Searching for alternative high DNA-yielding bone types for DNA analysis of aged skeletal remains.

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Crnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Crnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

Bone of contention: Intra-element variability in remodelling of human femora based on histomorphometric and isotope analyses.

The volume of human carbon (δ13C) and nitrogen (δ15N) isotope data produced in archaeological research has increased markedly in recent years. However, knowledge of bone remodelling, its impact on isotope variation, and the temporal resolution of isotope data remains poorly understood. Varied remodelling rates mean different elements (e.g., femur and rib) produce different temporal signals but little research has examined intra-element variability. This study investigates human bone remodelling using osteon population density and the relationship with carbon and nitrogen isotope data at a high resolution, focusing on variation through femoral cross-sections, from periosteal to endosteal surfaces. Results demonstrate considerable differences in isotope values between cross-sectional segments of a single fragment, by up to 1.3‰ for carbon and 1.8‰ for nitrogen, illustrating the need for standardised sampling strategies. Remodelling also varies between bone sections, occurring predominantly within the endosteal portion, followed by the midcortical and periosteal. Therefore, the endosteal portion likely reflects a shorter period of life closer to the time of death, consistent with expectations. By contrast, the periosteal surface provides a longer average, though there were exceptions to this. Results revealed a weak negative correlation between osteon population density and δ15N or δ13C, confirming that remodelling has an effect on isotope values but is not the principal driver. However, a consistent elevation of δ15N and δ13C (0.5‰ average) was found between the endosteal and periosteal regions, which requires further investigation. These findings suggest that, with further research, there is potential for single bone fragments to reconstruct in-life dietary change and mobility, thus reducing destructive sampling.

DNA preservation in compact and trabecular bone.

Significant variation exists in the molecular structure of compact and trabecular bone. In compact bone full dissolution of the bone powder is required to efficiently release the DNA from hydroxyapatite. In trabecular bone where soft tissues are preserved, we assume that full dissolution of the bone powder is not required to release the DNA from collagen. To investigate this issue, research was performed on 45 Second World War diaphysis (compact bone)-epiphysis (trabecular bone) femur pairs, each processed with a full dissolution (FD) and partial dissolution (PD) extraction method. DNA quality and quantity were assessed using qPCR PowerQuant analyses, and autosomal STRs were typed to confirm the authenticity of isolated DNA. Our results support different mechanisms of DNA preservation in compact and trabecular bone because FD method was more efficient than PD method only in compact bone, and no difference in DNA yield was observed in trabecular bone, showing no need for full dissolution of the bone powder when trabecular bone tissue is processed. In addition, a significant difference in DNA yield was observed between compact and trabecular bone when PD was applied, with more DNA extracted from trabecular bone than compact bone. High suitability of trabecular bone processed with PD method is also supported by the similar quantities of DNA isolated by FD method when applied to both compact and trabecular bone. Additionally similar quantities of DNA were isolated when compact bone was extracted with FD method and trabecular bone was extracted with PD method. Processing trabecular bone with PD method in routine identification of skeletonized human remains shortens the extraction procedure and simplifies the grinding process.

Validation process of automatic DNA extraction from bone material using a new advanced protocol for the EZ2 Connect instrument.

Identification of human remains using genetic methods is an important task of forensic science. DNA markers are proving essential in the identification of unknown human remains. However, environmental factors can lead to poor preservation of DNA, including in bone material. The aim of this study was therefore to compare two methods of DNA isolation from bone material: the traditional organic method and the new protocol using the EZ2 Connect instrument. The study involved three types of bone material, namely molars/premolars, petrous parts of the temporal bone and femurs, all with an estimated PMI of 70-80 years. Importantly, the biological material was obtained from three different environments, categorized as preserving, neutral and degrading, based on basic physico-chemical tests and the potential impact on the bone. The results obtained show that the DNA was best preserved in the petrous bone, followed by the teeth, and the femur. DNA extraction using the EZ2 Connect instrument with a new protocol gave slightly better results for the petrous bone, comparable results for the teeth and worse results for the femur compared to the organic method. Several protocol modifications were tested and optimal conditions for DNA isolation were proposed for the EZ2 protocol. Furthermore, the use of an automated method facilitated the effective accumulation of isolates and increased the chances of successful identification of unknown human remains.

