Acidosis Inhibits Bone Formation by Osteoblasts In Vitro by Preventing Mineralization

The negative effect of acidosis on the skeleton has been known for almost a century. Bone mineral serves an important pathophysiologic role as a reserve of hydroxyl ions to buffer systemic protons if the kidneys and lungs are unable to maintain acid-base balance within narrow physiologic limits. Extracellular hydrogen ions are now thought to be the primary activation signal for osteoclastic bone resorption, and osteoclasts are very sensitive to small changes in pH within the pathophysiologic range. Herein, we investigated the effects of acidosis on osteoblast function by using mineralized bone nodule-forming primary osteoblast cultures. Osteoblasts harvested from neonatal rat calvariae were cultured up to 21 days in serum-containing medium, with ascorbate, beta-glycerophosphate and dexamethasone. pH was manipulated by addition of 5 to 30 mmol/L HCl and monitored by blood gas analyzer. Abundant, matrix-containing mineralized nodules formed in osteoblast cultures at pH 7.4, but acidification progressively reduced mineralization of bone nodules, with complete abolition at pH 6.9. Osteoblast proliferation and collagen synthesis, assessed by 3H-thymidine and 3H-proline incorporation, respectively, were unaffected by pH in the range 7.4 to 6.9; no effect of acidification on collagen ultrastructure and organization was evident. The apoptosis rate of osteoblasts, assessed by the enrichment of nucleosomes in cell lysates, was also unaffected by pH within this range. However, osteoblast alkaline phosphatase activity, which peaked strongly near pH 7.4, was reduced eight-fold at pH 6.9. Reducing pH to 6.9 also downregulated messenger ribonucleic acid (mRNA) for alkaline phosphatase, but upregulated mRNA for matrix Gla protein, an inhibitor of mineralization. The same pH reduction is associated with two-and four-fold increases in Ca2+ and PO4(3-) solubility for hydroxyapatite, respectively. Our results show that acidosis exerts a selective, inhibitory action on matrix mineralization that is reciprocal with the osteoclast activation response. Thus, in uncorrected acidosis, the deposition of alkaline mineral in bone by osteoblasts is reduced, and osteoclast resorptive activity is increased in order to maximize the availability of hydroxyl ions in solution to buffer protons.     

Unique roles of phosphorus in endochondral bone formation and osteocyte maturation

The mechanisms by which inorganic phosphate (P_i) homeostasis controls bone biology are poorly understood. Here we used Dmp1 null mice, a hypophosphatemic rickets/osteomalacia model, combined with a metatarsal organ culture and an application of neutralizing fibroblast growth factor 23 (FGF‐23) antibodies to gain insight into the roles of P_i in bone biology. We showed (1) that abnormal bone remodeling in Dmp1 null mice is due to reduced osteoclast number, which is secondary to a reduced ratio of RANKL/OPG expressed by osteoclast supporting cells and (2) that osteoblast extracellular matrix mineralization, growth plate maturation, secondary ossification center formation, and osteoblast differentiation are phosphate‐dependent. Finally, a working hypothesis is proposed to explain how phosphate and DMP1 control osteocyte maturation. © 2011 American Society for Bone and Mineral Research.     

Effect of metabolic acidosis on hyperlipidemia in uremia

Nine patients (aged 18±1 years) on maintenance hemodialysis with metabolic acidosis and hyperlipidemia were studied before and after 2 weeks of oral sodium bicarbonate (NaHCO₃) treatment to correct the acidosis. To control for the effect of additional sodium, they were also studied after 2 weeks of an equivalent amount of oral sodium chloride (NaCl). Oral NaHCO₃ treatment led to significant increases in venous pH, serum bicarbonate, and serum 1,25-dihydroxyvitamin D₃ concentrations, but no significant change in total and ionized calcium, phosphate, sodium, potassium, creatinine, blood urea nitrogen, and intact parathyroid hormone concentrations. Oral NaCl did not change any of the biochemical parameters. Before treatment of acidosis, these uremic patients had high serum triglycerides, low serum high-density lipoprotein (HDL) cholesterol, but normal total cholesterol compared with controls. Following 2 weeks of NaHCO₃ treatment, there was a significant decrease in the serum concentrations of triglycerides (P<0.01). HDL and total cholesterol did not change. There were no changes in triglycerides, HDL or total cholesterol from baseline values following 2 weeks of NaCl. Thus treatment of metabolic acidosis ameliorated hypertriglyceridemia but had no effect on HDL and total cholesterol in patients with uremia on hemodialysis. The underlying mechanism may involve 1,25-dihydroxyvitamin D3. Received: 3 August 1998 / Revised: 30 November 1998 / Accepted: 2 December 1998     

