Influence of Particle Size of Deproteinized Bovine Bone Mineral on New Bone Formation and Implant Stability after Simultaneous Sinus Floor Elevation: A Histomorphometric Study in Minipigs

BACKGROUND Deproteinized bovine bone mineral (DBBM) is one of the best-documented bone substitute materials for sinus floor elevation (SFE). PURPOSE DBBM is available in two particle sizes. Large particles are believed to facilitate improved neoangiogenesis compared with small ones. However, their impact on the rate of new bone formation, osteoconduction, and DBBM degradation has never been reported. In addition, the implant stability quotient (ISQ) has never been correlated to bone-to-implant contact (BIC) after SFE with simultaneous implant placement. MATERIALS AND METHODS Bilateral SFE with simultaneous implant placement was performed in 10 Göttingen minipigs. The two sides were randomized to receive large or small particle size DBBM. Two groups of 5 minipigs healed for 6 and 12 weeks, respectively. ISQ was recorded immediately after implant placement and at sacrifice. Qualitative histological differences were described and bone formation, DBBM degradation, BIC and bone-to-DBBM contact (osteoconduction) were quantified histomorphometrically. RESULTS DBBM particle size had no qualitative or quantitative impact on the amount of newly formed bone, DBBM degradation, or BIC for either of the healing periods (p > 0.05). Small-size DBBM showed higher osteoconduction after 6 weeks than large-size DBBM (p < 0.001). After 12 weeks this difference was compensated. There was no significant correlation between BIC and ISQ. CONCLUSION Small and large particle sizes were equally predictable when DBBM was used for SFE with simultaneous implant placement.

Periodontal healing after application of enamel matrix derivative in surgical supra/infrabony periodontal defects in rats with streptozotocin-induced diabetes

BACKGROUND AND OBJECTIVE Epidemiologic and clinical studies have indicated that diabetes is a risk factor for periodontal disease progression and healing. The aim of the present study was to evaluate short-term healing after enamel matrix derivative (EMD) application in combined supra/infrabony periodontal defects in diabetic rats. MATERIAL AND METHODS Thirty male Wistar rats were initially divided into two groups, one with streptozotocin-induced diabetes and another one with healthy (non-diabetic) animals. Bony defects were surgically created on the mesial root of the first maxillary molars. After root surface planing and EDTA conditioning, EMD was applied to the roots at one side of the maxillae, while those on the contralateral sides were left untreated. Animals were killed 3 wk after surgery, and block sections were prepared for histologic and histomorphometric analysis. RESULTS There was statistically significant more gingival recession in diabetic animals than in non-diabetic animals. The length of the junctional epithelium was significantly shorter in the EMD-treated sites in both diabetic and normoglycemic rats. Sulcus depth and length of supracrestal soft connective tissue showed no statistically significant differences between groups. In all animals, new bone formation was observed. Although new bone occurred more frequently in healthy animals, the extent of new bone was not significantly different between groups. In none of the teeth, a layer of new cementum was detectable. EMD had no influence on bone or cementum regeneration. Adverse reactions such as excessive inflammation due to bacterial root colonization, ankylosis and bone fractures were exclusively observed in diabetic animals, irrespective of EMD treatment. CONCLUSION Within the limits of the present study, it can be concluded that periodontal healing was impaired in streptozotocin-induced diabetic rats. EMD had no beneficial effects on new bone and cementum formation during short-term healing in this defect model and could not ameliorate the adverse effects in the systemically compromised animals.

Long-term clinical, microbiological, and radiographic outcomes of Brånemark™ implants installed in augmented maxillary bone for fixed full-arch rehabilitation