Detection of methamphetamine in mouse femurs exposed to high temperature.

Bone samples are valuable for examining the cause of death and circumstance leading up to death when body fluids are not available for forensic toxicological analysis. Examined were heat-induced changes in methamphetamine and amphetamine concentrations in femurs removed from methamphetamine-injected mice to determine if the burned bones could be used for toxicology testing. The femurs were heated at 100°C, 300°C, or 500°C for 10 or 30 min. The tissue structure of the heated femurs was preserved at 100°C for 30 min but was destructed at higher temperatures. Methamphetamine and amphetamine were detected in femurs heated at 100°C for 10 min, 100°C for 30 min, and 300°C for 10 min (with methamphetamine and amphetamine concentrations ranging from 0.36 to 35 µg/g and 0.54 to 47 µg/g, respectively). Methamphetamine and amphetamine were detectable when heated above their decomposition temperature as a result of limited heat transfer do to protection provide by the femoral muscle. Thus, the bone could be a useful analytical sample in cases of burn-related deaths, where it is difficult to collect body fluids.

The effects of orthodontic anchor screw inserted into the femur of growth-phase or mature rats -Osteoid formation, bone mineral density, collagen fiber bundles, biological apatite crystal orientation.

The purpose of this study is to investigate the effect of orthodontic anchor screws (OASs) inserted into the femur of growth-phase or mature rats using histological observation and bone structure analysis. The experimental animals are growth-phase (6-week-old) or mature (25-week-old) male Wistar rats. OAS was placed into the point one-third of the femoral length from the proximal end of the femur, and the response of the surrounding bone was observed and measured. The results showed at the OAS bone interface, in growth-phase rats, bone mineral density (BMD) was reduced and the running angle of collagen fiber bundles varied significantly. In mature rats, more osteoid was observed and biological apatite (BAp) crystals showed a different orientation. It was suggested that after the insertion of OASs, bone volume and quality are decreased, but after a sufficient healing period, a new bone micro/nano structure, different from the original structure, are reconstructed.

The importance of bone sample selection when using radiocarbon analysis in cases of unidentified human remains.

The radiocarbon (14C) analysis of skeletonised human remains can provide vital information regarding the time since death. This study analysed the 14C content in both cortical and trabecular bone from the femur and rib of 18 human remains donated to the Australian Facility for Taphonomic Experimental Research. To determine the most appropriate bone to sample for 14C analysis when estimating the time since death, differences in turnover time between the bone types, and the difference between environmental 14C during the year of death (YOD) and the level of 14C found within the bone (i.e., the lag time) were examined. Overall, the average lag time for all donors was 25 years, with the cortical bone from femur samples providing the largest lag time (39 years) and the trabecular bone from rib samples containing the lowest level of 14C (most modern), with an average lag time of 13 years. The results of this study suggest that in cases of unidentified human remains, the analysis of trabecular bone from a rib may be preferred sample, as this bone contains the most recent 14C, and can provide an estimate of both the time since death, and a broad approximation of YOD.

Estimation of the post-mortem interval in human bones by infrared spectroscopy.

In forensic anthropology, there is an inherent difficulty in estimating the post-mortem interval (PMI). This study aimed to assess whether there is a correlation between changes in the bone mineral component and the PMI estimation. Samples of femur and humerus from 80 identified individuals with known post-mortem interval were analyzed. Infrared spectroscopy in attenuated total reflectance mode (FTIR-ATR) was applied for this analysis, several indices having been obtained from the infrared bands most representative of the bone's relative contents in carbonate and phosphate. Specific relationships between sex/age and PMI were attained: for larger PMIs, there was an increase in the amount of B-type carbonate (BPI), A-type carbonates (API), and in the carbonate/phosphate (C/P) ratio, and a decrease of the crystallinity index (CI) and in the carbonate ratio (C/C). Two particular infrared indices (CI and C/C) were identified as the most suitable for post-mortem interval estimation, especially in females, controlling the effects of sex and age (in the statistical analysis).