Carbon dioxide pneumoperitoneum causes severe peritoneal acidosis,unaltered by heating,humidification, or bicarbonate in a porcine model

Background Carbon dioxide (CO₂) is the most common gas used for insufflation in laparoscopy, but its effects on peritoneal physiology are poorly understood. This study looks at the changes in peritoneal and bowel serosal pH during CO₂ pneumoperitoneum, and whether heating and humidification with or without bicarbonate alters the outcomes.Methods Twenty-one pigs divided into four groups as follows: (1) standard (STD) laparoscopy (n = 5); (2) heated and humidified (HH) laparoscopy (n = 6); (3) heated and humidified with bicarbonate (HHBI) laparoscopy (n = 5); and (4) laparotomy (n = 5). Peritoneal pH, bowel serosal pH, and arterial blood gas (ABG) were obtained at 15-min intervals for 3 h.Results Severe peritoneal acidosis (pH range 6.59–6.74) was observed in all laparoscopy groups, and this was unaltered by heating and humidification or the addition of bicarbonate. Bowel serosal acidosis was observed in all laparoscopy groups with onset of pneumoperitoneum, but it recovered after 45 minutes. No significant changes in peritoneal or bowel serosal pH were observed in the laparotomy group.Conclusion CO₂ pneumoperitoneum resulted in severe peritoneal acidosis that was unaltered by heating and humidification with or without bicarbonate. Alteration in peritoneal pH may conceivably be responsible for providing an environment favorable for tumor-cell implantation during laparoscopy.     

Effect of Bicarbonate Administration on Cyclosporine-Induced Nephrotoxicity in Rats

Background

Cyclosporine (CsA) is known to cause metabolic and distal tubular acidosis. There is some evidence that CsA reduces net HCO₃⁻ absorption. The aim of this study was to elucidate whether bicarbonate administration prevented CsA-induced functional or structural nephrotoxicity.

Methods

Seven days after uninephrectomy, 20 rats were divided into 4 groups. NaHCO₃ (0.28 mol/L) was added in drinking water for 7 days, whereas control rats received regular tap water. The bicarbonate pretreated rats were administered either CsA (50 mg/kg intraperitoneally) or vehicle daily for a week. At the end of the procedure, animals were placed in metabolic cages for 24 hours after which we measured creatinine clearance (Ccr), urinary total proteins, pH, and N-acetyl-β-D-glucosaminidase (NAG) activity. The kidney was fixed in formaldehyde.

Results

Ccr was significantly affected by the administration of CsA. The effects of CsA on serum pH and HCO₃⁻ concentration were prevented by pretreatment with NaHCO₃. However, it did not affect the CsA-induced increased urinary NAG activity or decreased Ccr. There was no protection of CsA-induced changes in renal tissues by NaHCO₃.

Conclusion

Overall NaHCO₃ administration did not prevent CsA-induced changes in Ccr and NAG activity. These data suggested involvement of factors other than acid-base status in CsA-induced nephrotoxicity. However, correction of acidosis should still be considered for patients receiving CsA because acidosis exacerbates tissue damage.     

