Repair and remodeling of partial-weightbearing, uninstrumented long bone fracture model in mice treated with low intensity vibration therapy.

BACKGROUND: While vibration therapy has shown encouraging results across many fields of medicine in the last decade, its role as originally envisioned for bone health remains uncertain. Especially regarding its efficacy in promoting fracture healing, mixed and incomplete outcomes suggest a need to clarify its potential. In particular, the definitive effect of vibration, when isolated from the confounding mechanical inputs of gait and stabilizing instrumentation, remains largely unknown. METHODS: Four cohorts of C57BL/6 male mice underwent single-leg, open fibula fracture. Vibration was applied at 0.3 g to two groups for 20 min/d. At 3 and 6 weeks, fibulae were harvested for microcomputed tomography and 3-point bending to failure. FINDINGS: In bone volume and tissue volume, the groups at each healing time point were statistically not different. At 3 weeks, however, the ratio of bone-to-tissue volume was lower for the vibrated group than control. Likewise, while bone mineral density did not differ, tissue volume density was lowest with vibration. At 6 weeks, mean differences were nominal. Biomechanically, vibration consistently trended ahead of control in strength and stiffness, but did not achieve statistical significance. INTERPRETATION: At this stage of therapeutic development, vibration therapy in isolation does not demonstrate a clear efficacy for bone healing, although further treatment permutations and translational uses remain open for investigation.

Ingestion of gastrolith mineralized matrix increases bone volume and tissue volume in mouse long bone fracture model.

PURPOSE: Fracture healing often requires extended convalescence as the bony fragments consolidate into restored viable tissue for load-bearing. Development of interventions to improve healing remains a priority for orthopaedic research. The goal of this study was to evaluate the ability of a naturally occurring matrix of amorphous calcium carbonate to affect fracture healing in an uninstrumented long bone model. METHODS: Complete transverse fracture was induced in the fibula of mature mice, followed by daily gavage of crushed gastrolith from crayfish at doses of 0 (control), 1 (1 MG), and 5 (5 MG) mg/kg. At Day 17, bones and sera were harvested. RESULTS: Morphologically, the 1 MG treated group had greater bone volume (BV), and both 1 MG and 5 MG had greater tissue volume (TV) than control (p < 0.05), as determined by µCT; BV/TV and mineral density did not yield a statistical difference. Histologically, regional variations in mineralized matrix were evident in all specimens, indicating a broad continuum of healing within the callus. Among serum proteins, bone-specific alkaline phosphatase, indicative of active mineralization, was greater in 5 MG than control (p < 0.05). Sclerostin, an inhibitor of osteogenesis, was lower in 5 MG than control (p < 0.05), also suggestive of enhanced healing. CONCLUSIONS: An increase in bone volume, tissue volume and cellular signaling for osteogenesis at 17 days following fibula fracture in this mouse model suggests that gastrolith treatment holds potential for improving fracture healing. Further study at subsequent time points is warranted to determine the extent to which the increase in callus size with gastrolith treatment may accelerate restoration of tissue integrity.

Whole-Body Vibration Mimics the Metabolic Effects of Exercise in Male Leptin Receptor-Deficient Mice.

Whole-body vibration (WBV) has gained attention as a potential exercise mimetic, but direct comparisons with the metabolic effects of exercise are scarce. To determine whether WBV recapitulates the metabolic and osteogenic effects of physical activity, we exposed male wild-type (WT) and leptin receptor-deficient (db/db) mice to daily treadmill exercise (TE) or WBV for 3 months. Body weights were analyzed and compared with WT and db/db mice that remained sedentary. Glucose and insulin tolerance testing revealed comparable attenuation of hyperglycemia and insulin resistance in db/db mice following TE or WBV. Both interventions reduced body weight in db/db mice and normalized muscle fiber diameter. TE or WBV also attenuated adipocyte hypertrophy in visceral adipose tissue and reduced hepatic lipid content in db/db mice. Although the effects of leptin receptor deficiency on cortical bone structure were not eliminated by either intervention, exercise and WBV increased circulating levels of osteocalcin in db/db mice. In the context of increased serum osteocalcin, the modest effects of TE and WBV on bone geometry, mineralization, and biomechanics may reflect subtle increases in osteoblast activity in multiple areas of the skeleton. Taken together, these observations indicate that WBV recapitulates the effects of exercise on metabolism in type 2 diabetes.