Efficacy of a novel bone preprocessing method for better DNA yield.

In cases involving identification of missing persons, mass disasters and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA profiling. Standard methods for extraction of DNA from such samples involve grinding of the bone and teeth samples. Here, we present an extremely efficient protocol for recovery of DNA from bone samples by a method of scrapping. The study was carried out on 25 samples and it was found that the quantity of DNA isolated by the scrapping method was up to 1.131 ng/µl with a success rate of 93% as compared to a much lower yield of 0.359 ng/µl DNA isolated with a success rate of 28% through the grinding method. The scrapping method of DNA extraction has been proven to be extremely useful in forensic examination of challenging samples that had multiple failures using the traditional grinding method.

Irisin Has a Protective Role against Osteoporosis in Ovariectomized Rats.

The reduction in estrogen levels results in a decrease in bone density at menopause. Irisin is a myokine that modulates the benefits of exercise, which may include bone health. This study was planned to examine irisin's impact in preventing osteoporosis after ovariectomy. 4 groups of female albino rats (10 rats/group): control, sham-operated, ovariectomized (OVX-control), and OVX-irisin-treated. Serum levels of bone markers [osteocalcin (OC), bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), calcium (Ca++), phosphorus (P)], glucose, and insulin were being measured. Body mass index, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), dry and ash femur weight, and bone contents of Ca++ and P were investigated. The femur was examined histopathologically. The OVX-control group showed an increase in serum levels of OC, BALP, TRAP, calcium, phosphorus, BMI, glucose, insulin, and HOMA-IR (P < 0.05) and a reduction in dry and ash weight of the femur, the concentration of calcium and phosphorus content in bone ash (P < 0.05). The OVX-irisin-treated group exhibited a decrease in serum levels of OC, BALP and TRAP, calcium, phosphorus, BMI, glucose, insulin, HOMA-IR (P < 0.05), and a rise in dry and ash weight of the femur, the concentration of calcium and phosphorus in bone ash (P < 0.05). Histological examination of the distal femur diaphysis of the OVX-irisin-treated group exhibited proper bone architecture and density compared with that of the OVX-control group. It is concluded that irisin treatment in the OVX rats safeguarded the regular bone architecture and normal levels of serum bone biomarkers. Irisin may be a possible novel target in the prohibition of postmenopausal osteoporosis.

Temperature effect in physicochemical and bioactive behavior of biogenic hydroxyapatite obtained from porcine bones.

Biogenic hydroxyapatite (BHAp) is a widely used material in the biomedical area due to its similarities with the bone tissue mineral phase. Several works have been spotlighted on the thermal behavior of bone. However, little research has focused on determining the influence of calcination temperature in the physicochemical and bioactive properties of BHAp. In this work, a study of the physicochemical properties' changes and bioactive response of BHAp produced from porcine femur bones using calcination temperatures between 900 to 1200 °C was conducted. The samples' structural, morphological, and compositional changes were determined using XRD, SEM, and FTIR techniques. XRD results identified three temperature ranges, in which there are structural changes in BHAp samples and the presence of additional phases. Moreover, FTIR results corroborated that B-type substitution is promoted by increasing the heat treatment temperature. Likewise, samples were immersed in a simulated biological fluid (SBF), following the methodology described by Kokubo and using ISO 23317:2014 standard, for 3 and 7 days. FTIR and SEM results determined that the highest reaction velocity was reached for samples above 1000 °C, due to intensity increasing of phosphate and carbonate bands and bone-like apatite morphologies, compared to other temperatures evaluated.

A collagen extraction and deuterium oxide stable isotope tracer method for the quantification of bone collagen synthesis rates in vivo.

