Tissue sodium content in patients with systemic lupus erythematosus: association with disease activity and markers of inflammation.

OBJECTIVES: Sodium (Na+) is stored in the skin and muscle and plays an important role in immune regulation. In animal models, increased tissue Na+ is associated with activation of the immune system, and high salt intake exacerbates autoimmune disease and worsens hypertension. However, there is no information about tissue Na+ and human autoimmune disease. We hypothesized that muscle and skin Na+ content is (a) higher in patients with systemic lupus erythematosus (SLE) than in control subjects, and (b) associated with blood pressure, disease activity, and inflammation markers (interleukin (IL)-6, IL-10 and IL-17 A) in SLE. METHODS: Lower-leg skin and muscle Na+ content was measured in 23 patients with SLE and in 28 control subjects using 23Na+ magnetic resonance imaging. Demographic and clinical information was collected from interviews and chart review, and blood pressure was measured. Disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Plasma inflammation markers were measured by multiplex immunoassay. RESULTS: Muscle Na+ content was higher in patients with SLE (18.8 (16.7-18.3) mmol/L) than in control subjects (15.8 (14.7-18.3) mmol/L; p < 0.001). Skin Na+ content was also higher in SLE patients than in controls, but this difference was not statistically significant. Among patients with SLE, muscle Na+ was associated with SLEDAI and higher concentrations of IL-10 after adjusting for age, race, and sex. Skin Na+ was significantly associated with systolic blood pressure, but this was attenuated after covariate adjustment. CONCLUSION: Patients with SLE had higher muscle Na+ content than control subjects. In patients with SLE, higher muscle Na+ content was associated with higher disease activity and IL-10 concentrations.

Real-world electronic health record identifies antimalarial underprescribing in patients with lupus nephritis.

Antimalarials (AMs) reduce disease activity and improve survival in patients with systemic lupus erythematosus (SLE), but studies have reported low AM prescribing frequencies. Using a real-world electronic health record cohort, we examined if patient or provider characteristics impacted AM prescribing. We identified 977 SLE cases, 94% of whom were ever prescribed an AM. Older patients and patients with SLE nephritis were less likely to be on AMs. Current age (odds ratio = 0.97, p < 0.01) and nephritis (odds ratio = 0.16, p < 0.01) were both significantly associated with ever AM use after adjustment for sex and race. Of the 244 SLE nephritis cases, only 63% were currently on AMs. SLE nephritis subjects who were currently prescribed AMs were more likely to be followed by a rheumatologist than a nephrologist and less likely to have undergone dialysis or renal transplant (both p < 0.001). Non-current versus current SLE nephritis AM users had higher serum creatinine (p < 0.001), higher urine protein (p = 0.05), and lower hemoglobin levels (p < 0.01). As AMs reduce disease damage and improve survival in patients with SLE, our results demonstrate an opportunity to target future efforts to improve prescribing rates among multi-specialty providers.

Increased blood pressure visit-to-visit variability in patients with systemic lupus erythematosus: association with inflammation and comorbidity burden.

BACKGROUND: Blood pressure visit-to-visit variability is a novel risk factor for deleterious long-term cardiac and renal outcomes in the general population. We hypothesized that patients with systemic lupus erythematosus (SLE) have greater blood pressure visit-to-visit variability than control subjects and that blood pressure visit-to-visit variability is associated with a higher comorbidity burden. METHODS: We studied 899 patients with SLE and 4172 matched controls using de-identified electronic health records from an academic medical center. We compared blood pressure visit-to-visit variability measures in patients with SLE and control subjects and examined the association between blood pressure visit-to-visit variability and patients' characteristics. RESULTS: Patients with SLE had higher systolic blood pressure visit-to-visit variability 9.7% (7.8-11.8%) than the control group 9.2% (7.4-11.2%), P < 0.001 by coefficient of variation. Additional measures of systolic blood pressure visit-to-visit variability (i.e. standard deviation, average real variation, successive variation and maximum measure-to-measure change) were also significantly higher in patients with SLE than in control subjects. In patients with SLE, blood pressure visit-to-visit variability correlated significantly with age, creatinine, CRP, triglyceride concentrations and the Charlson comorbidity score (all P < 0.05). Hydroxychloroquine use was associated with reduced blood pressure visit-to-visit variability (P < 0.001), whereas the use of antihypertensives, cyclophosphamide, mycophenolate mofetil and corticosteroids was associated with increased blood pressure visit-to-visit variability (P < 0.05). CONCLUSION: Patients with SLE had higher blood pressure visit-to-visit variability than controls, and this increased blood pressure visit-to-visit variability was associated with greater Charlson comorbidity scores, several clinical characteristics and immunosuppressant medications. In particular, hydroxychloroquine prescription was associated with lower blood pressure visit-to-visit variability.