PURPOSE The purpose of this study was to document the long-term outcome of Brånemark implants installed in augmented maxillary bone and to identify parameters that are associated with peri-implant bone level. MATERIAL AND METHODS Patients of a periodontal practice who had been referred to a maxillofacial surgeon for iliac crest bone grafting in the atrophic maxilla were retrospectively recruited. Five months following grafting, they received 7-8 turned Brånemark implants. Following submerged healing of another 5 months, implants were uncovered and restorative procedures for fixed rehabilitation were initiated 2-3 months thereafter. The primary outcome variable was bone level defined as the distance from the implant-abutment interface to the first visible bone-to-implant contact. Secondary outcome variables included plaque index, bleeding index, probing depth, and levels of 40 species in subgingival plaque samples as identified by means of checkerboard DNA-DNA hybridization. RESULTS Nine out of 16 patients (eight females, one male; mean age 59) with 71 implants agreed to come in for evaluation after on average 9 years (SD 4; range 3-13) of function. One implant was deemed mobile at the time of inspection. Clinical conditions were acceptable with 11% of the implants showing pockets ≥ 5 mm. Periodontopathogens were encountered frequently and in high numbers. Clinical parameters and bacterial levels were highly patient dependent. The mean bone level was 2.30 mm (SD 1.53; range 0.00-6.95), with 23% of the implants demonstrating advanced resorption (bone level > 3 mm). Regression analysis showed a significant association of the patient (p < .001) and plaque index (p = .007) with bone level. CONCLUSIONS The long-term outcome of Brånemark implants installed in iliac crest-augmented maxillary bone is acceptable; however, advanced peri-implant bone loss is rather common and indicative of graft resorption. This phenomenon is patient dependent and seems also associated with oral hygiene.

Influence of platform switching on bone-level alterations: a three-year randomized clinical trial

The concept of platform switching has been introduced to implant dentistry based on clinical observations of reduced peri-implant crestal bone loss. However, published data are controversial, and most studies are limited to 12 months. The aim of the present randomized clinical trial was to test the hypothesis that platform switching has a positive impact on crestal bone-level changes after 3 years. Two implants with a diameter of 4 mm were inserted crestally in the posterior mandible of 25 patients. The intraindividual allocation of platform switching (3.3-mm platform) and the standard implant (4-mm platform) was randomized. After 3 months of submerged healing, single-tooth crowns were cemented. Patients were followed up at short intervals for monitoring of healing and oral hygiene. Statistical analysis for the influence of time and platform type on bone levels employed the Brunner-Langer model. At 3 years, the mean radiographic peri-implant bone loss was 0.69 ± 0.43 mm (platform switching) and 0.74 ± 0.57 mm (standard platform). The mean intraindividual difference was 0.05 ± 0.58 mm (95% confidence interval: -0.19, 0.29). Crestal bone-level alteration depended on time (p < .001) but not on platform type (p = .363). The present randomized clinical trial could not confirm the hypothesis of a reduced peri-implant crestal bone loss, when implants had been restored according to the concept of platform switching.

The influence of yield surface shape and damage in the depth-dependent response of bone tissue to nanoindentation using spherical and Berkovich indenters

Prevention and treatment of osteoporosis rely on understanding of the micromechanical behaviour of bone and its influence on fracture toughness and cell-mediated adaptation processes. Postyield properties may be assessed by nonlinear finite element simulations of nanoindentation using elastoplastic and damage models. This computational study aims at determining the influence of yield surface shape and damage on the depth-dependent response of bone to nanoindentation using spherical and conical tips. Yield surface shape and damage were shown to have a major impact on the indentation curves. Their influence on indentation modulus, hardness, their ratio as well as the elastic-to-total work ratio is well described by multilinear regressions for both tip shapes. For conical tips, indentation depth was not statistically significant (p<0.0001). For spherical tips, damage was not a significant parameter (p<0.0001). The gained knowledge can be used for developing an inverse method for identification of postelastic properties of bone from nanoindentation.

Finite element analysis for prediction of bone strength

Article preview View full access options BoneKEy Reports | Review Print Email Share/bookmark Finite element analysis for prediction of bone strength Philippe K Zysset, Enrico Dall'Ara, Peter Varga & Dieter H Pahr Affiliations Corresponding author BoneKEy Reports (2013) 2, Article number: 386 (2013) doi:10.1038/bonekey.2013.120 Received 03 January 2013 Accepted 25 June 2013 Published online 07 August 2013 Article tools Citation Reprints Rights & permissions Abstract Abstract• References• Author information Finite element (FE) analysis has been applied for the past 40 years to simulate the mechanical behavior of bone. Although several validation studies have been performed on specific anatomical sites and load cases, this study aims to review the predictability of human bone strength at the three major osteoporotic fracture sites quantified in recently completed in vitro studies at our former institute. Specifically, the performance of FE analysis based on clinical computer tomography (QCT) is compared with the ones of the current densitometric standards, bone mineral content, bone mineral density (BMD) and areal BMD (aBMD). Clinical fractures were produced in monotonic axial compression of the distal radii, vertebral sections and in side loading of the proximal femora. QCT-based FE models of the three bones were developed to simulate as closely as possible the boundary conditions of each experiment. For all sites, the FE methodology exhibited the lowest errors and the highest correlations in predicting the experimental bone strength. Likely due to the improved CT image resolution, the quality of the FE prediction in the peripheral skeleton using high-resolution peripheral CT was superior to that in the axial skeleton with whole-body QCT. Because of its projective and scalar nature, the performance of aBMD in predicting bone strength depended on loading mode and was significantly inferior to FE in axial compression of radial or vertebral sections but not significantly inferior to FE in side loading of the femur. Considering the cumulated evidence from the published validation studies, it is concluded that FE models provide the most reliable surrogates of bone strength at any of the three fracture sites.