Effect of metabolic acidosis on branched-chain amino acids in uremia

 Fasting plasma concentrations of branched-chain amino acids (BCAA) valine, leucine, and isoleucine were measured in 20 young patients (aged 18±2 years) with end-stage renal disease just before initiation of dialysis and compared with 7 healthy controls (aged 19±1 years). Plasma valine, leucine, and isoleucine were all lower than control values (P<0.01 in all 3 cases). Plasma valine, but not leucine and isoleucine, correlated with venous pH (P<0.02). Plasma valine, leucine, or isoleucine did not correlate with blood urea nitrogen or serum creatinine. Seven patients (aged 18±1 years) on maintenance hemodialysis with metabolic acidosis were then studied before and after 2 weeks of oral sodium bicarbonate (NaHCO₃) treatment to correct the acidosis. To control for the effect of additional sodium, they were also studied after 2 weeks of an equivalent amount of oral sodium chloride (NaCl). Oral NaHCO₃ treatment led to significant increases in venous pH and serum bicarbonate concentrations, but no significant change in total and ionized calcium, phosphate, sodium, potassium, creatinine, blood urea nitrogen, and intact parathyroid hormone concentrations. Oral NaCl did not change any of the biochemical parameters. Fasting plasma concentrations of BCAA were measured. Before treatment of acidosis, uremic patients had low plasma concentrations of valine, leucine, and isoleucine compared with controls. Following 2 weeks of NaHCO₃ treatment, there were significant increases in the plasma concentrations of valine and leucine (P<0.01), although the values did not normalize. There were no changes in plasma concentrations of valine and leucine following 2 weeks of NaCl. The plasma concentration of isoleucine was not different during baseline (acidotic) and treatment periods with NaHCO₃ and NaCl. Thus treatment of metabolic acidosis ameliorated abnormalities in plasma concentrations of valine and leucine in patients with uremia on hemodialysis. Received: 18 February 1998 / Revised: 22 June 1998 / Accepted: 26 June 1998     

The syndrome of renal tubular acidosis and nerve deafness. Discordant manifestations in dizygotic twin brothers

The syndrome of renal tubular acidosis (RTA) and nerve deafness is a distinct nosological entity that is inherited as an autosomal recessive trait. We studied a pair of dizygotic twin brothers both with nerve deafness but only one with RTA. Distal RTA was diagnosed in twin A because of inappropriately high urinary pH (6.9) and low net acid excretion (40.0 Eq/min per 1.73 m²) in the presence of hyperchloraemic metablic acidosis, and fractional bicarbonate excretion of 1.6% at a normal serum bicarbonate concentration. The urine minus bloodPCO₂ differences (U-BPCO₂) during a neutral sodium phosphate load and in alkaline urine induced by bicarbonate supplementation were: 11 and 0 mm Hg, respectively. Twin A developed nephrocalcinosis and, after a 9.5-year follow-up period, was 5.3 cm taller than his brother. Twin B remained asymptomatic. Periodic determinations of blood pH and serum bicarbonate were normal and urine pH decreased to 4.6 in the face of ammonium chloride-induced metabolic acidosis. The U-BPCO₂ assessed in alkaline urine was 33.5 mm Hg. Audiograms demonstrated bilateral nerve deafness in both brothers. The presence of deafness without RTA has not been previously reported in this syndrome. This report also shows that a primary distal acidification defect is responsible for the RTA observed in this syndrome.     

DOES CORRECTION OF ACIDOSIS INFLUENCE MICROCIRCULATORY BLOOD FLOW DURING CARDIOPULMONARY BYPASS?

We studied 15 patients undergoing cardiopulmonary bypass (CPB)to examine the effect of response to correction of acidosison microcirculatory blood flow. Acidosis was defined when baseexcess was less than-10.0 mmol litre^–1 while carbon dioxidepartial pressure was within the normal range. CPB was carriedout at almost normothermic temperature (smallest rectal temperature35.2 (SD) 0.4°C). Sodium bicarbonate (NaHCO₃) was givento correct acidosis during steady state CPB. Skin microcirculatoryblood flow was assessed using a double-channel laser Dopplerflow (LDF) monitor. LDF was measured on the patient's foreheadand forearm before infusion of NaHCO₃ (baseline) and 30 s, 1,3, 5, 7, 10 and 15 min later. Skin, blood, rectal and nasopharyngealtemperatures did not change during the investigation. Plasmaviscosity, haemoglobin and carbon dioxide partial pressure alsoremained unchanged. Mean arterial pressure (MAP) and systemicvascular resistance (SVR) decreased slightly after infusionof NaHCO₃ (MAP—29%; SVR—32%). A total of NaHCO₃99.4 (4.4) mmol litre^–1 was given for correction of acidosis.pH and HCO₃ were within the normal range shortly after the infusionof NaHCO₃ LDF measured on both the forehead (+ 49%) and theforearm (+ 29%) increased significantly after infusion of NaHCO₃.Changes in pH correlated positively with changes in LDF (analysesof co-variance, P<0.02), but haemodynamic and other laboratoryvalues did not correlate with LDF. We conclude that the microcirculatoryresponse to correction of acidosis with NaHCO₃ during CPB canbe monitored using skin laser Doppler flowmet Infusion of NaHCO₃resulted in a significant improvement in skin microcirculatoryperfusion.     