The effect of genetic variation in PCSK9 on the LDL-cholesterol response to statin therapy.

Statins (HMG-CoA reductase inhibitors) lower low-density lipoprotein cholesterol (LDL-C) and prevent cardiovascular disease. However, there is wide individual variation in LDL-C response. Drugs targeting proprotein convertase subtilin/kexin type 9 (PCSK9) lower LDL-C and will be used with statins. PCSK9 mediates the degradation of LDL receptors (LDLRs). Therefore, a greater LDL-C response to statins would be expected in individuals with PCSK9 loss-of-function (LOF) variants because LDLR degradation is reduced. To examine this hypothesis, the effect of 11 PCSK9 functional variants on statin response was determined in 669 African Americans. One LOF variant, rs11591147 (p.R46L) was significantly associated with LDL-C response to statin (P=0.002). In the three carriers, there was a 55.6% greater LDL-C reduction compared with non-carriers. Another functional variant, rs28362261 (p.N425S), was marginally associated with statin response (P=0.0064).The effect of rs11591147 was present in individuals of European ancestry (N=2388, P=0.054). The therapeutic effect of statins may be modified by genetic variation in PCSK9.

Association of estimated sodium and potassium intake with blood pressure in patients with systemic lupus erythematosus.

Sodium and potassium intake are modifiable determinants of hypertension in the general population but have not been studied in patients with systemic lupus erythematosus (SLE). We examined the relationship between urinary excretion of sodium and potassium, as an estimate of intake, and blood pressure in patients with SLE. We studied 178 SLE patients and 86 controls, matched for age, sex, and race. Urine sodium (Na+) and potassium (K+) were measured by flame photometry. Blood pressure was the average of two resting measurements. The associations between systolic (SBP) and diastolic blood pressures (DBP) and estimated 24-hour urinary Na+, K+, and Na+:K+ ratio were tested. The estimated mean 24-hour urinary K+ excretion was lower, and the Na+:K+ ratio was higher in patients with SLE than controls. There were no significant differences in the estimated 24-hour urinary Na+. In patients with SLE, a higher urinary Na+:K+ ratio was associated with higher SBP (ß coefficient = 4.01, p = 0.023) and DBP (ß coefficient = 4.41, p = 0.002) after adjusting for age, sex, and race. SLE patients had significantly lower estimated 24-hour urinary K+ and higher estimated 24-hour urinary Na+: K+ ratio than controls. The urinary Na+:K+ ratio was significantly associated with SBP and DBP.

GlycA, a novel marker of inflammation, is elevated in systemic lupus erythematosus.

BACKGROUND: GlycA is a novel marker of systemic inflammation detected by nuclear magnetic resonance (NMR) spectroscopy. In the general population, GlycA is correlated with inflammatory markers such as C-reactive protein (CRP) and associated with coronary heart disease and diabetes. The utility of GlycA in patients with systemic lupus erythematosus (SLE) has not been defined. Therefore, we tested the hypothesis that GlycA concentrations are elevated in patients with SLE and associated with other markers of inflammation and coronary atherosclerosis. METHODS: We compared concentrations of GlycA, detected by NMR, in 116 patients with SLE and 84 control subjects frequency-matched for age, sex, and race. SLE disease activity index (SLEDAI) and the SLE Collaborating Clinics damage index (SLICC) were calculated. Acute phase reactants, a panel of cytokines, and a lipid panel were measured. Electron beam computer tomography (EBCT) was used to quantify coronary artery calcification, a measure of coronary artery atherosclerosis. RESULTS: Patients with SLE had higher concentrations of GlycA (398 (350-445)) than control subjects (339 (299-391)) µmol/L, p < 0.001. In patients with SLE, concentrations of GlycA were significantly associated with sedimentation rate (rho = 0.43), C-reactive protein (rho = 0.59), e-selectin (rho = 0.28), intracellular adhesion molecule-1 (rho = 0.30), triglycerides (rho = 0.45), all p < 0.0023 to account for multiple comparisons, but not with creatinine, SLEDAI, SLICC, or coronary calcium scores. CONCLUSIONS: Concentrations of GlycA are higher in patients with SLE than control subjects and associated with markers of inflammation but not with SLE disease activity or chronicity scores or coronary artery calcification.