Impact of bone graft harvesting techniques on bone formation and graft resorption: a histomorphometric study in the mandibles of minipigs.

BACKGROUND: Harvesting techniques can affect cellular parameters of autogenous bone grafts in vitro. Whether these differences translate to in vivo bone formation, however, remains unknown. OBJECTIVE: The purpose of this study was to assess the impact of different harvesting techniques on bone formation and graft resorption in vivo. MATERIAL AND METHODS: Four harvesting techniques were used: (i) corticocancellous blocks particulated by a bone mill; (ii) bone scraper; (iii) piezosurgery; and (iv) bone slurry collected from a filter device upon drilling. The grafts were placed into bone defects in the mandibles of 12 minipigs. The animals were sacrificed after 1, 2, 4 and 8 weeks of healing. Histology and histomorphometrical analyses were performed to assess bone formation and graft resorption. An explorative statistical analysis was performed. RESULTS: The amount of new bone increased, while the amount of residual bone decreased over time with all harvesting techniques. At all given time points, no significant advantage of any harvesting technique on bone formation was observed. The harvesting technique, however, affected bone formation and the amount of residual graft within the overall healing period. Friedman test revealed an impact of the harvesting technique on residual bone graft after 2 and 4 weeks. At the later time point, post hoc testing showed more newly formed bone in association with bone graft processed by bone mill than harvested by bone scraper and piezosurgery. CONCLUSIONS: Transplantation of autogenous bone particles harvested with four techniques in the present model resulted in moderate differences in terms of bone formation and graft resorption.

Increased vascularization during early healing after biologic augmentation in repair of chronic rotator cuff tears using autologous leukocyte- and platelet-rich fibrin (L-PRF): a prospective randomized controlled pilot trial

HYPOTHESIS We hypothesized that arthroscopic rotator cuff repairs using leukocyte- and platelet-rich fibrin (L-PRF) in a standardized, modified protocol is technically feasible and results in a higher vascularization response and watertight healing rate during early healing. METHODS Twenty patients with chronic rotator cuff tears were randomly assigned to 2 treatment groups. In the test group (N = 10), L-PRF was added in between the tendon and the bone during arthroscopic rotator cuff repair. The second group served as control (N = 10). They received the same arthroscopic treatment without the use of L-PRF. We used a double-row tension band technique. Clinical examinations including subjective shoulder value, visual analog scale, Constant, and Simple Shoulder Test scores and measurement of the vascularization with power Doppler ultrasonography were made at 6 and 12 weeks. RESULTS There have been no postoperative complications. At 6 and 12 weeks, there was no significant difference in the clinical scores between the test and the control groups. The mean vascularization index of the surgical tendon-to-bone insertions was always significantly higher in the L-PRF group than in the contralateral healthy shoulders at 6 and 12 weeks (P = .0001). Whereas the L-PRF group showed a higher vascularization compared with the control group at 6 weeks (P = .001), there was no difference after 12 weeks of follow-up (P = .889). Watertight healing was obtained in 89% of the repaired cuffs. DISCUSSION/CONCLUSIONS Arthroscopic rotator cuff repair with the application of L-PRF is technically feasible and yields higher early vascularization. Increased vascularization may potentially predispose to an increased and earlier cellular response and an increased healing rate.