Determination of joint loads using new sensate scaffolds for regenerating large cartilage defects in the knee.

Two complete unicondylar surface replacement scaffold designs to support tissue-engineered cartilage growth that utilized adult endogenous stem cells were 3D printed and tested in a dog stifle model. Integrated rosette strain gauges were calibrated and used to determine shear loading within stifle joints for up to 12 months. An activity index that compared extent of daily activity with tissue formation showed differences in the extent and quality of new tissue with the most active animal having the most new tissue formation. Shear loads were highest early and decreased with time indicating that cartilage tissue formation begins while tissues experience high shear loads and continues as the loads decrease toward normal physiological levels. Scaffolds with biomimetic support pegs facilitated the most rapid bone ingrowth and were noted to have more cartilage formation with better quality cartilage as measured using both indentation testing and histology. Comparison of implant placement depth to previous studies suggested that placement depth affects the amount of tissue formation. This study provides measurements of loading patterns and cartilage regeneration on a complete medial condylar surface replacement that can be used for preclinical testing of a tissue engineering approach for the most common form of early stage osteoarthritis, unicondylar disease. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1409-1421, 2017.

In vivo telemetric determination of shear and axial loads on a regenerative cartilage scaffold following ligament disruption.

Recent interest in repair of chondral and osteochondral cartilage defects to prevent osteoarthritis caused by ligament disruption has led to the research and development of biomimetic scaffolds combined with cell-based regeneration techniques. Current clinical focal defect repair strategies have had limited success. New scaffold-based approaches may provide solutions that can repair extensive damage and prevent osteoarthritis. This study utilized a novel scaffold design that accommodated strain gauges for shear and axial load monitoring in the canine stifle joint through implantable telemetry technology. Loading changes induced by ligament disruption are widely implicated in the development of injury-related osteoarthritis. Seeding the scaffold end with progenitor cells resulted in higher shear stress than without cell seeding and histology showed significantly more bone and cartilage formation. Biomechanically, the effect of transecting the anterior cruciate ligament was a significant reduction in braking load in shear, but no change axially, and conversely a significant reduction in push-off load axially, but no change in shear. This is the first study to report shear loads measured directly in knee joint tissue. Further, advances of these measurement techniques are critical to developing improved regeneration strategies and personalizing reliable rehabilitation protocols.

Human periodontal ligament fibroblast responses to compression in chronic periodontitis.

AIM: Test whether human periodontal ligament fibroblasts (PDLFs) retain homeostatic responses to a physiological compressive force during chronic periodontitis. MATERIAL AND METHODS: Six cell lines were established from periodontally healthy individuals (H-PDLFs) and another six were cultured from patients diagnosed with chronic periodontitis (D-PDLFs). Compressive force at 150 psi was applied to H-and D-PDLFs for 3 h on 2 consecutive days. After compression, comparisons between H-and D-PDLFs were performed by gene expression analysis of IL-6, proteases and 84 inflammation-related targets using real-time PCR. RESULTS: Compression of H-PDLFs resulted in a significant increase only in MMP-1 mRNA. In contrast, the same compressive force on D-PDLFs produced significant increases in the expression of MMPs-1,-7,-9 and -16. Moreover, compression of H-PDLFs resulted in down-regulation of IL-6, while IL-6 was significantly up-regulated in compressed D-PDLFs. Compression of H-PDLFs slightly up-regulated 3 and significantly down-regulated 15 inflammation-related genes, while the same treatment strongly up-regulated 21 inflammation-related genes in D-PDLFs. CONCLUSION: These results suggest a fundamental difference in the inflammatory response of healthy versus diseased PDLFs under physiological compression. Maintenance of these characteristics in vitro suggests that these cells may be at least partly responsible for the persistence of inflammation and localized susceptibility in chronic periodontitis.

Sodium-coupled vitamin C transporter (SVCT2): expression, function, and regulation in intervertebral disc cells.

