The study was designed as a multicenter radiological and anatomical investigation of an intervention group. Group allocation was based on the position of the vertebrae in the individual portions of the spine.

All of the body donors included in the study had provided their written consent during their lifetime and made their bodies available for anatomical and clinical research after their demise. All cadavers were obtained from the body donor program at the Medical Faculty of the University of Rostock. The methods used to obtain human tissue were humane and decent and met the ethical standards of the Declaration of Helsinki. The study was approved by the regional ethics committee for medical research (registration number A2017-0072).

The inclusion criterion for the investigation was the presence of 24 free vertebrae in each extracted spine. Exclusion criteria were bone metastases, tumors, Paget’s disease, fusion of vertebrae or the formation of block vertebrae and previous operations with foreign material in the spine. If the sample could not be harvested in one connected piece, contained compact bone or was not complete due to a prosthesis, it was excluded.

About 10–12 liters of a 96% ethanol solution (Ethanol 96%, MEK denatured, Walter-CMP, Kiel, Germany) at 0.5 bar were perfused into the cadaver through the left femoral artery and then stored in a 0.5% aqueous phenol solution in free-floating form in cuvettes, in order to prevent air pockets in the spinal canal or vessel stumps. Through preparation we aimed to conserve the swinging of the vertebral column in order to be able to calculate forces. After exposure of the superficially migrated spinal muscles (the trapezius muscle, the rhomboid major and rhomboid minor muscles, the levator scapulae muscle, and the latissimus dorsi muscle) and the autochthonous spinal muscles, the ribs were separated over a breadth of about four inches in the paravertebral aspect. The separation was performed in a way that the ligaments of the spine stayed fully intact and the short deep local spinal muscles (the long and short rotator muscles, interspinal muscles, and intertransverse muscles) were also fully preserved. Transection of the posterior atlantooccipital membrane from the dorsal aspect was followed by disarticulation of the atlantooccipital joint. The prevertebral muscles (musculus longus capitis, musculus rectus capitis anterior, lateralis, superior, posterior minor, obliquus capitis superior) between the base of the skull and the ventral portion of the cervical spine were transected and the ventral vertebrae were bluntly separated from the tissue of the throat through the retropharyngeal space. The parietal pleura was undermined bluntly from the ventral vertebral bodies. The pairwise branches of the aorta and venous stems (posterior intercostal arteries and intercostal vein) were separated and the subsequent portion of the spine to the psoas muscle was exposed. In the region of the sacrum, approximately at the level of the joint space of the sacroiliac joint, a saw cut was made on both sides and the entire spine was removed from the dorsal aspect (Fig. 1). Great effort was made to minimize mechanical manipulation of the spine in order to preserve the natural curvature (lordosis, kyphosis and scoliosis) of the respective portions. Therefore, the specimens were preserved in free-floating form in 70% ethanol at 4°C in recyclable synthetic tubes until imaging investigations were performed and cancellous bone was taken by puncture. The latter was performed from the ventral aspect in the middle of the vertebral body and the correct position was checked on X-ray investigations.

IBM SPSS Statistics for Windows, Version 23.0 (IBM Co., Armonk, NY, USA) was used for data analysis. Quantitative characteristics were expressed as means (M), standard deviation (SD), and numbers (n) of available observations, using the interval of M±SD. The Kruskal-Wallis test or the one-way ANOVA was used for group comparisons. This was selected according to the effect of the Shapiro-Wilk test on normal distribution. In the event of significant results, we performed pairwise comparisons or a post-hoc test. We used the Mann-U-Whitney rank-sum test to compare two groups for non-normally distributed values. On the other hand, the independent t-test was used to compare two groups with normally distributed values. Correlation analyses were also performed depending on the scale level. All P-values are the result of two-sided statistical tests. The level of significance was set to P<0.05.

The mean bone mineral density of the 10 spines, measured at the lumbar vertebrae 1 to 3, was 54.5±20.0 mg/cm³ (range, 24.2–87.5), which indicates apronounced osteoporosis (values below 80 mg/cm³ are considered to be a sign of osteoporosis, values below 60 mg/cm³ reflect a higher risk of fractures) [16, 17]. Table 1 summarizes additional data on the numbers and shapes of vertebral fractures based on the registered vertebral deformities. The correlation between number of fracture and bone density was very significant (r=–0.808, P=0.005).

The present investigation allowed a comparison of trabecular bone from all sections of the human spine, obtained from 10 body donors aged 73 to 102 years. All of the investigated probands had osteoporosis on bone densitometry.

In case of reduced bone density, vertebral body fractures were also found. These mainly concern T7, 8 and 12 and L1 [2].