High resolution quantitative computed tomography-based assessment of trabecular microstructure and strength estimates by finite-element analysis of the spine, but not DXA, reflects vertebral fracture status in men with glucocorticoid-induced osteoporosis

High-resolution quantitative computed tomography (HRQCT)-based analysis of spinal bone density and microstructure, finite element analysis (FEA), and DXA were used to investigate the vertebral bone status of men with glucocorticoid-induced osteoporosis (GIO). DXA of L1–L3 and total hip, QCT of L1–L3, and HRQCT of T12 were available for 73 men (54.6±14.0years) with GIO. Prevalent vertebral fracture status was evaluated on radiographs using a semi-quantitative (SQ) score (normal=0 to severe fracture=3), and the spinal deformity index (SDI) score (sum of SQ scores of T4 to L4 vertebrae). Thirty-one (42.4%) subjects had prevalent vertebral fractures. Cortical BMD (Ct.BMD) and thickness (Ct.Th), trabecular BMD (Tb.BMD), apparent trabecular bone volume fraction (app.BV/TV), and apparent trabecular separation (app.Tb.Sp) were analyzed by HRQCT. Stiffness and strength of T12 were computed by HRQCT-based nonlinear FEA for axial compression, anterior bending and axial torsion. In logistic regressions adjusted for age, glucocorticoid dose and osteoporosis treatment, Tb.BMD was most closely associated with vertebral fracture status (standardized odds ratio [sOR]: Tb.BMD T12: 4.05 [95% CI: 1.8–9.0], Tb.BMD L1–L3: 3.95 [1.8–8.9]). Strength divided by cross-sectional area for axial compression showed the most significant association with spine fracture status among FEA variables (2.56 [1.29–5.07]). SDI was best predicted by a microstructural model using Ct.Th and app.Tb.Sp (r2=0.57, p<0.001). Spinal or hip DXA measurements did not show significant associations with fracture status or severity. In this cross-sectional study of males with GIO, QCT, HRQCT-based measurements and FEA variables were superior to DXA in discriminating between patients of differing prevalent vertebral fracture status. A microstructural model combining aspects of cortical and trabecular bone reflected fracture severity most accurately.

Bone mineral density (BMD) and computer tomographic measurements of the equine proximal phalanx in correlation with breaking strength

Despite the fact that bone mineral density (BMD) is an important fracture risk predictor in human medicine, studies in equine orthopedic research are still lacking. We hypothesized that BMD correlates with bone failure and fatigue fractures of this bone. Thus, the objectives of this study were to measure the structural and mechanical properties of the proximal phalanx with dual energy X-ray absorptiometry (DXA), to correlate the data obtained from DXA and computer tomography (CT) measurements to those obtained by loading pressure examination and to establish representative region of interest (ROI) for in vitro BMD measurements of the equine proximal phalanx for predicting bone failure force. DXA was used to measure the whole bone BMD and additional three ROI sites in 14 equine proximal phalanges. Following evaluation of the bone density, whole bone, cortical width and area in the mid-diaphyseal plane were measured on CT images. Bones were broken using a manually controlled universal bone crusher to measure bone failure force and reevaluated for the site of fractures on follow-up CT images. Compressive load was applied at a constant displacement rate of 2 mm/min until failure, defined as the first clear drop in the load measurement. The lowest BMD was measured at the trabecular region (mean +/- SD: 1.52 +/- 0.12 g/cm2; median: 1.48 g/cm2; range: 1.38-1.83 g/cm2). There was a significant positive linear correlation between trabelcular BMD and the breaking strength (P = 0.023, r = 0.62). The trabecular region of the proximal phalanx appears to be the only significant indicator of failure of strength in vitro. This finding should be reassessed to further reveal the prognostic value of trabecular BMD in an in vivo fracture risk model.

Effects of zoledronate versus placebo on spine bone mineral density and microarchitecture assessed by the trabecular bone score in postmenopausal women with osteoporosis: a three-year study