BACKGROUND CONTEXT: Vitamin C (ascorbic acid [AA]) is essential for the synthesis of collagen and also acts as an antioxidant in the intervertebral disc (IVD). However, there is very little information currently available on the identity of the transporter that facilitates AA entry into IVD cells and the factors that mediate the transport process. PURPOSE: To investigate the expression of the two known isoforms of Na+ -coupled vitamin C transporter, SVCT1 and SVCT2, in IVD cells and its regulation by insulin-like growth factor 1 (IGF-1) and the steroid hormone dexamethasone. STUDY DESIGN: To identify the expression and functional activity of the sodium-dependent vitamin C transporter (SVCT) in the IVD. METHODS: Uptake studies were carried out with rabbit annulus fibrosis and nucleus pulposus cells in 24-well plates using [14C]-AA. To characterize SVCT transporter, uptake was done in the presence and absence of Na+ in the uptake buffer. Time dependency, Na+ activation kinetics, saturation kinetics, and substrate selectivity studies were performed. Regulatory studies were performed in the presence of IGF-1 and the steroid hormone dexamethasone. Gene expression was analyzed by quantitative polymerase chain reaction. RESULTS: Our real-time polymerase chain reaction results showed the presence of SVCT2 but not SVCT1 in IVD cells. Uptake of vitamin C in IVD cells is Na+ dependent and saturable. The Michaelis constant for the process is 96±11 µM. The activation of vitamin C uptake by Na+ exhibits a sigmoidal relationship, indicating involvement of more than one Na+ in the activation process. The uptake system does not recognize any other water-soluble vitamin as a substrate. Immunocytochemical analysis shows robust expression of SVCT2 protein in IVD cells. The growth factors IGF-1 and the steroid hormone dexamethasone upregulate the expression of SVCT2 in IVD cells. CONCLUSIONS: Our studies demonstrate that the active SVCT2 is expressed in IVD cells and that the expression of this transporter is regulated by growth factors IGF-1 and dexamethasone.

New technology applications: Knotless barbed suture for tracheal resection anastomosis.

OBJECTIVES/HYPOTHESIS: Tracheal resection anastomoses are often under tension and can be technically challenging. New suture materials such as V-loc (barbed, knotless wound closure device) may offer advantages over conventional methods. The objective of this study is to determine if a running V-loc suture is of comparable tensile strength to conventional closure. STUDY DESIGN: Laboratory based study of human cadaveric tissue. METHODS: Fresh human cadaveric tracheas were dissected and incised into segments. Anastomosis of adjacent segments was then performed with either submucosal interrupted 3-0 Vicryl, or a running submucosal 3-0 V-loc suture. Anastomosed specimens were stretched to failure on an Instron force tension machine. Surgeon satisfaction was recorded by visual analog scale (VAS). RESULTS: The tensile strength of 12 tracheal anastomoses was tested. Video documentation of V-loc suture technique and anastomosis failure was recorded. In both Vicryl (80%) and V-loc (100%) anastomoses, failure occurred at the membranous intercartilaginous region. In 20% of the Vicryl anastomoses, the suture was noted to break prior to tissue failure. Anastomoses with V-loc suture had equivalent failure force (mean, 59 N) compared to interrupted Vicryl (51 N), with P = .57. On VAS, surgeons were more satisfied with V-loc suture closure compared to interrupted Vicryl closure (paired t test, P = .003). CONCLUSIONS: Tracheal anastomosis with running v-loc suture is a feasible alternative to conventional closure with interrupted Vicryl suture. V-loc suture provided a surgical advantage by improved ease of use.

Pneumatic pressure bioreactor for cyclic hydrostatic stress application: mechanobiology effects on periodontal ligament cells.

A bioreactor system was developed to provide high-amplitude cyclic hydrostatic compressive stress (cHSC) using compressed air mixed commercially as needed to create partial pressures of oxygen and carbon dioxide appropriate for the cells under investigation. Operating pressures as high as 300 psi are achievable in this system at cyclic speeds of up to 0.2 Hz. In this study, ligamentous fibroblasts from human periodontal ligaments (n = 6) were compressed on two consecutive days at 150 psi for 3 h each day, and the mRNA for families of extracellular matrix protein and protease isoforms was evaluated by real-time PCR array. Several integrins were significantly upregulated, most notably alpha-3 (6.4-fold), as was SPG7 (12.1-fold). Among the collagens, Col8a1 was highly upregulated at 53.5-fold, with Col6a1, Col6a2, and Col7a1 also significantly upregulated 4.4- to 8.5-fold. MMP-1 was the most affected at 122.9-fold upregulation. MMP-14 likewise increased 17.8-fold with slight reductions for the gelatinases and a significant increase of TIMP-2 at 5.8-fold. The development of this bioreactor system and its utility in characterizing periodontal ligament fibroblast mechanobiology in intermediate-term testing hold promise for better simulating the conditions of the musculoskeletal system and the large cyclic compressive stresses joints may experience in gait, exertion, and mastication.