A possible explanation for this fracture distribution along the spine could be the predominant curvature of the spine [18, 19]. The turning point of the thoracic kyphosis is found in the middle area of the thoracic spine (T7, T8). Due to the increased curvature, higher bending moments and compression forces occur. This theory is also supported by the fact that an increase in the radius of curvature, equated with more severe kyphosis, which forms in the course of life and is of particular importance in osteoporosis, results in increased bending stress. The increased risk of fractures in the transition T12 - L1 can also be explained by increased mobility in the lumbar vertebral area and the resulting higher compression loads [19]. According to the current state of knowledge, it is assumed that the overall mobility in flexion/extension decreases with increasing age [20-22]. This could contribute to local changes in the micro-architecture. No fractures were discovered in the cervical spine (CS) in the present examination.

Above T4, especially in the CS, osteoporotic fractures are only described in individual case reports [3, 4]–with exception of the Dens-axis fracture [23, 24]. No fractures in the CS were discovered in the present examination.

Conspicuous findings are, compared to thoracic spine (TS) and lumbar spine (LS), the significantly higher BVF of the trabecular microarchitecture of CS.

Grote et al. [11] provide evidence that the CS differs from the other spinal column sections. In their study, they were able to show that the loss of bone volume with age is the least in the CS. Furthermore, no significant age-related loss of trabecular structural density was observed in the cervical vertebral body 3/4. The individual vertebral bodies of the CS show a difference in terms of their microarchitecture compared to the rest of the spinal column and therefore clearly occupies a special position of the individual movement segments. In contrast to Grote et al. [11], no histomorphometric but rather imaging evidence using micro-CT was obtained in this present examination. Methodologically comparable results are described in the case report by Schröder et al. [5] and in an article by Schober et al. [25]. Interesting in this context is the study by Acquaah et al. [26]. They already observed significantly lower BVF in the lumbar spine compared to the cervical spine during development at the age of 7 months, 1.2 and 2.5 years. The authors also compared their data on early bone development with the BVF data from literature. Their conclusion was that the loss of bone mass that occurs during the first year of life is never fully compensated. According to the authors, early development could therefore be important for the development of bone strength, but more likely due to structural changes in trabecular microarchitecture rather than bone mass itself. BVF was lowest in the over-80s group and significantly higher in all subgroups of the CS than in other spinal segments, which supports Acquaah et al. [26] conclusions. The decline in BVF with age for women and men [27] is similar. Chen et al. [28] assume after completion of their animal investigation that the BMD and BVF decreased from the cervical to lumbar spine. Cancellous bone of vertebra is metabolically more active than cortical bone and trabecular BMD may act as an initial predictor of spinal osteoporotic fracture [1]. Low BMD is correlated with increased fracture of the spine. Vertebral BMD decreases from cervical to lumbar spine, with the lowest BMD at the third lumbar vertebra [29].

Awareness of the different regional microstructural properties is of crucial importance for the assessment of age- and gender-related bone loss in vertebrae. This knowledge also provides better insights into the pathological mechanisms of spinal osteoporosis and the associated risk of fractures [30]. The risk of fractures is associated with age-related micro-damage to trabecular bone–among other effects–which is partly caused by reduced BVF [31]. And BVF on the other hand is determined by Tb.N and Tb.Sp. Reduction of Tb.N in advanced age increases Tb.Sp [27]. Tb.N was significantly higher in the CS than in other spine sections. In the present study we found not only more but thicker trabecular bone in the cervical spine and the trabeculae were significantly closer to each other. This indicates the greater biomechanical competence of cervical vertebrae.

In general, the thickness of vertical struts in the trabecular bone of vertebrae is 100 to 200 µm, whereas the Tb.Sp of vertical trabeculae achieve 600 to 900 µm [32-34]. At this point our values agree with already published data. In our investigation DA was significantly lower in the cervical spine than in other sections of the spine. The more anisotropic structure is caused by the worsening of trabecular architecture in vertebrae, which in turn means a greater tendency to fractures [32].

The significant difference between men and women is interesting in this context. The higher DA of men in the TS potentially explains their higher fracture risk in this area [2].

The data registered in the present investigation underline the specific features of the cervical spine. Reasons for these features must be determined in future investigations, especially regarding the greater bone density of the cervical spine and its morphological changes.

The puncture needle was selected on the basis of the data reported by Uhl et al. [35], who stated that the 8-G Jamshidi (Becton Dickinson GmbH, Heidelberg, Germany) needle is easy to use and also provides samples of good histological quality. Furthermore, biopsies obtained in animal experiments showed that a large drill specimen is useful for the assessment of trabecular bone volume, trabecular thickness, and the number of trabeculae [36].

The location of sample collection is very important for the interpretation. Chen et al. [27] and Gong et al. [37] report that the central and anterosuperior portions of the vertebral body have less BVF than the posterior portions. Banse et al. [33] also believe that the region with the least density is primarily located in the upper and anterior half of the vertebral body. Considering these facts, we obtained our samples from the center of the vertebral body and from the anterior aspect. It may be assumed that we sampled the structures with the lowest density in order to solve the question whether all structures of the vertebral column show rectified changes.

In summary osteoporosis is a major disease in aged women, increasing the risk for fractures accompanied by changes in the microarchitecture [38]. Further studies on non-osteoporotic spine should follow to evaluate changes in microarchitecture.