Genetic variation and coronary atherosclerosis in patients with systemic lupus erythematosus.

Coronary artery disease is the major cause of mortality in patients with systemic lupus erythematosus (SLE). Increased cardiovascular risk in SLE is not explained by traditional risk factors. We examined the hypothesis that genetic variation contributes to the presence of coronary atherosclerosis in patients with SLE. The genotypes of single-nucleotide polymorphisms (SNP) in 152 candidate genes linked with autoimmune or cardiovascular risk were determined in 125 patients with SLE. Coronary artery calcium (CAC), a measure of coronary atherosclerosis, was detected in 32 patients (26%) by electron-beam computed tomography. Polymorphism in 20 of the candidate genes (ADAM33, ADIPOQ, CCL5, CCR7, CDKN2B, CSF1, IL4, IL12A, IL23R, INS, IRF5, MIF, MS4A1, PTGS1, PTPN22, RETN, SELE, TNFSF4, TNFRSF11B, and VCAM1) were nominally associated with the presence of CAC (p-values = 0.001-0.047 after adjustment for age, sex and race). Some of these are known susceptibility genes for SLE and others have been implicated in cardiovascular disease in other populations. No association withstood false discovery rate adjustment. Replication studies in additional cohorts of patients with SLE may be informative.

Age- and Sex-Different Associations between Cognitive Performance and Inflammatory Biomarkers in Community Dwelling Older Adults: towards Precision Preventive Strategies.

BACKGROUND: Studies have demonstrated associations between inflammatory biomarkers and cognitive function in people with dementia or stroke, but little is known regarding these associations in healthy middle-aged and older populations. OBJECTIVES: This study aims to examine associations between inflammatory biomarkers (both vascular and systemic) and cognitive performance in stroke- and dementia-free middle-aged and older adults without apolipoprotein E4 (ApoE ε4) allele carriers. DESIGN: A cross-sectional study. SETTING: Social Environment and Biomarkers of Aging Study (SEBAS) 2006. PARTICIPANTS: A total of 983 participants aged 53 years and older. MEASUREMENTS: Composite cognitive function assessment, including the Short Portable Mental Status Questionnaire, the Rey Auditory Verbal Learning Test, and the Wechsler Adult Intelligence Scale. Overnight venous blood sampling for 6 inflammatory biomarkers (C-reactive protein, interleukin-6, fibrinogen, homocysteine, intercellular adhesion molecule-1 and E-selectin) and ApoE genotyping. RESULTS: Among 983 participants (mean age: 65.8±9.5 years), 808 were non-ApoE e4 allele carriers and were stroke- and dementia-free. Higher log fibrinogen was associated with poorer cognitive function after adjustment for potential confounding factors in non-ApoE e4 allele carriers and stroke- and dementia-free populations (unstandardized coefficients ß= -1.553, P value= 0.003). In participants aged 65 years or older, both of elevated fibrinogen and homocysteine were associated with poorer cognitive function (ß= -2.288, P value= 0.015; ß= -1.331, P value= 0.012, respectively). Elevated log CRP was significantly associated with lower cognitive function only in women (ß= -0.514, P value= 0.024). CONCLUSION: Higher serum levels of fibrinogen were negatively associated with cognitive function, which was independent of ApoE genotyping and prior cerebrovascular events in dementia-free community-dwelling older adults. Further studies are needed to validate the roles of fibrinogen in the pathophysiology of dementia and elucidate the underlying mechanisms.

Physio-Cognitive Decline Syndrome as the Phenotype and Treatment Target of Unhealthy Aging.