Effect of whole-body vibration on bone properties in aging mice.

Recent studies suggest that whole-body vibration (WBV) can improve measures of bone health for certain clinical conditions and ages. In the elderly, there also is particular interest in assessing the ability of physical interventions such as WBV to improve coordination, strength, and movement speed, which help prevent falls and fractures and maintain ambulation for independent living. The current study evaluated the efficacy of WBV in an aging mouse model. Two levels of vibration--0.5 and 1.5g--were applied at 32Hz to CB57BL/6 male mice (n=9 each) beginning at age 18 months and continuing for 12 weeks, 30 min/day, in a novel pivoting vibration device. Previous reports indicate that bone parameters in these mice begin to decrease substantially at 18 months, equivalent to mid-fifties for humans. Micro-computed tomography (micro-CT) and biomechanical assessments were made in the femur, radius, and lumbar vertebra to determine the effect of these WBV magnitudes and durations in the aging model. Sera also were collected for analysis of bone formation and breakdown markers. Mineralizing surface and cell counts were determined histologically. Bone volume in four regions of the femur did not change significantly, but there was a consistent shift toward higher mean density in the bone density spectrum (BDS), with the two vibration levels producing similar results. This new parameter represents an integral of the conventional density histogram. The amount of high density bone statistically improved in the head, neck, and diaphysis. Biomechanically, there was a trend toward greater stiffness in the 1.5 g group (p=0.139 vs. controls in the radius), and no change in strength. In the lumbar spine, no differences were seen due to vibration. Both vibration groups significantly reduced pyridinoline crosslinks, a collagen breakdown marker. They also significantly increased dynamic mineralization, MS/BS. Furthermore, osteoclasts were most numerous in the 1.5 g group (p≤ 0.05). These findings suggest that some benefits of WBV found in previous studies of young and mature rodent models may extend to an aging population. Density parameters indicated 0.5 g was more effective than 1.5 g. Serological markers, by contrast, favored 1.5 g, while biomechanically and histologically the results were mixed. Although the purported anabolic effect of WBV on bone homeostasis may depend on location and the parameter of interest, this emerging therapy at a minimum does not appear to compromise bone health by the measures studied here.

Periodontal ligament fibroblasts sustain destructive immune modulators of chronic periodontitis.

BACKGROUND: In healthy periodontal tissue, innate immune responses effectively confine and suppress a bacterial insult. However, a disruption of the host-bacterial equilibrium may produce an overexpression of cytokines and lead to permanent, host-mediated tissue damage. Although such periodontal destruction primarily results from activated immune mechanisms, the site-specific damage suggests that local tissues participate in these pathologic changes. Periodontal ligament fibroblasts (PDLFs) are prominent in the periodontium and are critical in homeostasis and regeneration because they have the ability to produce multiple cytokines in response to a bacterial insult. These cells could play a role in the local pathogenesis of periodontal disease. METHODS: We studied alkaline phosphatase (ALP) activity, interleukin (IL)-6 production, and morphologic characteristics of cultured PDLFs that were isolated from periodontally healthy sites (H-PDLFs) and diseased sites (D-PDLFs) in humans. Quantitative analyses of 84 genes that are related to inflammation were performed using real-time polymerase chain reaction arrays. RESULTS: A mineralizing medium induced a significant increase of ALP in H-PDLFs, but no significant enzymatic changes were detected in D-PDLFs after such treatment. The protein and gene expression of IL6 showed a significant upregulation in D-PDLFs, which also demonstrated a significant upregulation of 54% of genes in the inflammatory gene arrays. CONCLUSIONS: To our knowledge, these results represent the first biologic evidence that D-PDLFs retain uniquely inflammatory phenotypes that could maintain localized destructive signals in periodontitis. The overexpression of proinflammatory cytokines by PDLFs could amplify local inflammation by the continuous triggering of immune responses. In addition, the location of these cells could be critical in the progression of the inflammatory front into the deeper tissues.

