Improvement in Protective Sensation: Clinical Evidence From a Randomized Controlled Trial for Treatment of Painful Diabetic Neuropathy With 10 kHz Spinal Cord Stimulation.

BACKGROUND: Painful diabetic neuropathy (PDN) can result in the loss of protective sensation, in which people are at twice the likelihood of foot ulceration and three times the risk of lower extremity amputation. Here, we evaluated the long-term effects of high-frequency (10 kHz) paresthesia-independent spinal cord stimulation (SCS) on protective sensation in the feet and the associated risk of foot ulceration for individuals with PDN. METHODS: The SENZA-PDN clinical study was a randomized, controlled trial in which 216 participants with PDN were randomized to receive either conventional medical management (CMM) alone or 10 kHz SCS plus CMM, with optional treatment crossover after 6 months. At study visits (baseline through 24 months), 10-g monofilament sensory assessments were conducted at 10 locations per foot. Two published methods were used to evaluate protective sensation via classifying risk of foot ulceration. RESULTS: Participants in the 10 kHz SCS group reported increased numbers of sensate locations as compared to CMM alone (P < .001) and to preimplantation (P < .01) and were significantly more likely to be at low risk of foot ulceration using both classification methods. The proportion of low-risk participants approximately doubled from preimplantation to 3 months postimplantation and remained stable through 24 months (P ≤ .01). CONCLUSIONS: Significant improvements were observed in protective sensation from preimplantation to 24 months postimplantation for the 10 kHz SCS group. With this unique, disease-modifying improvement in sensory function, 10 kHz SCS provides the potential to reduce ulceration, amputation, and other severe sequelae of PDN. TRIAL REGISTRATION: The SENZA-PDN study is registered on ClinicalTrials.gov with identifier NCT03228420.

Health care resource utilization and costs in patients with painful diabetic neuropathy treated with 10 kHz spinal cord stimulation therapy.

BACKGROUND: Diabetic peripheral neuropathy, a common comorbidity of diabetes, is a neurodegenerative disorder that targets sensory, autonomic, and motor nerves frequently associated with painful diabetic neuropathy (PDN). PDN carries an economic burden as the result of reduced work and productivity. A recent multicenter randomized controlled trial, SENZA-PDN (NCT03228420), assessed the impact of high-frequency (10 kHz) spinal cord stimulation (SCS) on pain relief. The effects of high-frequency SCS on health care resource utilization and medical costs are not known. OBJECTIVE: To evaluate the effect of high-frequency (10 kHz) SCS on health care resource utilization (HRU) and medical costs in patients with PDN using data from the SENZA-PDN trial. METHODS: Participants with PDN were randomly assigned 1:1 to receive either 10 kHz SCS plus conventional medical management (CMM) (SCS treatment group) or CMM alone (CMM treatment group). Patient outcomes and HRU up to the 6-month follow-up are reported here. Costs (2020 USD) for each service was estimated based on publicly available Medicare fee schedules, Medicare claims data, and literature. HRU metrics of inpatient and outpatient contacts and costs are reported as means and SDs. Univariate and bivariate analyses were used to compare SCS and CMM treatment groups at 6 months. RESULTS: At 6-month follow up, the SCS arm experienced approximately half the mean rate of hospitalizations per patient compared with the CMM treatment group (0.08 vs 0.15; P = 0.066). The CMM treatment group's total health care costs per patient were approximately 51% higher compared with the SCS treatment group (equivalent to mean annual cost per patient of $9,532 vs $6,300). CONCLUSIONS: Our analysis of the SENZA-PDN trial indicates that the addition of 10 kHz SCS therapy results in lower rates of hospitalization and consequently lower health care costs among patients with PDN compared with those receiving conventional management alone.

Long-term efficacy of high-frequency (10 kHz) spinal cord stimulation for the treatment of painful diabetic neuropathy: 24-Month results of a randomized controlled trial.