The trabecular bone score (TBS) is an index of bone microarchitectural texture calculated from anteroposterior dual-energy X-ray absorptiometry (DXA) scans of the lumbar spine (LS) that predicts fracture risk, independent of bone mineral density (BMD). The aim of this study was to compare the effects of yearly intravenous zoledronate (ZOL) versus placebo (PLB) on LS BMD and TBS in postmenopausal women with osteoporosis. Changes in TBS were assessed in the subset of 107 patients recruited at the Department of Osteoporosis of the University Hospital of Berne, Switzerland, who were included in the HORIZON trial. All subjects received adequate calcium and vitamin D3. In these patients randomly assigned to either ZOL (n = 54) or PLB (n = 53) for 3 years, BMD was measured by DXA and TBS assessed by TBS iNsight (v1.9) at baseline and 6, 12, 24, and 36 months after treatment initiation. Baseline characteristics (mean ± SD) were similar between groups in terms of age, 76.8 ± 5.0 years; body mass index (BMI), 24.5 ± 3.6 kg/m(2) ; TBS, 1.178 ± 0.1 but for LS T-score (ZOL-2.9 ± 1.5 versus PLB-2.1 ± 1.5). Changes in LS BMD were significantly greater with ZOL than with PLB at all time points (p < 0.0001 for all), reaching +9.58% versus +1.38% at month 36. Change in TBS was significantly greater with ZOL than with PLB as of month 24, reaching +1.41 versus-0.49% at month 36; p = 0.031, respectively. LS BMD and TBS were weakly correlated (r = 0.20) and there were no correlations between changes in BMD and TBS from baseline at any visit. In postmenopausal women with osteoporosis, once-yearly intravenous ZOL therapy significantly increased LS BMD relative to PLB over 3 years and TBS as of 2 years.

Microstructural Parameters of Bone Evaluated Using HR-pQCT Correlate with the DXA-Derived Cortical Index and the Trabecular Bone Score in a Cohort of Randomly Selected Premenopausal Women.

BACKGROUND Areal bone mineral density is predictive for fracture risk. Microstructural bone parameters evaluated at the appendicular skeleton by high-resolution peripheral quantitative computed tomography (HR-pQCT) display differences between healthy patients and fracture patients. With the simple geometry of the cortex at the distal tibial diaphysis, a cortical index of the tibia combining material and mechanical properties correlated highly with bone strength ex vivo. The trabecular bone score derived from the scan of the lumbar spine by dual-energy X-ray absorptiometry (DXA) correlated ex vivo with the micro architectural parameters. It is unknown if these microstructural correlations could be made in healthy premenopausal women. METHODS Randomly selected women between 20-40 years of age were examined by DXA and HR-pQCT at the standard regions of interest and at customized sub regions to focus on cortical and trabecular parameters of strength separately. For cortical strength, at the distal tibia the volumetric cortical index was calculated directly from HR-pQCT and the areal cortical index was derived from the DXA scan using a Canny threshold-based tool. For trabecular strength, the trabecular bone score was calculated based on the DXA scan of the lumbar spine and was compared with the corresponding parameters derived from the HR-pQCT measurements at radius and tibia. RESULTS Seventy-two healthy women were included (average age 33.8 years, average BMI 23.2 kg/m(2)). The areal cortical index correlated highly with the volumetric cortical index at the distal tibia (R  =  0.798). The trabecular bone score correlated moderately with the microstructural parameters of the trabecular bone. CONCLUSION This study in randomly selected premenopausal women demonstrated that microstructural parameters of the bone evaluated by HR-pQCT correlated with the DXA derived parameters of skeletal regions containing predominantly cortical or cancellous bone. Whether these indexes are suitable for better predictions of the fracture risk deserves further investigation.

Cortical bone loss at the tibia in postmenopausal women with osteoporosis is associated with incident non-vertebral fractures: Results of a randomized controlled ancillary study of HORIZON

BACKGROUND In postmenopausal women, yearly intravenous zoledronate (ZOL) compared to placebo (PLB) significantly increased bone mineral density (BMD) at lumbar spine (LS), femoral neck (FN), and total hip (TH) and decreased fracture risk. The effects of ZOL on BMD at the tibial epiphysis (T-EPI) and diaphysis (T-DIA) are unknown. METHODS A randomized controlled ancillary study of the HORIZON trial was conducted at the Department of Osteoporosis of the University Hospital of Berne, Switzerland. Women with ≥1 follow-up DXA measurement who had received ≥1 dose of either ZOL (n=55) or PLB (n=55) were included. BMD was measured at LS, FN, TH, T-EPI, and T-DIA at baseline, 6, 12, 24, and 36 months. Morphometric vertebral fractures were assessed. Incident clinical fractures were recorded as adverse events. RESULTS Baseline characteristics were comparable with those in HORIZON and between groups. After 36 months, BMD was significantly higher in women treated with ZOL vs. PLB at LS, FN, TH, and T-EPI (+7.6%, +3.7%, +5.6%, and +5.5%, respectively, p<0.01 for all) but not T-DIA (+1.1%). The number of patients with ≥1 incident non-vertebral or morphometric fracture did not differ between groups (9 ZOL/11 PLB). Mean changes in BMD did not differ between groups with and without incident fracture, except that women with an incident non-vertebral fracture had significantly higher bone loss at predominantly cortical T-DIA (p=0.005). CONCLUSION ZOL was significantly superior to PLB at T-EPI but not at T-DIA. Women with an incident non-vertebral fracture experienced bone loss at T-DIA.