Tensile strain-induced Ets-2 phosphorylation by CaMKII and the homeostasis of cranial sutures.

BACKGROUND: Mechanotransduction underpins the homeostasis of musculoskeletal tissues, including cranial sutures. Intracellular calcium, [Ca 2+]ic, and protein phosphorylation are two intermediate variables in signal relay during mechanotransduction. This project establishes a chain of cause and effect, linking cellular strain to substrate phosphorylation, and identifies the agent and target sites of phosphorylation. METHODS: Cyclic tensile force (0.5 N at 1 Hz) was applied to 1-day-old rat sagittal sutures. [Ca 2+]ic was measured by FURA-2. Ets-2 phosphorylation by CaMKII was tested using Western blot autoradiography. Peptide array was constructed to determine the precise sites of phosphorylation. The results were confirmed with mass spectroscopy and Western blots using phospho-specific antibodies. RESULTS: [Ca 2+]ic increased rapidly in response to tensile stress. In the presence of Ca2+, CaMKII caused Ets-2 phosphorylation. Of the three possible sites for phosphorylation of Ets-2 by CaMKII, RVPS, FESF, RLSS, Serine 246, 310, and 313 were the targets. Furthermore, the contiguous sequence modified this effect. Mass spectroscopy showed 80 Da (molecular weight of phosphate group, -PO3) right shifts consistent with phosphorylation. There was cytosolic translocation of Ets-2 on tensile deformation of suture cells. CaMKII binding of Ets-2 occurred within 30 minutes after the onset of tensile strain. CONCLUSIONS: Cranial suture cells can respond to tensile forces by increasing [Ca 2+]ic, which causes CaMKII to phosphorylate Ets-2, thus altering Ets-2 binding to its downstream promoters. Of note, Ets-2 is at the intersection of three key pathways important in craniosynostosis: fibroblast growth factor-2, transforming growth factor-beta, and mechanotransduction.

Effects of facetectomy and crosslink augmentation on motion segment flexibility in posterior lumbar interbody fusion.

STUDY DESIGN: Biomechanical assessment using calf lumbar motion segments. OBJECTIVE: To determine whether facetectomy affects the primary stability of posterior lumbar interbody fusion. SUMMARY OF BACKGROUND DATA: To improve visualization and access to the disc space, the facet joints frequently are removed. Previous biomechanical studies have indicated a fundamental role for the facet joints in maintaining spinal segment stability. METHODS: Single motion segments from calf lumbar spines were tested for pure-moment flexibility in flexion-extension (FE), lateral bending (LB), and axial rotation (AR). After testing intact, an interbody cage and pedicle screw system were implanted. Next, a bilateral facetectomy was performed, and finally a crosslink was added. Flexibility testing was repeated at each stage of implantation. Data are reported for range of motion (ROM), neutral zone (NZ), and a new compliance parameter (COM), based on the slopes of the moment-angle curve in the neutral and elastic regions. RESULTS: With posterior lumbar interbody fusion implantation, ROM in FE was reduced 82% +/- 4% (mean +/- standard deviation) and NZ 78% +/- 7% over intact (P < 0.015: Wilcoxon). Reduction in LB was slightly more, whereas reduction in AR was considerably less and did not achieve statistical significance for NZ. After facetectomy, ROM in FE increased 0.3 degrees (P < 0.05), on average, and NZ did not change. In LB neither changed significantly. In AR, ROM increased 0.6 degrees (P < 0.05), and NZ increased 0.2 degrees (P < 0.05). The addition of a crosslink changed ROM and NZ less than 0.1 degrees in FE and LB, whereas in AR it restored half of the stability lost due to facetectomy in ROM (P < 0.05), and had a similar trendwise effect on NZ. The new compliance measure, COM, was found to agree with the direction of change in ROM more consistently than did NZ. CONCLUSION: Facetectomy causes a nominal increase in ROM and NZ in FE and LB, which are not affected by the addition of a crosslink. Although the effect of facetectomy is greater in AR-and crosslink has a measurable restoring effect-all differences are within a few tenths of a degree under this loading paradigm. Thus, the clinical utility of adding a crosslink may not be justified based on these small biomechanical changes. COM can serve as a complement to ROM and NZ, or even as a surrogate when its 2 components are reported together, as it shows strong agreement with ROM, effectively distinguishes between lax and elastic region behaviors, and provides a measure of flexibility independent of the load range.