AIMS: To evaluate the long-term efficacy of high-frequency (10 kHz) spinal cord stimulation (SCS) for treating refractory painful diabetic neuropathy (PDN). METHODS: The SENZA-PDN study was a prospective, multicenter, randomized controlled trial that compared conventional medical management (CMM) alone with 10 kHz SCS plus CMM (10 kHz SCS+CMM) in 216 patients with refractory PDN. After 6 months, participants with insufficient pain relief could cross over to the other treatment. In total, 142 patients with a 10 kHz SCS system were followed for 24 months, including 84 initial 10 kHz SCS+CMM recipients and 58 crossovers from CMM alone. Assessments included pain intensity, health-related quality of life (HRQoL), sleep, and neurological function. Investigators assessed neurological function via sensory, reflex, and motor tests. They identified a clinically meaningful improvement relative to the baseline assessment if there was a significant persistent improvement in neurological function that impacted the participant's well-being and was attributable to a neurological finding. RESULTS: At 24 months, 10 kHz SCS reduced pain by a mean of 79.9% compared to baseline, with 90.1% of participants experiencing ≥50% pain relief. Participants had significantly improved HRQoL and sleep, and 65.7% demonstrated clinically meaningful neurological improvement. Five (3.2%) SCS systems were explanted due to infection. CONCLUSIONS: Over 24 months, 10 kHz SCS provided durable pain relief and significant improvements in HRQoL and sleep. Furthermore, the majority of participants demonstrated neurological improvement. These long-term data support 10 kHz SCS as a safe and highly effective therapy for PDN. TRIAL REGISTRATION: ClincalTrials.gov Identifier, NCT03228420.

Osteoblast activity on collagen-GAG scaffolds is affected by collagen and GAG concentrations.

Optimization of a tissue engineering scaffold for use in bone tissue engineering requires control of many factors such as pore size, porosity, permeability and, as this study shows, the composition of the matrix. The collagen-glycosaminoglycan (GAG) scaffold variants were fabricated by varying the collagen and GAG content of the scaffold. Scaffolds were seeded with MC3T3 osteoblasts and cultured for up to 7 days. During the culture period, osteoblastic activity was evaluated by measuring metabolic activity, cell number, and spatial distribution. Collagen and GAG concentrations both affected osteoblast viability, proliferation, and spatial distribution within the scaffold. Scaffolds containing 1% collagen (w/v) and 0.088% GAG (w/v) were found to have a porosity of approximately 99%, high cell metabolic activity and cell number, and good cell infiltration over the 7 days in culture. Taken together, these results indicate the need to tailor the parameters of a biological substrate for use in a specific tissue application, in this case bone tissue engineering.

A comparative study of shear stresses in collagen-glycosaminoglycan and calcium phosphate scaffolds in bone tissue-engineering bioreactors.

The increasing demand for bone grafts, combined with their limited availability and potential risks, has led to much new research in bone tissue engineering. Current strategies of bone tissue engineering commonly use cell-seeded scaffolds and flow perfusion bioreactors to stimulate the cells to produce bone tissue suitable for implantation into the patient's body. The aim of this study was to quantify and compare the wall shear stresses in two bone tissue engineering scaffold types (collagen-glycosaminoglycan (CG) and calcium phosphate) exposed to fluid flow in a perfusion bioreactor. Based on micro-computed tomography images, three-dimensional numerical computational fluid dynamics (CFD) models of the two scaffold types were developed to calculate the wall shear stresses within the scaffolds. For a given flow rate (normalized according to the cross-sectional area of the scaffolds), shear stress was 2.8 times as high in the CG as in the calcium-phosphate scaffold. This is due to the differences in scaffold geometry, particularly the pore size (CG pore size approximately 96 microm, calcium phosphate pore size approximately 350 microm). The numerically obtained results were compared with those from an analytical method that researchers use widely experimentalists to determine perfusion flow rates in bioreactors. Our CFD simulations revealed that the cells in both scaffold types were exposed to a wide range of wall shear stresses throughout the scaffolds and that the analytical method predicted shear stresses 12% to 21% greater than those predicted using the CFD method. This study demonstrated that the wall shear stresses in calcium phosphate scaffolds (745.2 mPa) are approximately 40 times as high as in CG scaffolds (19.4 mPa) when flow rates are applied that have been experimentally used to stimulate the release of prostaglandin E(2). These findings indicate the importance of using accurate computational models to estimate shear stress and determine experimental conditions in perfusion bioreactors for tissue engineering.

