Citrate Supplementation Restores the Impaired Mineralisation Resulting from the Acidic Microenvironment: An In Vitro Study.

Chronic metabolic acidosis leads to bone-remodelling disorders based on excessive mineral matrix resorption and inhibition of bone formation, but also affects the homeostasis of citrate, which is an essential player in maintaining the acid-base balance and in driving the mineralisation process. This study aimed to investigate the impact of acidosis on the osteogenic properties of bone-forming cells and the effects of citrate supplementation in restoring the osteogenic features impaired by the acidic milieu. For this purpose, human mesenchymal stromal cells were cultured in an osteogenic medium and the extracellular matrix mineralisation was analysed at the micro- and nano-level, both in neutral and acidic conditions and after treatment with calcium citrate and potassium citrate. The acidic milieu significantly decreased the citrate release and hindered the organisation of the extracellular matrix, but the citrate supplementation increased collagen production and, particularly calcium citrate, promoted the mineralisation process. Moreover, the positive effect of citrate supplementation was observed also in the physiological microenvironment. This in vitro study proves that the mineral matrix organisation is influenced by citrate availability in the microenvironment surrounding bone-forming cells, thus providing a biological basis for using citrate-based supplements in the management of bone-remodelling disorders related to chronic low-grade acidosis.

Role of Citrate in Pathophysiology and Medical Management of Bone Diseases.

Citrate is an intermediate in the "Tricarboxylic Acid Cycle" and is used by all aerobic organisms to produce usable chemical energy. It is a derivative of citric acid, a weak organic acid which can be introduced with diet since it naturally exists in a variety of fruits and vegetables, and can be consumed as a dietary supplement. The close association between this compound and bone was pointed out for the first time by Dickens in 1941, who showed that approximately 90% of the citrate bulk of the human body resides in mineralised tissues. Since then, the number of published articles has increased exponentially, and considerable progress in understanding how citrate is involved in bone metabolism has been made. This review summarises current knowledge regarding the role of citrate in the pathophysiology and medical management of bone disorders.

Potassium Citrate Supplementation Decreases the Biochemical Markers of Bone Loss in a Group of Osteopenic Women: The Results of a Randomized, Double-Blind, Placebo-Controlled Pilot Study.

The relationship involving acid-base imbalance, mineral metabolism and bone health status has previously been reported but the efficacy of the alkalizing supplementation in targeting acid overload and preventing bone loss has not yet been fully elucidated. In this randomized, double-blind, placebo-controlled study, the hypothesis that potassium citrate (K citrate) modifies bone turnover in women with postmenopausal osteopenia was tested. Three hundred and ten women were screened; 40 women met the inclusion criteria and were randomly assigned to the treatment or the placebo group. They were treated with K citrate (30 mEq day-1) or a placebo in addition to calcium carbonate (500 mg day-1) and vitamin D (400 IU day-1). At baseline and time points of 3 and 6 months, serum indicators of renal function, electrolytes, calciotropic hormones, serum bone turnover markers (BTMs), tartrate-resistant acid phosphatase 5b (TRACP5b), carboxy-terminal telopeptide of type I collagen (CTX), bone alkaline phosphatase (BAP), procollagen type 1 N terminal propeptide (PINP)), and urine pH, electrolytes, and citrate were measured. The follow-up was completed by 17/20 patients in the "K citrate" group and 18/20 patients in the "placebo" group. At baseline, 90% of the patients exhibited low potassium excretion in 24 h urine samples, and 85% of cases had at least one urine parameter associated with low-grade acidosis (low pH, low citrate excretion). After treatment, CTX and BAP decreased significantly in both groups, but subjects with evidence of low-grade acidosis gained significant benefits from the treatment compared to the placebo. In patients with low 24h-citrate excretion at baseline, a 30% mean decrease in BAP and CTX was observed at 6 months. A significant reduction was also evident when low citrate (BAP: -25%; CTX: -35%) and a low pH (BAP: -25%; CTX: -30%) were found in fasting-morning urine. In conclusion, our results suggested that K citrate supplementation improved the beneficial effects of calcium and vitamin D in osteopenic women with a documented potassium and citrate deficit, and a metabolic profile consistent with low-grade acidosis.

