. These observations open a fundamentally new paradigm for clinical approaches that include in vivo stone dissolution and identify high-frequency layering of organic matter and minerals as a template for biomineralization in natural and engineered settings.

High frequency and wide range of human kidney papillary crystalline plugs.

Most of kidney stones are supposed to originate from Randall's plaque at the tip of the papilla or from papillary tubular plugs. Nevertheless, the frequency and the composition of crystalline plugs remain only partly described. The objective was to assess the frequency, the composition and the topography of papillary plugs in human kidneys. A total of 76 papillae from 25 kidneys removed for cancer and without stones were analysed by immunohistochemistry combined with Yasue staining, field emission-scanning electron microscopy and Fourier transformed infrared micro-spectroscopy. Papillary tubular plugs have been observed by Yasue staining in 23/25 patients (92%) and 52/76 papillae (68%). Most of these plugs were made of calcium phosphate, mainly carbonated apatite and amorphous calcium phosphate, and rarely octacalcium phosphate pentahydrate. Calcium and magnesium phosphate (whitlockite) have also been observed. Based upon immunostaining coupled to Yasue coloration, most of calcium phosphate plugs were located in the deepest part of the loop of Henle. Calcium oxalate monohydrate and dihydrate tubular plugs were less frequent and stood in collecting ducts. At last, we observed calcium phosphate plugs deforming and sometimes breaking adjacent collecting ducts. Papillary tubular plugging, which may be considered as a potential first step toward kidney stone formation, is a very frequent setting, even in kidneys of non-stone formers. The variety in their composition and the distal precipitation of calcium oxalate suggest that plugs may occur in various conditions of urine supersaturation. Plugs were sometimes associated with collecting duct deformation.

[Features of ontogenesis of uroliths].

The aim of the study was investigation of urolith growth process using the method of ontogenetic analysis. A total of 435 autochtonic uroliths obtained after surgery or spontaneous elimination from 422 patients with urolithiasis were studied with roentgenophasic analysis on diffractometer DRON-3, IR-spectroscopy, electron-probe. The concrements incorporate several zones with a nucleus as one of the zones. In some cases spherolith consisted of several splitted individuums with different orientation. Their nuclei contained phosphates and organic substance resultant from renal inflammation. In the other cases lithogenesis of the spherolith started from monocrystal microscopic nucleus which arrived from the kidney. The number of spherolith emergences can be interpreted as a rhythmic course of the disease. A prolonged macrorhythm characterizes stable disease. Formation of cut surfaces of the spherolith defines interruption of rhythmic zonality. A change in the composition of the crystal medium does not stop concrement growth, it provokes replacement of one mineral phase for the other. The ontogenetic analysis has detected that in the position of the concrement in the kidney mineral substance of its deep zones undergoes phasic transformations and morphological alterations. In changing medium conditions there are recovery of surfaces of solution with a change of a mineral phase, formation of new growth rhythms, aggregation and compression of the particles. This explains mixed composition of most of uroliths.

Microstructures of Randall's plaques and their interfaces with calcium oxalate monohydrate kidney stones reflect underlying mineral precipitation mechanisms.

Randall's plaques (RP) are preferred sites for the formation of calcium oxalate monohydrate (COM) kidney stones. However, although processes of interstitial calcium phosphate (CaP) plaque formation are not well understood, the potential of plaque microstructures as indicators of CaP precipitation conditions received only limited attention. We investigated RP-associated COM stones for structural details of the calcified tissues and microstructural features of plaque-stone interfaces as indicators of the initial processes of stone formation. Significantly increased CaP supersaturation can be expected for interstitial fluid, if reabsorbed ions from the tubular system continuously diffuse into the collagenous connective tissue. Densely packed, fine-grained CaP particles were found in dense textures of basement membranes while larger, laminated particles were scattered in coarse-meshed interstitial tissue, which we propose to be due to differential spatial confinements and restrictions of ion diffusion. Particle morphologies suggest an initial precipitation as metastable amorphous calcium phosphate (ACP). Morphologies and arrangements of first COM crystals at the RP-stone interface ranged from stacked euhedral platelets to skeletal morphologies and even porous, dendritic structures, indicating, in this order, increasing levels of COM supersaturation. Furthermore, these first COM crystals were often coated with CaP. On this basis, we propose that ions from CaP-supersaturated interstitial fluid may diffuse through porous RP into the urine, where a resulting local increase in COM supersaturation could trigger crystal nucleation and, hence, initiate stone formation. Ion-depleted fluid in persistent pores of initial COM layers may get replenished from interstitial fluid, leading to CaP precipitation in porous COM.