Applications of atomic force microscopy for the assessment of nanoscale morphological and mechanical properties of bone

Scanning probe microscopy (SPM) has been in use for 30 years, and the form of SPM known as atomic force microscopy (AFM) has been around for 25 of those years. AFM has been used to produce high resolution images of a variety of samples ranging from DNA to carbon nanotubes. Type I collagen and many collagen-based tissues (including dentin, tendon, cartilage, skin, fascia, vocal cords, and cornea) have been studied with AFM, but comparatively few studies of bone have been undertaken. The purpose of this review is to introduce the general principles of AFM operation, demonstrate what AFM has been used for in bone research, and discuss the new directions that this technique can take the study of bone at the nanoscale

Bone Quality and Quantity are Mediated by Mechanical Stimuli

Prevention of fracture through improved bone mechanical strength is of great importance given the large number of bone disease-related fractures each year, the decreased quality of life associated with fractures, and the large anticipated increase in fracture incidence over the upcoming years due to the aging population. Exercise and other forms of mechanical stimulation have been shown to increase bone mass, suggesting improved strength. However, while bone mass is a good indicator of strength, other components (such as bone quality) also contribute to bone mechanical integrity. While increased bone mass has been explored considerably using both exercise and targeted loading models, the role of mechanical stimulation in altering bone quality has been explored to a lesser degree. Understanding how to improve both the quantity and quality of bone is critical to increasing fracture resistance. Herein, we discuss quantity and quality-based improvements that have been observed using both exercise and targeted loading models of bone adaptation

β-Aminopropionitrile-induced reduction in enzymatic crosslinking causes in vitro changes in collagen morphology and molecular composition

Type I collagen morphology can be characterized using fibril D-spacing, a metric which describes the periodicity of repeating bands of gap and overlap regions of collagen molecules arranged into collagen fibrils. This fibrillar structure is stabilized by enzymatic crosslinks initiated by lysyl oxidase (LOX), a step which can be disrupted using β-aminopropionitrile (BAPN). Murine in vivo studies have confirmed effects of BAPN on collagen nanostructure and the objective of this study was to evaluate the mechanism of these effects in vitro by measuring D-spacing, evaluating the ratio of mature to immature crosslinks, and quantifying gene expression of type I collagen and LOX. Osteoblasts were cultured in complete media, and differentiated using ascorbic acid, in the presence or absence of 0.25mM BAPN-fumarate. The matrix produced was imaged using atomic force microscopy (AFM) and 2D Fast Fourier transforms were performed to extract D-spacing from individual fibrils. The experiment was repeated for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Fourier Transform infrared spectroscopy (FTIR) analyses. The D-spacing distribution of collagen produced in the presence of BAPN was shifted toward higher D-spacing values, indicating BAPN affects the morphology of collagen produced in vitro, supporting aforementioned in vivo experiments. In contrast, no difference in gene expression was found for any target gene, suggesting LOX inhibition does not upregulate the LOX gene to compensate for the reduction in aldehyde formation, or regulate expression of genes encoding type I collagen. Finally, the mature to immature crosslink ratio decreased with BAPN treatment and was linked to a reduction in peak percent area of mature crosslink hydroxylysylpyridinoline (HP). In conclusion, in vitro treatment of osteoblasts with low levels of BAPN did not induce changes in genes encoding LOX or type I collagen, but led to an increase in collagen D-spacing as well as a decrease in mature crosslinks. © 2016 Canelón, Wallace. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Substrate strain mitigates effects of β-aminopropionitrile-induced reduction in enzymatic crosslinking

Enzymatic crosslinks stabilize type I collagen and are catalyzed by lysyl oxidase (LOX), a step interrupted through β-aminopropionitrile (BAPN) exposure. This study evaluated dose-dependent effects of BAPN on osteoblast gene expression of type I collagen, LOX, and genes associated with crosslink formation. The second objective was to characterize collagen produced in vitro after exposure to BAPN, and to explore changes to collagen properties under continuous cyclical substrate strain. To evaluate dose-dependent effects, osteoblasts were exposed to a range of BAPN dosages (0–10 mM) for gene expression analysis and cell proliferation. Results showed significant upregulation of BMP-1, POST, and COL1A1and change in cell proliferation. Results also showed while the gene encoding LOX was unaffected by BAPN treatment, other genes related to LOX activation and matrix production were upregulated. For the loading study, the combined effects of BAPN and mechanical loading were assessed. Gene expression was quantified, atomic force microscopy was used to extract elastic properties of the collagen matrix, and Fourier Transform infrared spectroscopy was used to assess collagen secondary structure for enzymatic crosslinking analysis. BAPN upregulated BMP-1 in static samples and BAPN combined with mechanical loading downregulated LOX when compared to control-static samples. Results showed a higher indentation modulus in BAPN-loaded samples compared to control-loaded samples. Loading increased the mature to immature crosslink ratios in control samples, and BAPN increased the height ratio in static samples. In summary, effects of BAPN (upregulation of genes involved in crosslinking, mature/immature crosslinking ratios, upward trend in collagen elasticity) were mitigated by mechanical loading