Potassium citrate prevents increased osteoclastogenesis resulting from acidic conditions: Implication for the treatment of postmenopausal bone loss.

The extracellular acidic milieu in bones results in activation of osteoclasts (OC) and inhibition of osteoblasts (OB) causing a net loss of calcium from the skeleton and the deterioration of bone microarchitecture. Alkalinization through supplementation with potassium citrate (K citrate) has been proposed to limit the osteopenia progression, even though its pharmacological activity in bone microenvironment is not well defined. We evaluated if K citrate was able to prevent the adverse effects that acidic milieu induces on bone cells. OC and OB were maintained in neutral (pH 7.4) versus acidic (pH 6.9) culture medium, and treated with different K citrate concentrations. We evaluated the OC differentiation at seven days, by counting of multinucleated cells expressing tartrate-resistant acid phosphatase, and the activity of mature OC at 14 days, by quantifying of collagen degradation. To evaluate the effects on OB, we analyzed proliferation, mineralization, and expression of bone-related genes. We found that the low pH increased OC differentiation and activity and decreased OB function. The osteoclastogenesis was also promoted by RANKL concentrations ineffective at pH 7.4. Non-cytotoxic K citrate concentrations were not sufficient to steadily neutralize the acidic medium, but a) inhibited the osteoclastogenesis, the collagen degradation, and the expression of genes involved in RANKL-mediated OC differentiation, b) enhanced OB proliferation and alkaline phosphatase expression, whereas it did not affect the in vitro mineralization, and c) were effective also in OC cultures resistant to alendronate, i.e. the positive control of osteoclastogenesis inhibition. In conclusion, K citrate prevents the increase in OC activity induced by the acidic microenvironment, and the effect does not depend exclusively on its alkalizing capacity. These data provide the biological basis for the use of K citrate in preventing the osteopenia progression resulting from low-grade acidosis.

Alpha-lipoic Acid After Median Nerve Decompression at the Carpal Tunnel: A Randomized Controlled Trial.

PURPOSE: The postoperative course of median nerve decompression in carpal tunnel syndrome may be associated with complications. The aim of this study was to explore the possible effects of alpha-lipoic acid (ALA) in the postoperative period after surgical decompression of the median nerve at the wrist. METHODS: We conducted a double-blind prospective, randomized, controlled trial. A total of 64 patients with proven carpal tunnel syndrome were enrolled and randomly assigned into 1 of 2 groups: group A (n = 32) patients had surgical decompression of the median nerve followed by ALA for 40 days, and group P (n = 32) patients had surgical decompression followed by placebo. The primary end point of the study was a comprehensive indicator of sensory and motor nerve conduction velocity (electrophysiology score) at 3 months after surgery, Other end points were static 2-point discrimination, Boston Carpal Tunnel score, presence or absence of pillar pain, and use of analgesics beyond the second postoperative day. RESULTS: Alpha-lipoic acid did not improve nerve conduction velocity or Boston Carpal Tunnel score significantly. However, a statistically significant reduction in the postoperative incidence of pillar pain was noted in the ALA group. In addition, static 2-point discrimination improved in both groups. CONCLUSIONS: Postoperative administration of ALA for 40 days after median nerve decompression may result in a lower incidence of pillar pain. This treatment is relatively well tolerated, which may support its value as standard postoperative supplementation after carpal tunnel decompression if further studies on larger samples confirm these preliminary findings. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic I.