In this era of unprecedented longevity, healthy aging is an important public health priority. Avoiding or shortening the period of disability or dementia before death is critical to achieving the defining objectives of healthy aging, namely to develop and maintain functional capabilities that enable wellbeing in older age. The first step is to identify people who are at risk and then to implement effective primary interventions. Geriatricians have identified a distinct clinical phenotype of concurrent physical frailty and cognitive impairment, which predicts high risk of incident dementia and disability and is potentially reversible. Differing operational definitions for this phenotype include "cognitive frailty", "motoric cognitive risk syndrome" and the recently proposed "physio-cognitive decline syndrome (PCDS)". PCDS is defined as concurrent mobility impairment no disability (MIND: slow gait or/and weak handgrip) and cognitive impairment no dementia (CIND: ≥1.5 SD below the mean for age-, sex-, and education-matched norms in any cognitive domain but without dementia). By these criteria, PCDS has a prevalence of 10-15% among community-dwelling older persons without dementia or disability, who are at increased risk for incident disability (HR 3.9, 95% CI 3.0-5.1), incident dementia (HR 3.4, 95% CI 2.4-5.0) and all-cause mortality (HR 6.7, 95% CI 1.8-26.1). Moreover, PCDS is associated with characteristic neuroanatomic changes in the cerebellum and hippocampus, and their neurocircuitry, which are distinct from neuroimaging features in normal aging and common dementia syndromes. Basic research and longitudinal clinical studies also implicate a hypothetical muscle-brain axis in the pathoetiology of PCDS. Most important, community-dwelling elders with PCDS who participated in a multidomain intervention had significant improvements in global cognitive function, and especially in the subdomains of naming and concentration. Our proposed operational definition of PCDS successfully identifies an appreciable population of at-risk older people, establishes a distinct phenotype with an apparently unique pathoetiology, and is potentially reversible. We now need further studies to elucidate the pathophysiology of PCDS, to validate neuroimaging features and muscle-secreted microRNA biomarkers, and to evaluate the effectiveness of sustained multidomain interventions.

Changing patterns of medication use in patients with rheumatoid arthritis in a Medicaid population.

OBJECTIVE: To examine changes in patterns of medication utilization in patients with RA. METHODS: Data from Tennessee Medicaid (TennCare) databases (1995-2004) were used to identify adults with both a diagnosis of RA and at least one DMARD prescription each year. Annual age-specific utilization of DMARDs, glucocorticoids, NSAIDs and narcotics was measured on the last day of each year to determine the point prevalence of use of these agents. RESULTS: Records from 23 342 patients with treated RA were analysed. Most patients were females (78%) and white (74%). The median age was 57 yrs (interquartile range: 48-65). The proportion of patients who had a current DMARD prescription on the index date increased from 62% in 1995 to 71% in 2004 (P < 0.001). MTX was the most commonly used DMARD. By the end of 2004, 22% of patients had a current prescription for a biologic, and etanercept represented 51% of all biologic therapies. During the study period, the overall utilization of glucocorticoids decreased from 46% to 38% (P < 0.001), whereas NSAID utilization increased from 33% to 38% (P < 0.001), and use of narcotics increased from 38% to 55% (P < 0.001). A secondary analysis that identified RA patients based on diagnosis codes alone, showed similar patterns, but lower DMARD utilization which increased from 33% to 52% overall and from 0% to 16% for biologics. CONCLUSIONS: The utilization of DMARDs increased in TennCare patients with RA, and by 2004, use of biologics was substantial. Although glucocorticoid utilization decreased, use of both NSAIDs and narcotics increased.

Platelet derived growth factor releasing chitosan sponge for periodontal bone regeneration.

With an aim of improving bone regeneration, chitosan sponge containing platelet-derived growth factor-BB (PDGF-BB) were developed. For fabrication of chitosan sponge, chitosan solution was freeze-dried, crosslinked and freeze-dried again. PDGF-BB was incorporated into the chitosan sponge by soaking chitosan sponge into the PDGF-BB solution. Release kinetics of PDGF-BB, cell attachment, proliferation capacity and bony regenerative potentials of PDGF-BB-loaded chitosan sponge were investigated. Prepared chitosan sponge retained porous structure with 100 microm pore diameter that was suitable for cellular migration and growth. Release rate of PDGF-BB could be controlled by varying initial loading content of PDGF-BB to obtain optimal therapeutic efficacy. PDGF-BB-loaded chitosan sponge induced significantly high cell attachment and proliferation level, which indicated good cellular adaptability. PDGF-BB-loaded chitosan sponge demonstrated marked increase in new bone formation and rapid calcification. Degradation of the chitosan sponge was proceeded at defect site and subsequently replaced with new bone. Histomorphometric analysis confirmed that PDGF-BB-loaded chitosan sponge significantly induced new bone formation. These results suggested that chitosan sponge and PDGF-BB-loaded chitosan sponge may be beneficial to enhance periodontal bone regeneration.

Controlled release of platelet-derived growth factor from porous poly(L-lactide) membranes for guided tissue regeneration.