The development of safe and practical strategies to prevent weakening of bone tissue is vital, yet attempts to achieve this have been hindered by a lack of understanding of the short-term (days-weeks) physiology of bone collagen turnover. To address this, we have developed a method to quantify bone collagen synthesis in vivo, using deuterium oxide (D2 O) tracer incorporation techniques combined with gas chromatography pyrolysis isotope-ratio mass spectrometry (GC-pyrolysis-IRMS). Forty-six male and female rats from a selectively bred model ingested D2 O for 3 weeks. Femur diaphyses (FEM), tibia proximal (T-PRO), and distal (T-DIS) epiphyses-metaphyses and tibia mid-shaft diaphyses (T-MID) were obtained from all rats after necropsy. After demineralisation, collagen proteins were isolated and hydrolysed and collagen fractional synthetic rates (FSRs) determined by incorporation of deuterium into protein-bound alanine via GC-pyrolysis-IRMS. The collagen FSR for the FEM (0.131 ± 0.078%/day; 95% CI [0.106-0.156]) was greater than the FSR at T-MID (0.055 ± 0.049%/day; 95% CI [0.040-0.070]; p < 0.001). The T-PRO site had the highest FSR (0.203 ± 0.123%/day; 95% CI [0.166-0.241]) and T-DIS the lowest (0.027 ± 0.015%/day; 95% CI [0.022-0.031]). The three tibial sites exhibited different FSRs (p < 0.001). Herein, we have developed a sensitive method to quantify in vivo bone collagen synthesis and identified site-specific rates of synthesis, which could be applicable to studies of human bone collagen turnover.

Bone fragment or bone powder? ATR-FTIR spectroscopy-based comparison of chemical composition and DNA preservation of bones after 10 years in a freezer.

Freezing bone samples to preserve their biomolecular properties for various analyses at a later time is a common practice. Storage temperature and freeze-thaw cycles are well-known factors affecting degradation of molecules in the bone, whereas less is known about the form in which the tissue is most stable. In general, as little intervention as possible is advised before storage. In the case of DNA analyses, homogenization of the bone shortly before DNA extraction is recommended. Because recent research on the DNA yield from frozen bone fragments and frozen bone powder indicates better DNA preservation in the latter, the aim of the study presented here was to investigate and compare the chemical composition of both types of samples (fragments versus powder) using ATR-FTIR spectroscopy. Pairs of bone fragments and bone powder originating from the same femur of 57 individuals from a Second World War mass grave, stored in a freezer at - 20 °C for 10 years, were analyzed. Prior to analysis, the stored fragments were ground into powder, whereas the stored powder was analyzed without any further preparation. Spectroscopic analysis was performed using ATR-FTIR spectroscopy. The spectra obtained were processed and analyzed to determine and compare the chemical composition of both types of samples. The results show that frozen powdered samples have significantly better-preserved organic matter and lower concentrations of B-type carbonates, but higher concentrations of A-type carbonates and stoichiometric apatite. In addition, there are more differences in the samples with a low DNA degradation index and less in the samples with a high DNA degradation index. Because the results are inconsistent with the current understanding of bone preservation, additional research into optimal preparation and long-term storage of bone samples is necessary.

Effects of high-intensity interval training in more or less active mice on biomechanical, biophysical and biochemical bone parameters.

High-intensity interval training (HIIT) is of scientific interest due its role in improving physical fitness, but the effects of HIIT on bone health need be carefully explored. Further, it is necessary to know whether HIIT effects on bone health are dependent on the physical activity levels. This may be experimentally tested since we have built a large cage (LC) that allows animals to move freely, promoting an increase of spontaneous physical activity (SPA) in comparison to a small cage (SC). Thus, we examined the effects of HIIT on biophysical, biomechanical and biochemical parameters of bone tissue of C57BL/6J mice living in cages of two different sizes: small (SC) or large (LC) cages with 1320 cm2 and 4800 cm2 floor space, respectively. Male mice were subdivided into two groups within each housing type: Control (C) and Trained (T). At the end of the interventions, all mice were euthanized to extract the femur bone for biophysical, biomechanical and biochemical analyses. Based a significant interaction from two-way ANOVA, trained mice kept in large cage (but not for trained mice housed in SC) exhibited a reduction of tenacity and displacement at failure in bone. This suggests that long-term HIIT program, in addition with a more active lifestyle correlates with exerts negative effects on the bone of healthy mice. A caution must also be raised about the excessive adoption of physical training, at least regarding bone tissue. On the other hand, increased calcium was found in femur of mice housed in LC. In line with this, LC-C mice were more active (i.e. SPA) than other groups. This implies that an active lifestyle without long-term high intensity physical training seems to play a role in promoting benefits to bone tissue. Our data provides new insights for treatment of osteo-health related disorders.