The Role of Osteoblast Density and Endogenous Interleukin-6 Production in Osteoclast Formation From the Hemopoietic Stem Cell Line FDCP-MIX C2GM in Coculture With Primary Osteoblasts

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C₂GM was shown to be strongly dependent on osteoblast density. In cocultures of C₂GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 × 10⁴ cells/cm²), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 × 10⁴ cells/cm²). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear. Received: 26 June 1997 / Accepted: 14 November 1997     

Ferric ion could facilitate osteoclast differentiation and bone resorption through the production of reactive oxygen species

Iron overload is widely regarded as a risk factor for osteoporosis. It has been demonstrated that iron can inhibit osteoblast differentiation. However, the effects of iron on osteoclast differentiation and bone resorption remain controversial. In this study, we found that ferric ion promoted Receptor Activator of Nuclear Factor κ B Ligand (RANKL)‐induced osteoclast (OC) formation in both RAW264.7 cells and bone marrow‐derived macrophages (BMMs), and this effect was accompanied by elevated levels of reactive oxygen species (ROS) and oxidative stress. Moreover, this effect was attenuated by the administration of antioxidant N‐acetyl‐L ‐cysteine (NAC). Therefore, we conclude that ferric ion can promote osteoclast differentiation and bone resorption through the production of ROS. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1843–1852, 2012     

Effects of cobalt and chromium ions at clinically equivalent concentrations after metal-on-metal hip replacement on human osteoblasts and osteoclasts: implications for skeletal health

Metal-on-metal hip replacement (MOMHR) using large diameter bearings has become a popular alternative to conventional total hip arthroplasty, but is associated with elevated local tissue and circulating levels of chromium (Cr) and cobalt (Co) ions that may affect bone health. We examined the effects of acute and chronic exposure to these metals on human osteoblast and osteoclast formation and function over a clinically relevant concentration range previously reported in serum and within hip synovial fluid in patients after MOMHR. SaOS-2 cells were cultured with Co²⁺, Cr³⁺ and Cr⁶⁺ for 3 days after which an MTS assay was used to assess cell viability, for 13 days after which alkaline phosphatase and cell viability were assessed and for 21 days after which nodule formation was assessed. Monocytes were isolated from human peripheral blood and settled onto dentine disks then cultured with M-CSF and RANKL plus either Co²⁺, Cr³⁺ or Cr⁶⁺ ions for 21 days from day 0 or between days 14 and 21. Cells were fixed and stained for TRAP and osteoclast number and amount of resorption per dentine disk determined. Co²⁺ and Cr³⁺ did not affect osteoblast survival or function over the clinically equivalent concentration range, whilst Cr⁶⁺ reduced osteoblast survival and function at concentrations within the clinically equivalent serum range after MOMHR (IC₅₀ = 2.2 μM). In contrast, osteoclasts were more sensitive to metal ions exposure. At serum levels a mild stimulatory effect on resorption in forming osteoclasts was found for Co²⁺ and Cr³⁺, whilst at higher serum and synovial equivalent concentrations, and with Cr⁶⁺, a reduction in cell number and resorption was observed. Co²⁺ and Cr⁶⁺ within the clinical range reduced cell number and resorption in mature osteoclasts. Our data suggest that metal ions at equivalent concentrations to those found in MOMHR affect bone cell health and may contribute to the observed bone-related complications of these prostheses.     

Vitamin B12 Deficiency Stimulates Osteoclastogenesis via Increased Homocysteine and Methylmalonic Acid

The risk of nutrient deficiencies increases with age in our modern Western society, and vitamin B₁₂ deficiency is especially prevalent in the elderly and causes increased homocysteine (Hcy) and methylmalonic acid (MMA) levels. These three factors have been recognized as risk factors for reduced bone mineral density and increased fracture risk, though mechanistic evidence is still lacking. In the present study, we investigated the influence of B₁₂, Hcy, and MMA on differentiation and activity of bone cells. B₁₂ deficiency did not affect the onset of osteoblast differentiation, maturation, matrix mineralization, or adipocyte differentiation from human mesenchymal stem cells (hMSCs). B₁₂ deficiency caused an increase in the secretion of Hcy and MMA into the culture medium by osteoblasts, but Hcy and MMA appeared to have no effect on hMSC osteoblast differentiation. We further studied the effect of B₁₂, Hcy, and MMA on the formation of multinucleated tartrate-resistant acid phosphatase–positive osteoclasts from mouse bone marrow. We observed that B₁₂ did not show an effect on osteoclastogenesis. However, Hcy as well as MMA were found to induce osteoclastogenesis in a dose-dependent manner. On the basis of these results, we conclude that B₁₂ deficiency may lead to decreased bone mass by increased osteoclast formation due to increased MMA and Hcy levels.     