Matrix remodeling expression in anulus cells subjected to increased compressive load.

STUDY DESIGN: Mechanobiology study of gene expression changes as a result of compressive overload of anular fibrochondrocytes. OBJECTIVE: To test hypotheses regarding phenotype shift in genes coding for representative extracellular matrix (ECM) proteins and matrix modulators. SUMMARY OF THE BACKGROUND DATA: In degenerative disc disease, the transfer of compressive load through the disc shifts largely from the nucleus onto the anulus. In vivo models simulating this condition have shown derangement of the collagenous ultrastructure in the anulus. In vitro models of cultured anulus cells subjected to static compressive stress generally suggest a down-regulation of synthesis. This study evaluated the expression of specific isomers of genes responsible for mechanical viability and metabolism of the disc under cyclic compressive loads. METHODS: Fibrochondrocytes were digested from the anuli of 3, 2-week-old pigs, embedded in 1.5% alginate gel, and hydrostatically compressed at 0.5 Hz for 3 hours to amplitudes of 10 and 30 atm. These levels represented nominal load transfer through the healthy disc and high load transfer through the degenerative disc. Ribonucleic acid was isolated, reverse transcribed, and evaluated by real-time polymerase chain reaction for expression of type I (C-I) and type II (C-II) collagen, aggrecan, the matrix metalloproteinase (MMP-1), and the transforming growth factor beta (TGFbeta-1). Results were expressed at percentages of uncompressed controls. RESULTS: The lower pressure of 10 atm resulted in up-regulation of all ECM protein genes. C-I and C-II both averaged 141%, and aggrecan 121% of controls (P < 0.05). MMP-1 and TGFbeta-1 were essentially unchanged. With the pressure increased to 30 atm, C-II remained approximately at the level expressed under lower pressure, but C-I was reduced to 42% of controls (P < 0.05), indicating a phenotype shift. MMP-1 and TGFbeta-1 also were down-regulated to 71% and 54% of controls, respectively (P < 0.05). CONCLUSIONS: The up-regulation of the ECM genes with nominal pressure highlights the mechanobiological importance of common activity in fibrocartilage homeostasis. Differential regulation of the 2 primary collagen types with high pressure indicates a capacity of the anulus to remodel according to pathomechanical conditions.

Annular bulge contours from an axial photogrammetric method.

OBJECTIVE: To determine the characteristic contour of annular bulge in the lumbar spine under compression using a non-contacting technique. DESIGN: Transverse bulge of the annulus in lumbar motion segments was measured from photographs taken through an acrylic load plate. BACKGROUND: Annular bulge is considered a potential factor in nerve root impingement. However, consensus is lacking regarding which annular region bulges most. Previous studies have relied on markers in discrete locations or on contacting techniques, both of which limit the accuracy of bulge measurements. Reporting of continuous contours around the periphery of the disc has been limited to individual cases; characteristic behavior derived from a statistical treatment of the data is unknown. METHODS: Sixteen lumbar discal motion segments were loaded to 2500 N in compression. Axial photographs were taken in the pre-loaded and loaded states and digitized with an effective accuracy of 0.05 mm at 5 degrees intervals. The bulge was averaged over the sample of specimens at each interval to provide a mean-value plot in the transverse plane. Regional variations were evaluated statistically. RESULTS: It was found that the bulge was greatest posterolaterally (0.93 mm, SD 0.52 mm), and smallest anteriorly (0.37 mm, SD 0.26 mm; corrected p < 0.05), with an average of all data points of 0.67 mm, SD 0.42 mm. The restraining influence of the posterior longitudinal ligament, while not statistically significant, was evident in magnified view of the bulge contour. These regional differences were greater in the lower than in the upper lumbar segments. CONCLUSIONS: Herniation posterolaterally may be associated with relatively large annular bulge in that region, although normal bulge is only of the order of 1/100th the diameter of the disc.