Absorption and transport of a Mytilus edulis-derived peptide with the function of preventing osteoporosis.

The YPRKDETGAERT peptide (PME-1) identified from the Mytilus edulis proteins has been shown to promote the proliferation and differentiation of osteoblasts and it has good bone-forming activity in vitro. Further, PME-1 has been shown to prevent osteoporosis in vivo. PME-1 can be absorbed through the gastrointestinal tract, and the passing rate in monolayer Caco-2 cells was 6.57%. PME-1 can also enter the blood circulation and the concentration of PME-1 in serum reached the maximum, 61.06 ± 26.32 ng mL-1, 20 min after feeding. The multifunctional in vivo imager was used to further determine the distribution of the 5-FITC-(Acp)-YPRKDETGAERT peptide (PME-1-FITC) 2 h after feeding the peptide, and the result confirmed the above results and showed that a part of PME-1-FITC can affect bone in vivo. Therefore, PME-1 not only was easily absorbed in the gastrointestinal tract, but also has the potential beneficial effect on preventing osteoporosis.

Hydroxyapatite biomaterial production from chicken (femur and beak) and fishbone waste through a chemical less method for Cd2+ removal from shipbuilding wastewater.

Hydroxyapatite (HAp) powder was produced from chicken (femur and beak) and fishbone wastes and used as a green adsorbent to decrease Cd2+ from aqueous media. The HAp powder was generated at 900 °C and characterized using physicochemical techniques. Chicken femur' HAp (16.72 m2/g) had a higher surface compared to chicken beak and fishbone ones. The solution pH was the most important parameter in removing Cd2+. The highest Cd2+ removal was achieved at pH 6, temperature of 25 °C, contact time of 80 min, and adsorbent mass of 2 g/L. The Cd2+ adsorption data fitted well with the quasi-second-order model in kinetics and the Freundlich model in isotherm. The highest adsorption capacity of Cd2+ using HAp-chicken femur, HAp-fish bone, and HAp-chicken beak was determined 22.94 mg/g, 21.54 mg/g, and 21.45 mg/g, respectively. The Cd2+ adsorption using HAp powder was a spontaneous and exothermic process and accidental collisions at the liquid-solid interface were reduced. The decrease of Cd2+ adsorption efficiency was not significant after multiple recovery steps of the desired powders. In addition to Cd2+, other parameters of real wastewater (shipbuilding industry) were reduced by the proposed adsorbents. The utilization of hydroxyapatite powder is expected to be a cheap and eco-friendly method for eliminating metals such as Cd2+.

An Unanticipated Role for Sphingosine Kinase-2 in Bone and in the Anabolic Effect of Parathyroid Hormone.