A Class III Semaphorin (Sema3e) Inhibits Mouse Osteoblast Migration and Decreases Osteoclast Formation In Vitro

Originally identified as axonal guidance cues, semaphorins are expressed throughout many different tissues and regulate numerous non-neuronal processes. We demonstrate that most class III semaphorins are expressed in mouse osteoblasts and are differentially regulated by cell growth and differentiation: Sema3d expression is increased and Sema3e expression decreased during proliferation in culture, while expression of Sema3a is unaffected by cell density but increases in cultures of mineralizing osteoblasts. Expression of Sema3a, -3e, and -3d is also differentially regulated by osteogenic stimuli; inhibition of GSK3β decreased expression of Sema3a and -3e, while 1,25-(OH)₂D₃ increased expression of Sema3e. Parathyroid hormone had no effect on expression of Sema3a, -3b, or -3d. Osteoblasts, macrophages, and osteoclasts express the Sema3e receptor PlexinD1, suggesting an autocrine and paracrine role for Sema3e. No effects of recombinant Sema3e on osteoblast proliferation, differentiation, or mineralization were observed; but Sema3e did inhibit the migration of osteoblasts in a wound-healing assay. The formation of multinucleated, tartrate-resistant acid phosphatase–positive osteoclasts was decreased by 81% in cultures of mouse bone marrow macrophages incubated with 200 ng/mL Sema3e. Correspondingly, decreased expression of osteoclast markers (Itgb3, Acp5, Cd51, Nfatc1, CalcR, and Ctsk) was observed by qPCR in macrophage cultures differentiated in the presence of Sema3e. Our results demonstrate that class III semaphorins are expressed by osteoblasts and differentially regulated by differentiation, mineralization, and osteogenic stimuli. Sema3e is a novel inhibitor of osteoclast formation in vitro and may play a role in maintaining local bone homeostasis, potentially acting as a coupling factor between osteoclasts and osteoblasts.     

Renal tubular acidosis and osteopetrosis with carbonic anhydrase II deficiency: pathogenesis of impaired acidification

Renal tubular acidosis with osteopetrosis is an autosomal recessive disorder due to deficiency of carbonic anhydrase II (CAII). A 3.5-year-old Egyptian boy with osteopetrosis and cerebral calcification had a persistent normal anion gap type of metabolic acidosis (plasma pH 7.26) and a mild degree of hypokalemia. A baseline urine pH was 7.0; ammonium (NH₄ ⁺) excretion was low at 11 μmol/min per 1.73 m²; fractional excretion of bicarbonate HCO₃ (FE_HCO3) was high at 9%, when plasma HCO₃ was 20 mmol/l; citrate excretion rate was high for the degree of acidosis at 0.35 mmol/mmol creatinine. Intravenous administration of sodium bicarbonate led to a urine pH of 7.6, a FE_HCO3 of 14%, a urine-blood PCO₂ difference of 7 mmHg, NH₄ ⁺ excretion fell to close to nil, and citrate excretion remained at 0.38 mmol/mmol creatinine. Intravenous administration of arginine hydrochloride caused the urine pH to fall to 5.8, the FE_HCO3 to fall to 0, the NH₄ ⁺ excretion rate to rise to 43 μmol/min per 1.73 m², and citrate excretion to fall to <0.01 mmol/mmol creatinine. These results show that our patient had a low rate of NH₄ ⁺ excretion, a low urine minus blood PCO₂ difference in alkaline urine, and a low urinary citrate excretion, but only when he was severely acidotic. He failed to achieve a maximally low urine pH. These findings indicate that his renal acidification mechanisms were impaired in both the proximal and distal tubule, the result of his CA_II deficiency. Received October 24, 1996; received in revised form and accepted February 20, 1997