The effect of dehydrothermal treatment on the mechanical and structural properties of collagen-GAG scaffolds.

The mechanical properties of tissue engineering scaffolds are critical for preserving the structural integrity and functionality during both in vivo implantation and long-term performance. In addition, the mechanical and structural properties of the scaffold can direct cellular activity within a tissue-engineered construct. In this context, the aim of this study was to investigate the effects of dehydrothermal (DHT) treatment on the mechanical and structural properties of collagen-glycosaminoglycan (CG) scaffolds. Temperature (105-180 degrees C) and exposure period (24-120 h) of DHT treatment were varied to determine their effect on the mechanical properties, crosslinking density, and denaturation of CG scaffolds. As expected, increasing the temperature and duration of DHT treatment resulted in an increase in the mechanical properties. Compressive properties increased up to twofold, while tensile properties increased up to 3.8-fold. Crosslink density was found to increase with DHT temperature but not exposure period. Denaturation also increased with DHT temperature and exposure period, ranging from 25% to 60% denaturation. Crosslink density was found to be correlated with compressive modulus, whilst denaturation was found to correlate with tensile modulus. Taken together, these results indicate that DHT treatment is a viable technique for altering the mechanical properties of CG scaffolds. The enhanced mechanical properties of DHT-treated CG scaffolds improve their suitability for use both in vitro and in vivo. In addition, this work facilitates the investigation of the effects of mechanical properties and denaturation on cell activity in a 3D environment.

Mechanical loading by fluid shear is sufficient to alter the cytoskeletal composition of osteoblastic cells.

Many structural modifications have been observed as a part of the cellular response to mechanical loading in a variety of cell types. Although changes in morphology and cytoskeletal rearrangement have been widely reported, few studies have investigated the change in cytoskeletal composition. Measuring how the amounts of specific structural proteins in the cytoskeleton change in response to mechanical loading will help to elucidate cellular mechanisms of functional adaptation to the applied forces. Therefore, the overall hypothesis of this study was that osteoblasts would respond to fluid shear stress by altering the amount of specific cross-linking proteins in the composition of the cytoskeleton. Mouse osteoblast cell line MC3T3-E1 and human fetal osteoblasts (hFOB) were exposed to 2 Pa of steady fluid shear for 2 h in a parallel plate flow chamber, and then the amount of actin, vimentin, alpha-actinin, filamin, and talin in the cytoskeleton was measured using Western blot analyses. After mechanical loading, there was no change in the amount of actin monomers in the cytoskeleton, but the cross-linking proteins alpha-actinin and filamin that cofractionated with the cytoskeleton increased by 29% (P<0.01) and 18% (P<0.02), respectively. Localization of the cross-linking proteins by fluorescent microscopy revealed that they were more widely distributed throughout the cell after exposure to fluid shear. The amount of vimentin in the cytoskeleton also increased by 15% (P<0.01). These results indicate that osteoblasts responded to mechanical loading by altering the cytoskeletal composition, which included an increase in specific proteins that would likely enhance the mechanical resistance of the cytoskeleton.

Over-expression of alpha-actinin with a GFP fusion protein is sufficient to increase whole-cell stiffness in human osteoblasts.

Osteoblasts respond to shear stress by simultaneously increasing their whole-cell stiffness and up-regulating the cytoskeletal crosslinking protein alpha-actinin. The stiffness of reconstituted cytoskeletal networks increases following the addition of alpha-actinin, but the effect of alpha-actinin on whole-cell mechanical behavior has not been investigated. The hypothesis of this study was that increasing alpha-actinin in the cytoskeleton would be sufficient to increase whole-cell stiffness. hFOB osteoblasts were transfected with a plasmid for GFP-tagged alpha-actinin, resulting in a 150% increase in the amount of alpha-actinin. The GFP-alpha-actinin fusion protein co-fractionated with the cytoskeleton and co-localized to the same regions of the cytoskeleton as endogenous alpha-actinin. Whole-cell mechanical behavior was measured by atomic force microscopy using a 25 mum diameter microsphere as an indenter. The whole-cell stiffness of cells over-expressing GFP-alpha-actinin was 60% higher than cells expressing only endogenous alpha-actinin (p < 0.002), which was within the range of mechanical behavior observed in osteoblastic cells exposed to 1 and 2 Pa of fluid shear. These results indicate that the up-regulation of alpha-actinin synthesis in osteoblasts is sufficient to alter the whole-cell mechanical behavior and highlights the potential role of alpha-actinin to reinforce cells against mechanical loads.

