Nine participants with a complete spinal cord lesion grade A or B according to the American Spinal Injury Association Impairment Scale (AIS) as a component of the International Standards for Neurological Classification of SCI (ISNCSCI) and evidence of an acute NHO in the hip joint muscles were recruited for the study. In addition to obtaining a positive vote from the local ethics committee (Register No. 3712-10), an intervention-related patient insurance was granted, in case of a possible surgical complication, such as postoperative bleeding, infection, enhancement of NHO, and resulting costs for follow-up treatment. After detailed education and preparation, tissue was obtained and preserved from the hip joint tissue affected by NHO and from an unaffected calf, from the caput laterale of the M. gastrocnemius, as control by fine-needle biopsy under sonographic control. Both were subsequently examined histologically.

As part of a screening procedure for early identification of the development of NHO, every 14 days between weeks 2 and 12 after spinal trauma, all newly injured patients underwent a sonographic examination of the hip joints and a routine laboratory examination, which included C-reactive protein (CRP) and leukocyte count. CRP and leukocyte count were selected because in a myositic phase, before the start of mineralization/ossification, an inflammatory reaction is more likely to be observed and at this time, for example, the bone-specific alkaline phosphatase is generally not yet altered. At 12 weeks after trauma, if there was a clinical suspicion of acute NHO with redness and hyperthermia of the affected soft tissues around the hip joint, a blood test and sonography of the surrounding tissue of the joints were also performed as above. All participants had clinical symptoms of NHO for no longer than four weeks.

In case of sonographic evidence of edema and/or calcification in the tissues surrounding the hip joint, magnetic resonance imaging (MR) or computed tomography (CT) of the pelvis was performed if MRI was contraindicated for other medical reasons. MRI and CT were selected as diagnostic methods because they are good at visualizing acute neurogenic heterotopic ossification or ossifications that have already developed and are comparatively less time-consuming and invasive than, for example, a 3-phase nuclear bone scan. If a radiological diagnosis of incipient NHO was confirmed, individuals were screened for inclusion and exclusion criteria for the study, after a detailed explanation.

The inclusion criteria included a minimum age of 18 years, in addition to the participant’s written consent to the study. The neurological level of injury had to be at least at the level of dermatome TH12 or higher so that the puncture could not produce pain in the participants. Gender was not a factor. It did not matter whether the NHO involved the left, right, or both hip joints.

Exclusion and discontinuation criteria included a refusal to perform the study at any time on the part of the participant as well as an increased bleeding tendency at the time of puncture or skin irritations in the body area to be punctured. Furthermore, ventilated individuals in whom an uncontrollable respiratory insufficiency could not be excluded before or during the puncture were excluded. Due to the requirements of the ethics committee, we were allowed a maximum of 10 punctures.

Fine needle punction was performed using a BARD Magnum® biopsy system (Karlsruhe, Germany). The correct puncture site was found sonographically using a 5 MHz linear transducer. A 16 G (1.6 mm) × 25 cm punch needle was used for the involved hip musculature tissue puncture and a 16 G (1.6 mm) × 10 cm punch needle was used for the calf musculature puncture. The study participant was positioned on his contralateral side for this purpose. The gluteal and lower leg region to be punctured was washed several times with skin disinfectant (Softasept® N) and covered sterilely. A linear transducer was used to locate the region of the NHO, which appeared hypersonor due to edema formation and, in the case of incipient ossification, with partly calcareous inclusions. After finding the region to be punctured, the skin in the area of the puncture was incised over a length of approx. 0.3 cm with a puncture scalpel (No. 11) down to the subcutis. The puncture needle was then advanced through this puncture site to the area of the NHO for specimen collection under sonographic control. Finally, a sterile dressing was applied. On the day following the puncture, the puncture areas affected by NHO were subjected to irradiation with 7 Gy (J/kg) and 15 MeV photons to prevent further ossification. Furthermore, the irradiation served to prevent a possible increase in NHO activity due to the puncture itself. However, radiation was not part of the study and was performed as standard in the presence of NHO regardless of the puncture.

Puncture of the lower leg was performed in the middle third of the gastrocnemius muscle. For this purpose, the shorter puncture needles as above were used. One of each of the thus obtained, approx. 1 cm long tissue strips from the gluteal and calf region was placed in a sample tube filled with formaldehyde and glutaraldehyde and thus preserved for further histological examination.

Biopsy specimens from affected and unaffected body regions were processed for light microscopic examination using hematoxylin-eosin (HE) and elastica-van Gieson (EvG) staining.