Comparative "in vitro" evaluation of the antiresorptive activity residing in four Ayurvedic medicinal plants. Hemidesmus indicus emerges for its potential in the treatment of bone loss diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Four Indian plants, traditionally used in Ayurvedic medicine: Asparagus racemosus Willd., Emblica officinalis Gaertn., Hemidesmus indicus R. Br., and Rubia cordifolia L. were selected on the basis of their ethnobotanical use and of scientific evidence that suggests a potential efficacy in the treatment of bone-loss diseases. The antiresorptive properties of the four plants have been investigated. The aim was to provide adequate evidence for the exploitation of natural compounds as alternative therapeutics for the treatment of diseases caused by increased osteoclast activity. MATERIALS AND METHODS: Decoctions were prepared from dried plant material according to the traditional procedure and standardization by HPLC was performed using marker compounds for each species. Total polyphenols, flavonoids and radical scavenging activity of the decoctions were also determined. The bioactivity of the plant decoctions was evaluated in subsequent phases. (1) A cytotoxicity screening was performed on the mouse monocytic RAW 264.7 cell line to define the concentrations that could be utilized in the following step. (2) The antiresorptive properties of plant decoctions were compared with that of a "gold standard" drug (alendronate) by measuring osteoclastogenesis inhibition and osteoclast apoptosis. (3) The toxic effect on bone forming cells was excluded by evaluating the impact on the proliferation of osteogenic precursors (mesenchymal stem cells, MSC). RESULTS: All the decoctions inhibited osteoclastogenesis similarly to alendronate at the highest doses, but Hemidesmus indicus and Rubia cordifolia were also effective at lower concentrations. Apoptosis increased significantly when cells were exposed to the highest concentration of Emblica officinalis, Hemidesmus indicus, and Rubia cordifolia. All concentrations of Emblica officinalis tested inhibited the proliferation of osteogenic precursors, while only the highest doses of Asparagus racemosus and Rubia cordifolia were toxic. On the contrary, Hemidesmus indicus did not affect osteogenic precursor growth at any concentration tested. CONCLUSION: Among the medicinal plants included in the study, Hemidesmus indicus showed the greatest antiosteoclastic activity without toxic effect on osteogenic precursors. Therefore, Hemidesmus indicus exhibits the properties of an antiresorptive drug and represents the ideal candidate for further clinical investigations.

Proton pump inhibitor chemosensitization in human osteosarcoma: from the bench to the patients' bed.

BACKGROUND: Major goals in translational oncology are to reduce systemic toxicity of current anticancer strategies and improve effectiveness. An extremely efficient cancer cell mechanism to avoid and/or reduce the effects of highly cytotoxic drugs is the establishment of an acidic microenvironment, an hallmark of all malignant tumors. The H +-rich milieu that anticancer drugs meet once they get inside the tumor leads to their protonation and neutralization, therefore hindering their access into tumor cells. We have previously shown that proton pump inhibitors (PPI) may efficiently counterattack this tumor advantage leading to a consistent chemosensitization of tumors. In this study, we investigated the effects of PPI in chemosensitizing osteosarcoma. METHOD: MG-63 and Saos-2 cell lines were used as human osteosarcoma models. Cell proliferation after pretreatment with PPI and subsequent treatment with cisplatin was evaluated by using erythrosin B dye vital staining. Tumour growth was evaluated in xenograft treated with cisplatin after PPI pretreatment. Subsequently, a multi-centre historically controlled trial, was performed to evaluate the activity of a pre-treatment administration of PPIs as chemosensitizers during neoadjuvant chemotherapy based on methotrexate, cisplatin, and adriamycin. RESULTS: Preclinical experiments showed that PPI sensitize both human osteosarcoma cell lines and xenografts to cisplatin. A clinical study subsequently showed that pretreatment with PPI drug esomeprazole leads to an increase in the local effect of chemotherapy, as expressed by percentage of tumor necrosis. This was particularly evident in chondroblastic osteosarcoma, an histological subtype that normally shows a poor histological response. Notably, no significant increase in toxicity was recorded in PPI treated patients. CONCLUSION: This study provides the first evidence that PPI may be beneficially added to standard regimens in combination to conventional chemotherapy.

Is wear debris responsible for failure in alumina-on-alumina implants?