Scaling relations between trabecular bone volume fraction and microstructure at different skeletal sites

In this study, we investigated the scaling relations between trabecular bone volume fraction (BV/TV) and parameters of the trabecular microstructure at different skeletal sites. Cylindrical bone samples with a diameter of 8mm were harvested from different skeletal sites of 154 human donors in vitro: 87 from the distal radius, 59/69 from the thoracic/lumbar spine, 51 from the femoral neck, and 83 from the greater trochanter. μCT images were obtained with an isotropic spatial resolution of 26μm. BV/TV and trabecular microstructure parameters (TbN, TbTh, TbSp, scaling indices (< > and σ of α and αz), and Minkowski Functionals (Surface, Curvature, Euler)) were computed for each sample. The regression coefficient β was determined for each skeletal site as the slope of a linear fit in the double-logarithmic representations of the correlations of BV/TV versus the respective microstructure parameter. Statistically significant correlation coefficients ranging from r=0.36 to r=0.97 were observed for BV/TV versus microstructure parameters, except for Curvature and Euler. The regression coefficients β were 0.19 to 0.23 (TbN), 0.21 to 0.30 (TbTh), −0.28 to −0.24 (TbSp), 0.58 to 0.71 (Surface) and 0.12 to 0.16 (<α>), 0.07 to 0.11 (<αz>), −0.44 to −0.30 (σ(α)), and −0.39 to −0.14 (σ(αz)) at the different skeletal sites. The 95% confidence intervals of β overlapped for almost all microstructure parameters at the different skeletal sites. The scaling relations were independent of vertebral fracture status and similar for subjects aged 60–69, 70–79, and >79years. In conclusion, the bone volume fraction–microstructure scaling relations showed a rather universal character.

Effect of pulverized natural bone mineral on regeneration of three-wall intrabony defects. A preclinical study.

AIMS The objective of this study is to evaluate the effects of a paste-like bone substitute material with easy handling properties and improved mechanical stability on periodontal regeneration of intrabony defects in dogs. MATERIALS AND METHODS Mandibular and maxillary first and third premolars were extracted, and three-wall intrabony defects were created on second and fourth premolars. After a healing period of 3 months, acute type defects were filled with a paste-like formulation of deproteinized bovine bone mineral (DBBM) (particle size, 0.125-0.25 mm) in a collagenous carrier matrix (T1), pulverized DBBM (particle size, 0.125-0.25 mm) without the carrier (T2), or Bio-Oss® granules (particle size, 0.25-1.00 mm) as control (C). All defects were covered with a Bio-Gide® membrane. The dogs were sacrificed after 12 weeks, and the specimens were analyzed histologically and histometrically. RESULTS Postoperative healing of all defects was uneventful, and no histological signs of inflammation were observed in the augmented and gingival regions. New cementum, new periodontal ligament, and new bone were observed in all three groups. The mean vertical bone gain was 3.26 mm (T1), 3.60 mm (T2), and 3.81 mm (C). That of new cementum was 2.25 mm (T1), 3.88 mm (T2), and 3.53 mm (C). The differences did not reach statistical significance. The DBBM particles were both incorporated in new bone and embedded in immature bone marrow. CONCLUSIONS The results of this preclinical study showed that the 0.125-0.25-mm DBBM particles in a powder or paste formulation resulted in periodontal regeneration comparable to the commercially available DBBM. Osteoconductivity, in particular, was not affected by DBBM size or paste formulation. CLINICAL RELEVANCE The improved handling properties of the paste-like bone substitute consisting of small DBBM particles embedded in a collagen-based carrier hold promise for clinical applications.