Platelet-derived growth factor-BB (PDGF-BB) was incorporated into porous poly (L-lactide) (PLLA) membranes with an aim of improving early bone healing in guided tissue regeneration (GTR) therapy. Porous PDGF-BB loaded membranes were fabricated by coating PDGF-BB-dissolved PLLA methylene chloride-ethyl acetate solutions on polyglycolic acid (PGA) meshes. Release kinetics of PDGF-BB, biologic activity, degradability and guided tissue regenerative potentials of the membranes were investigated. Release of PDGF-BB could be controlled by adding bovine serum albumin that may provide porous diffusion channels for PDGF-BB release and by varying initial loading content of PDGF-BB. Biologic activity of PDGF-BB in the membranes was ascertained by fibroblast chemotaxis. PDGF-BB loaded membranes maintained proper degradation property for periodontal application. PDGF-BB loaded membrane markedly increased new bone formation in rat calvarial defects, and completed bony reunion after 2 weeks of implantation period. These results suggested that PDGF-BB loaded PLLA membrane might potentially enhance guided tissue regenerative efficacy.

Controlled release of platelet-derived growth factor-BB from chondroitin sulfate-chitosan sponge for guided bone regeneration.

Platelet-derived growth factor-BB (PDGF-BB) releasing porous chondroitin-4-sulfate (CS)-chitosan sponge was designed with an aim of controlling growth factor delivery in order to improve bone formation. Porous CS-chitosan sponge was fabricated by freeze drying and crosslinking aqueous CS-chitosan solution. PDGF-BB was incorporated into the CS-chitosan sponge by soaking CS-chitosan sponge into the PDGF-BB solution. CS-chitosan sponge retained a porous structure with a 150-200-microm pore diameter that was suitable for cellular migration and osteoid ingrowth. Release rate of PDGF-BB could be controlled by varying the composition of CS in the sponge or initial loading content of PDGF-BB. CS-chitosan sponge induced increased osteoblast migration and proliferation as compared with chitosan sponge alone. Furthermore, the release of PDGF-BB from CS-chitosan sponge significantly enhanced osteoblast proliferation. These results suggest that PDGF-BB-releasing CS-chitosan sponge may be beneficial to enhance bone cell adaptation and regenerative potential when applied in wound sites.

Longitudinal monitoring for disease progression of localized juvenile periodontitis.

The present study was performed to investigate the factors associated with disease progression in localized juvenile periodontitis (LJP) patients by longitudinal monitoring of microbiological changes. Following a 9-month period, 9 LJP patients were divided into 2 groups based upon attachment loss, progressing and non-progressing. Both groups received scaling, root planing, and modified Widman flaps. Clinical and microbiological data were obtained at baseline, following the observation period, and at 6 and 12 months post-treatment. At 6 and 12 months post-treatment significantly more cocci were persistent in the non-progressing group than in the progressing group. Actinobacillus actinomycetemcomitans was also more frequently isolated in the progressing group than in the non-progressing group initially and following the 9-month observation period. Also after treatment, A. actinomycetemcomitans recolonized earlier in the progressing group than in the non-progressing group. These studies suggest that A. actinomycetemcomitans may play a role in disease progression in LJP; however, they do not eliminate the possibility that other organisms may also play a role, since A. actinomycetemcomitans was not detected in all of the patients in whom disease progressed.

Bacterial antibody titers in ligature-induced periodontitis in beagle dogs.

Serum antibodies to indigenous bacteria in ten beagle dogs were examined over a 7-month period during the development of ligature-induced periodontitis. Gram-negative strains comprised approximately 75% of the cultivable periodontitis microflora with a predominance of black-pigmented Bacteroides species. A total of 44 bacterial strains representing the predominant cultivable subgingival beagle dog microflora was selected for antibody determination. The IgG and, in some cases, IgM serum antibody titers to these organisms were determined by indirect immunofluorescence. The antibody titers to most test strains remained unchanged during the experimental period. Gram-negative bacteria generally exhibited lower titers than the Gram-positive bacteria. Especially low titers were found for the black-pigmented Bacteroides. Four dogs that developed the most severe periodontitis showed about 2-fold higher IgG titers to some Gram-negative anaerobic rods in the pre-ligation period than dogs that developed a more moderate periodontitis. These data suggested a possible diagnostic value of such antibody determinations. However, the overall finding of the present study was that serum antibody titers to key periodontopathic organisms remained low throughout the experiment. This result may suggest that the rapid periodontal destruction in ligature-induced periodontitis is due in part to an inadequate antibody response against the infecting microorganisms and their pathogenic products.

Bacterial IgG and IgM antibody titers in acute necrotizing ulcerative gingivitis.