Mechanical stimulation of osteoblasts using steady and dynamic fluid flow.

In bone tissue engineering, flow perfusion bioreactors have shown great potential for accelerated production of functional constructs, but bioreactor culture conditions have not been optimized. The goal of this study was to investigate the short-term (1- 49 h) effects of intermittent steady, pulsatile, and oscillatory fluid flow (peak flow rate = 1.0 mL/min) on MC3T3-E1 osteoblast activity within a collagen-glycosaminoglycan scaffold. Bioreactor culture at a continuous low flow rate (0.05 mL/min) was also evaluated. Fluid flow exposure stimulated 8 to 51, 15 to 48, and 1.4 to 2.7 greater cyclooxygenase-2 (COX-2) expression, prostaglandin E2 (PGE2) production, and osteopontin expression, respectively, whereas membrane-associated prostaglandin E synthase-1 was 1.8 greater only under steady flow. Overall, intermittent flow (high flow rate) caused greater stimulation than a continuous low flow rate without a loss in cell number. Pulsatile and oscillatory fluid flow tripled COX-2 expression from 25 to 49 h (p < or =0.04), whereas under steady flow, PGE2 production dropped 52% at 49 h (p = 0.05). These results indicate that intermittent flow is advantageous for mechanically stimulating osteoblasts while maintaining cell viability. In addition, results at 49 h suggest that dynamic (pulsatile and oscillatory) flow may be more stimulatory than steady flow over long-term culture.

Design and validation of a dynamic flow perfusion bioreactor for use with compliant tissue engineering scaffolds.

In tissue engineering, flow perfusion bioreactors can be used to enhance nutrient diffusion while mechanically stimulating cells to increase matrix production. The goal of this study was to design and validate a dynamic flow perfusion bioreactor for use with compliant scaffolds. Using a non-permanent staining technique, scaffold perfusion was verified for flow rates of 0.1-2.0 mL/min. Flow analysis revealed that steady, pulsatile and oscillatory flow profiles were effectively transferred from the pump to the scaffold. Compared to static culture, bioreactor culture of osteoblast-seeded collagen-GAG scaffolds led to a 27-34% decrease in cell number but stimulated an 800-1200% increase in the production of prostaglandin E(2), an early-stage bone formation marker. This validated flow perfusion bioreactor provides the basis for optimisation of bioreactor culture in tissue engineering applications.

Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells.

Numerous cellular biochemical responses to mechanical loading are transient, indicating a cell's ability to adapt its behavior to a new mechanical environment. Since load-induced cellular deformation can initiate these biochemical responses, the overall goal of this study was to investigate the adaptation of global, or whole-cell, mechanical behavior, i.e., cellular deformability, in response to mechanical loading for osteoblastic cells. Confluent cell cultures were subjected to 1 or 2 Pa flow-induced shear stress for 2 h. Whole-cell mechanical behavior was then measured for individual cells using an atomic force microscope. Compared to cells maintained under static conditions, whole-cell stiffness was 1.36-fold (p=0.006) and 1.70-fold (p<0.001) greater for cells exposed to 1 and 2 Pa shear loading, respectively. The increase in shear stress magnitude from 1 to 2 Pa also caused a statistically significant, 1.25-fold increase in cell stiffness (p=0.02). Increases in cell stiffness were not altered in either flow group for 70 min after flow was terminated (p=0.15). Flow-induced rearrangement of the actin cytoskeleton was also maintained for at least 90 min after flow was terminated. Taken together, these findings support the hypothesis that cells become mechanically adapted to their mechanical environment via cytoskeletal modifications. Accordingly, cellular mechanical adaptation may play a key role in regulation of cellular mechanosensitivity and the related effects on tissue structure and function.