BACKGROUND AND PURPOSE: Ceramic-on-ceramic articulation is an attractive alternative to metal-on-polyethylene (PE) bearings, but little is known about the in vivo effects induced by dissemination of alumina wear debris in the periprosthetic tissues. We hypothesized that wear debris is not the main factor responsible for loosening and failure of the implant but that mechanical problems caused by incorrect surgical technique, prosthetic design, or trauma, may cause instability of the implants and result in production of wear debris. PATIENTS AND METHODS: Clinical, radiographic, laboratory, and microbiological data from 30 consecutive patients with failed alumina-on-alumina arthroplasties, 19 with screwed socket and 11 with press-fit socket, were systematically collected and evaluated. Retrieved peri-implant tissues and prosthesis wear were also analyzed. RESULTS AND INTERPRETATION: Loosening was due to malpositioning, primary mechanical instability, trauma, or infection. Bone stock was generally preserved, even if screwed implants showed higher levels of osteolysis. Variable implant wear and tissue macrophage reaction were present but activation of giant cells/osteoclasts was not induced, and no correlation between histocytic reaction and the level of osteolysis was found. These findings indicate that, in contrast to the situation with metal-on-PE bearings, wear debris and occasional osteolysis were the effect rather than the cause of failure of ceramic-on-ceramic implants, and that press-fit socket fixation was the socket fixation design of preference.

Paracrine inhibition of osteoblast differentiation induced by neuroblastoma cells.

The aim of our study was to investigate whether the defective function of osteogenic cells induced by neuroblastoma might play a role in the development of skeletal metastases. This mechanism has been extensively demonstrated for multiple myeloma, in which the blockage of osteoblast differentiation has been ascribed to the inhibitors of canonical Wingless pathway (Wnt), namely Dickkopf 1 (Dkk1). Our purpose was to verify if neuroblastoma cells derived from bone marrow metastases (SH-SY5Y, LAN1) or primaries (NB100, CHP212) hamper the differentiation of mesenchymal stem cells (hMSCs) into osteoblasts in a paracrine manner, and to test whether this ability depends on Dkk1 activity. We found that all neuroblastoma cells increased the proliferation of hMSCs collected from pediatric-aged donors, with a corresponding decrease in osteoblast differentiation markers, including alkaline phosphatase (ALP), analyzed as gene expression, enzymatic activity and number of ALP-positive colony forming units, osteoprotegerin (OPG) release, OPG and osteocalcin gene-expression. Dkk1 mRNA and protein were detectable in all cell lines, and the use of neutralizing anti-Dkk1 antibody reversed the effects induced by SH-SY5Y cells. Taken together, our results confirm that neuroblastoma hinders osteoblastogenesis, and that Dkk1 release seems to play a crucial role in blocking the differentiation of osteoprogenitor cells, though the ability to promote osteoclast activation remains an essential requirement for the development of skeletal metastases. Finally, our findings suggest that strategies regulating Wnt signaling and Dkk1 activity could be considered for adjuvant therapies in neuroblastoma metastasizing to the skeleton.

Molecular basis of osteoclastogenesis induced by osteoblasts exposed to wear particles.

In this study, we investigate the molecular mechanisms by which human osteoblasts (HOB) challenged with wear debris promote the differentiation of osteoclast precursors. HOB were obtained from trabecular bone and exposed to alumina (Al(2)O(3)) or 'ultra-high molecular weight polyethylene' (UHMWPE) particles for 24h. The supernatant (HOB-CM) was used for the immunoenzymatic detection of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), as well as for inducing the osteoclast differentiation from peripheral blood mononuclear cells (PBMC). The OPG-to-RANKL ratio was significantly decreased in the conditioned medium of UHMWPE-challenged HOB. Morphological and cytochemical analysis showed that HOB-CM induced by itself the osteoclast formation, but a large amount of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive giant cells were obtained when PBMCs were cultured with 1 microg/mL UHMWPE HOB-CM. The expression of genes involved in osteoclast differentiation and activation was evaluated, i.e. c-fms, RANK, c-src, c-fos, cathepsin-K (CATK), TRAP, and calcitonin R (CTR). The UHMWPE HOB-CM increases c-src expression, suggesting that polyethylene debris favour the paracrine activity of HOB in inducing the pathway involved in osteoclast polarization and adhesion. On the contrary, Al(2)O(3) HOB-CM downregulates c-fos expression, suggesting that the passage from macrophages into the osteoclast lineage is deviated. These results show that Al(2)O(3) wear debris is less active than UHMWPE in inducing osteoclast differentiation. Moreover, they provide new insight into the molecular basis of particle-induced osteoclastogenesis, that is the starting point for planning mode-specific targeting of periprosthetic osteolysis.