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Sphingosine kinase (SPHK) is the rate-limiting enzyme in S1P production and has 2 isoforms. To evaluate the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc-/-) and mice in which the SPHK2 gene was deleted in all tissues (SPHK2-/-). SPHK1-Oc-/- had normal bone mass. By contrast, SPHK2-/- female mice had a 14% lower spinal bone mineral density (BMD; P < 0.01) and males a 22% lower BMD at the same site (P < 0.001). SPHK2-/- and control mice were subsequently treated either with daily parathyroid hormone [PTH](1-34) or vehicle for 29 days. The response to PTH was significantly attenuated in the SPHK2-/-mice. The mean femoral bone volume to total volume fraction (BV/TV) increased by 24.8% in the PTH-treated female control animals vs 10.6% in the vehicle-treated female controls (P < 0.01). In contrast, in the SPHK2-/- female mice the difference in femoral trabecular BV/TV at the end of treatment was not significant (20.5 vs13.3%, PTH vs vehicle, P = NS). The anabolic response to PTH was significantly attenuated in the spine of male SPHK2-/- mice (29.7% vs 23.1%, PTH vs vehicle, in controls, P < 0.05; 26.9% vs 19.5% PTH vs vehicle in SPHK2-/- mice, P = NS). The spine responded normally in the SPHK2-/- female mice. Interestingly, suppression of sclerostin was blunted in the SPHK2-/- mice when those animals were treated with an anabolic PTH regimen. We conclude that SPHK2 has an important role in mediating both normal bone remodeling and the anabolic response to PTH.

Profiling of human burned bones: oxidising versus reducing conditions.

Complementary optical and neutron-based vibrational spectroscopy techniques (Infrared, Raman and inelastic neutron scattering) were applied to the study of human bones (femur and humerus) burned simultaneously under either aerobic or anaerobic conditions, in a wide range of temperatures (400 to 1000 °C). This is the first INS study of human skeletal remains heated in an oxygen-deprived atmosphere. Clear differences were observed between both types of samples, namely the absence of hydroxyapatite's OH vibrational bands in bone burned anaerobically (in unsealed containers), coupled to the presence of cyanamide (NCNH2) and portlandite (Ca(OH)2) in these reductive conditions. These results are expected to allow a better understanding of the heat effect on bone´s constituents in distinct environmental settings, thus contributing for an accurate characterisation of both forensic and archaeological human skeletal remains found in distinct scenarios regarding oxygen availability.

Is the 0.2%-Strain-Offset Approach Appropriate for Calculating the Yield Stress of Cortical Bone?

The 0.2% strain offset approach is mostly used to calculate the yield stress and serves as an efficient method for cross-lab comparisons of measured material properties. However, it is difficult to accurately determine the yield of the bone. Especially when computational models require accurate material parameters, clarification of the yield point is needed. We tested 24 cortical specimens harvested from six bovine femora in three-point bending mode, and 11 bovine femoral cortical specimens in the tensile mode. The Young's modulus and yield stress for each specimen derived from the specimen-specific finite element (FE) optimization method was regarded as the most ideal constitutive parameter. Then, the strain offset optimization method was used to find the strain offset closest to the ideal yield stress for the 24 specimens. The results showed that the 0 strain offsets underestimated (- 25%) the yield stress in bending and tensile tests, while the 0.2% strain offsets overestimated the yield stress (+ 65%) in three-point bending tests. Instead, the yield stress determined by 0.007 and 0.05% strain offset for bending and tensile loading respectively, can effectively characterize the biomechanical responses of the bone, thereby helping to build an accurate FE model.

Probing short and long-range interactions in native collagen inside the bone matrix by BioSolids CryoProbe.

Solid-state nuclear magnetic resonance is a promising technique to probe bone mineralization and interaction of collagen protein in the native state. However, many of the developments are hampered due to the low sensitivity of the technique. In this article, we report solid-state nuclear magnetic resonance (NMR) experiments using the newly developed BioSolids CryoProbe™ to access its applicability for elucidating the atomic-level structural details of collagen protein in native state inside the bone. We report here approximately a fourfold sensitivity enhancement in the natural abundance 13 C spectrum compared with the room temperature conventional solid-state NMR probe. With the advantage of sensitivity enhancement, we have been able to perform natural abundance 15 N cross-polarization magic angle spinning (CPMAS) and two-dimensional (2D) 1 H-13 C heteronuclear correlation (HETCOR) experiments of native collagen within a reasonable timeframe. Due to high sensitivity, 2D 1 H/13 C HETCOR experiments have helped in detecting several short and long-range interactions of native collagen assembly, thus significantly expanding the scope of the method to such challenging biomaterials.