Measurement and characterization of whole-cell mechanical behavior.

An understanding of whole-cell mechanical behavior can provide insight into cellular responses to mechanical loading and diseases in which such responses are altered. However, this aspect of cellular mechanical behavior has received limited attention. In this study, we used the atomic force microscope (AFM) in conjunction with several mechanical characterization methods (Hertz contact theory, an exponential equation, and a parallel-spring recruitment model) to establish a mechanically rigorous method for measuring and characterizing whole-cell mechanical behavior in the deformation range 0-500 nm. Using MC3T3-E1 osteoblasts, measurement repeatability was assessed by performing multiple loading cycles on individual cells. Despite variability in measurements, repeatability of the measurement technique was statistically confirmed. The measurement technique also proved acceptable since only 5% of the total variance across all measurements was due to variations within measurements for a single cell. The parallel-spring recruitment model, a single-parameter model, accurately described the measured nonlinear force-deformation response (R2>0.99) while providing a mechanistic explanation of whole-cell mechanical behavior. Taken together, the results should improve the capabilities of the AFM to probe whole-cell mechanical behavior. In addition, the success of the parallel-spring recruitment model provides insight into the micromechanical basis of whole-cell behavior.

The effects of morphology, confluency, and phenotype on whole-cell mechanical behavior.

Emerging evidence indicates that cellular mechanical behavior can be altered by disease, drug treatment, and mechanical loading. To effectively investigate how disease and mechanical or biochemical treatments influence cellular mechanical behavior, it is imperative to determine the source of large inter-cell differences in whole-cell mechanical behavior within a single cell line. In this study, we used the atomic force microscope to investigate the effects of cell morphological parameters and confluency on whole-cell mechanical behavior for osteoblastic and fibroblastic cells. For nonconfluent cells, projected nucleus area, cell area, and cell aspect ratio were not correlated with mechanical behavior (p>or=0.46), as characterized by a parallel-spring recruitment model. However, measured force-deformation responses were statistically different between osteoblastic and fibroblastic cells (p<0.001) and between confluent and nonconfluent cells (p<0.001). Osteoblastic cells were 2.3-2.8 times stiffer than fibroblastic cells, and confluent cells were 1.5-1.8 times stiffer than nonconfluent cells. The results indicate that structural differences related to phenotype and confluency affect whole-cell mechanical behavior, while structural differences related to global morphology do not. This suggests that cytoskeleton structural parameters, such as filament density, filament crosslinking, and cell-cell and cell-matrix attachments, dominate inter-cell variability in whole-cell mechanical behavior.

Biomechanical effects of intraspecimen variations in tissue modulus for trabecular bone.

Although recent nanoindentation studies have revealed the existence of substantial variations in tissue modulus within single specimens of trabecular bone, little is known regarding the biomechanical effects of such intraspecimen variations. In this study, high-resolution finite element modeling was used to investigate these effects. With limited literature information on the spatial distribution of intraspecimen variations in tissue modulus, two plausible spatial distributions were evaluated. In addition, three specimens (human femoral neck, human vertebral body, and bovine proximal tibia) were studied to assess the role of trabecular architecture. Results indicated that for all specimen/distribution combinations, the apparent modulus of the whole specimen decreased nonlinearly with increasing coefficient of variation (COV) of tissue modulus within the specimen. Apparent modulus decreased by <4% when tissue modulus COV was increased from 0% to 20% but decreased by 7-24%, depending on the assumed spatial distribution, for an increase in tissue modulus COV from 20% to 50%. For compressive loading to the elastic limit, increasing tissue modulus COV from 20% to 50% caused up to a 28-fold increase in the amount of failed tissue, depending on assumed spatial distribution and trabecular architecture. We conclude that intraspecimen variations in tissue modulus, if large, may have appreciable effects on trabecular apparent modulus and tissue-level failure. Since the observed effects depended on the assumed spatial distribution of the tissue modulus variations, a description of such distributions, particularly as a function of age, disease, and drug treatment, may provide new insight into trabecular bone structure-function relationships.