IgG AND IgM ANTIBODY TITERS to eight bacterial isolates were measured by indirect immunofluorescence and ELISA in sera from acute necrotizing ulcerative gingivitis (ANUG) patients during the acute phase, from ANUG patients during the convalescent phase, from patients with gingivitis and from subjects with normal gingiva. Subjects were matched with respect to age and sex. Compared to the gingivitis and healthy groups, the ANUG groups exhibited significantly higher IgG and IgM titers to intermediate-sized spirochetes and higher IgG titers to Bacteroides melaninogenicus subsp intermedius. The findings support recent studies showing that these organisms are major bacterial components in ANUG lesions. They also suggest that these bacteria proliferate above-normal levels several weeks or months prior to the clinical onset of ANUG.

Antibodies to Actinobacillus actinomycetemcomitans in a Korean population.

Previous studies demonstrated that serum antibodies to Actinobacillus actinomycetemcomitans strain Y4 are significantly elevated in sera from localized juvenile periodontitis (LJP) and postlocalized juvenile periodontitis (P-LJP) patients compared to normal controls in United States populations. This study examined the age of subjects in relation to the antibody levels to A. actinomycetemcomitans in a Korean population. Seven groups were investigated including sera from newborns, infants, children, periodontally normal puberty and adult groups and LJP and P-LJP groups. Antibody levels were determined by an enzyme-linked immunosorbent assay (ELISA) to A. actinomycetemcomitans strain SNUDC 10-1 (serotype C) isolated from a Korean LJP patient. In the healthy non-LJP and non-P-LJP subjects, IgG levels decreased from the newborn group to the 5-month-old group and then gradually increased through the adult group. The IgM levels in these groups continuously increased from birth until a plateau was reached in the 2- to 6-year group. Serum IgA levels to strain SNUDC 10-1 were too low to be measured by this assay.

Actinobacillus actinomycetemcomitans serotypes and leukotoxicity in Korean localized juvenile periodontitis.

Actinobacillus actinomycetemcomitans is though to play an important role in the pathogenesis of localized juvenile periodontitis (LJP). Preliminary data suggested that the serotype distribution of A. actinomycetemcomitans in Korea and the United States differ. This study evaluated A. actinomycetemcomitans prevalence, serotype distribution, and leukotoxicity in Korean LJP patients by culture, enzyme-linked immunosorbent assay, indirect immunofluorescence, and lactate dehydrogenase release from polymorphonuclear leukocytes exposed to A. actinomycetemcomitans. A. actinomycetemcomitans occurred in 75% of LJP lesions and 6% of normal sites with approximately equal distribution of serotype a, b, and c. Single serotypes were isolated from nine patients while three patients harbored two serotypes either in the same or different disease sites. A. actinomycetemcomitans leukotoxicity occurred in 22% isolates with a 69% prevalence. Individual sites harbored both leukotoxic and non-leukotoxic strains with no serotype association. The distribution of serotypes and leukotoxic strains of A. actinomycetemcomitans in Korean LJP patients differed from those reported in the United States. This suggests that serotype b may not be more important in the pathogenesis of LJP.

Tissue engineered bone formation using chitosan/tricalcium phosphate sponges.

BACKGROUND: Chitosan is a biodegradable natural polymer that has been shown to improve wound healing. This study aimed to develop chitosan/tricalcium phosphate (TCP) sponges as tissue engineering scaffolds for bone formation by three-dimensional osteoblast culture. METHODS: The sponges were prepared by freeze-drying and cross-linking a mixture of chitosan solution with TCP. Fetal rat calvarial osteoblastic cells were isolated, cultured, and seeded into the sponges. The cell-sponge constructs were cultured for 56 days. Cell proliferation, alkaline phosphatase (ALPase) activity, and calcium deposition in the cell-sponge constructs were measured at 1, 7, 14, 28, and 56 days. Histologic examination was performed with light microscopy and scanning electron microscopy. RESULTS: Chitosan/TCP sponges supported the proliferation of osteoblastic cells as well as their differentiation as indicated by high ALPase activities and deposition of mineralized matrices by the cells. Light and scanning electron microscopic examination indicated that seeded osteoblastic cells were well attached to sponge matrices and proliferated in a multi-layer fashion. Small bone-like spicules were observed on the sponge matrix at 14 days. Seeded cells appeared to be embedded in the newly formed tissue matrix, which is characteristic of the osteoblast differentiation and their progression into osteocytic cells. The amount of mineralized tissue formed in the sponge at 56 days was significant. CONCLUSIONS: These results suggest that the chitosan/TCP sponge is a feasible tool as a scaffolding material to grow osteoblast in a three-dimensional structure for transplantation into a site for bone regeneration.