Rural Surgery Training Programs in the United States: A Review of the Literature

RURAL SURGERY TRAINING PROGRAMS IN THE UNITED STATES: A REVIEW OF THE LITERATURE Abstract: Rural surgery training programs have been developed to train general surgeons for community and rural service. There are 10 rural surgery training programs in the United States. Rural surgery training began in 1974 in LaCrosse, Wisconsin to prepare general surgery residents for rural service. There is a national shortage of general surgeons which translates into a deficit of surgeons in rural areas. The terms “general surgeon” and “rural surgeon” are not synonymous as rural surgery is more extensive, more broad-based and more subspecialty with more trauma, burn care and wound care and far more endoscopic and laparoscopic procedures. There are many advantages to rural general surgery training programs. A well-trained rural surgeon with OB/GYN experience can handle 66% of all inpatient operations in a rural hospital. Rural surgery programs have been developed to address the above problems and attract graduating general surgeons to communities and rural areas. These programs have had high general surgery practice and high rural placement. Selection of appropriate campus, hospital and attending staff is critical for the educational experience to be maximal. Attending surgeons need to be general surgeons, rural surgeons and subspecialty surgeons. The American College of Surgeons (ACS) has developed the Advisory Council for Rural Surgery (ACRS). The ACRS has developed a repository of rural training experiences and training sites. There are five different types of training experience ranging from rotations to dedicated tracks to fellowships. Rural surgery training programs are a mechanism to attract more medical students to general surgery residencies, more graduating chief general surgery residents to private practice in community and rural areas, improve the case numbers and confidence of graduating surgery residents, and improve the quality of surgical care in this country

Short-Term Exercise In Mice Increases Tibial Post-Yield Mechanical Properties While Two Weeks of Latency Following Exercise Increases Tissue-Level Strength

We have previously shown that exercise during growth increases post-yield deformation in C57BL6/129 (B6;129) male tibiae at the expense of reduced pre-yield deformation and structural and tissue strength. Other research in the literature indicates that increased mineral content, cross-sectional geometry and structural strength due to exercise can be maintained or increased after exercise ends for as long as 14 weeks. It was therefore hypothesized that after our exercise protocol ended, effects of exercise on mechanical properties would persist, resulting in increased post-yield behavior and rescued strength versus age-matched control mice. Beginning at 8 weeks of age, exercise consisted of running on a treadmill (30 min/day, 12 m/min, 5° incline) for 21 consecutive days. At the end of running and 2 weeks later, in the cortical bone of the tibial mid-diaphyses of B6;129 male mice, changes due to exercise and latency following exercise were assayed by mechanical tests and analyses of cross-sectional geometry. Exercise increased structural post-yield deformation compared with weight-matched control mice, without changes in bone size or shape, suggesting that exercised-induced changes in pre-existing bone quality were responsible. Over the 2-week latency period, no growth-related changes were noted in control mice, but exercise-induced changes resulted in increased tissue stiffness and strength versus mice sacrificed immediately after exercise ended. Our data indicate that periods of exercise followed by latency can alter strength, stiffness, and ductility of bone independent of changes in size or shape, suggesting that exercise may be a practical way to increase the quality of the bone extracellular matrix

True Gold or Pyrite: A Review of Reference Point Indentation for Assessing Bone Mechanical Properties In Vivo

Although the gold standard for determining bones' mechanical integrity is the direct measure of mechanical properties, clinical evaluation has long relied on surrogates of mechanical properties for assessment of fracture risk. Nearly a decade ago, reference point indentation (RPI) emerged as an innovative way to potentially assess mechanical properties of bone in vivo. Beginning with the BioDent device, and then followed by the newer generation OsteoProbe, this RPI technology has been utilized in several publications. In this review we present an overview of the technology and some important details about the two devices. We also highlight select key studies, focused specifically on the in vivo application of these devices, as a way of synthesizing where the technology stands in 2015. The BioDent machine has been shown, in two clinical reports, to be able to differentiate fracture versus nonfracture patient populations and in preclinical studies to detect treatment effects that are consistent with those quantified using traditional mechanical tests. The OsteoProbe appears able to separate clinical cohorts yet there exists a lack of clarity regarding details of testing, which suggests more rigorous work needs to be undertaken with this machine. Taken together, RPI technology has shown promising results, yet much more work is needed to determine if its theoretical potential to assess mechanical properties in vivo can be realized