Disc cell therapy with bone-marrow-derived autologous mesenchymal stromal cells in a large porcine disc degeneration model.

PURPOSE: Disc regeneration through matrix-assisted autologous mesenchymal stromal cell therapy seems promising against disc degeneration with convincing results in small animal models. Whether these positive results can be transferred to larger animal models or humans is unclear. METHODS: Fibrin matrix-assisted autologous bone-marrow-derived mesenchymal stromal cell therapy was compared to acellular fibrin matrix therapy in a porcine in vivo model. First, disc degeneration was induced by annular puncture and partial nucleotomy with a large 16G-needle, and 12 weeks later, disc therapy was performed in a second surgery with a thinner 26G needle. Seventy-two lumbar discs from 12 aged adult pigs were evaluated by histology, micro-CT, and gene expression analysis 13 and 24 weeks after nucleotomy and 1 and 12 weeks after treatment, respectively. RESULTS: Radiologic disc height was not significantly different in both treatment groups. In the semi-quantitative histologic degeneration score, significant disc degeneration was still evident 1 week after treatment both in the mesenchymal stromal cell group and in the acellular fibrin matrix group. 12 weeks after treatment, degeneration was, however, not further increased and mesenchymal-stromal-cell-treated discs showed significantly less disc degeneration in the annulus fibrosus (p = 0.02), whereas reduction in the nucleus pulposus did not reach statistical significance. Cell treatment compared to matrix alone found less Col1 gene expression as a marker for fibrosis and more expression of the trophic factor BMP2 in the nucleus pulposus, whereas the inflammation marker IL1ß was reduced in the annulus fibrosus. CONCLUSIONS: Disc treatment with fibrin matrix-assisted autologous mesenchymal stromal cells reduced degenerative findings compared to acellular fibrin matrix alone. Regenerative changes, however, were not significant for all parameters showing limitations in a large biomechanically demanding model with aged discs. These slides can be retrieved under Electronic Supplementary Material.

[Postoperative implant-associated osteomyelitis of the shoulder: Hardware-retaining revision concept using temporary drainage]. / Postoperative implantatassoziierte Osteitis am Schultergürtel: Materialerhaltendes Revisionskonzept mit Einlage einer Dauerdrainage.

BACKGROUND: Posttraumatic and postoperative osteomyelitis (PPO) is a subgroup of bone infections with increasing importance. However, to date no standardized reoperation concept exists particularly for patients with PPO of the shoulder region. Therefore the purpose of this study was to evaluate a revision concept including débridement, irrigation, and insertion of temporary drainage with hardware retention until healing. PATIENTS AND METHODS: A total of 31 patients with PPO were included with a proximal humerus fracture (n = 14), clavicle fracture (n = 10), or AC-joint separation (n = 7). In all, 27 of these patients could be followed for > 1 year. RESULTS: Hardware retention until fracture or ligament healing could be achieved in > 83%. Six patients required follow-up débridement due to recurrent infections, but then were unremarkable. Clinical outcome showed excellent Constant scores (91.6 ± 2.8). CONCLUSION: A cost-efficient, simple, and successful revision concept for patients with PPO of the shoulder region is described.

Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

INTRODUCTION: Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. MATERIALS AND METHODS: NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. RESULTS: Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. CONCLUSIONS: 3D-culture caused a rapid loss of NC phenotype towards a CLC phenotype with disappearance of vacuoles, reduced cell size, increased proliferation, and gene-expression changes. These findings may be related to NC nutritional demands and support the latest hypothesis of NC maturation into CLC opposing the idea that NC get lost in human discs by cell death or apoptosis to be replaced by CLC from the inner annulus.

Short-term follow-up of disc cell therapy in a porcine nucleotomy model with an albumin-hyaluronan hydrogel: in vivo and in vitro results of metabolic disc cell activity and implant distribution.

PURPOSE: Cell therapy would be favorably performed immediately after nucleotomy, to restore intervertebral disc functionality and to slow down disc degeneration. Promising results were reported from small animal models but remaining problems, especially in larger animals, include loss of vital cells due to annular damage at the injection site and detrimental intradiscal conditions. The aim of the present study was to optimize cell-based disc therapy using a new albumin-hyaluronan hydrogel together with bone marrow-derived mesenchymal stem cells in a large porcine disc model. METHODS: Luciferase cell labeling was evaluated to follow-up stem cells metabolically up to 7 days in 3D cell cultures mimicking the harsh disc environment with low oxygen and glucose concentrations. As a pilot in vivo study, the implant was injected into porcine discs after removal of ~10% of nucleus volume and animals were killed immediately after surgery (n = 6) and 3 days later (n = 6). 24 discs were analyzed. Implant persistence and cell activity (luciferase + WST assay) were observed simultaneously. RESULTS: In vitro cell culture with reduction of glucose (20, 5, 0.5, 0 mM) and oxygen (21, 5, 2%) significantly decreased metabolic cell activity and luciferase activity after 3 days, with no recovery and a further decrease after 7 days, establishing luciferase activity as a metabolic sensor. During 3 days of 3D culture with disc-like conditions, luciferase activity decreased to 8%. In vivo, initial implant volume shrank to 61% at day 3 with evidence for hydrogel compression. Luciferase activity in vivo at day 3 was 2% without referencing but 23% after referencing to in vitro cell adaptation, and 38% after additional consideration of detected implant volume loss. CONCLUSION: In vitro analysis up to 7 days established for the first time luciferase activity as a metabolic sensor for mesenchymal stem cells used in regenerative disc therapy. Under the present protocol, short-term in vivo analysis after 3 days suggests improved implant retainment inside the disc and persistence of metabolically active cells; however, further studies will have to prove long-term in vivo outcome.

Core decompression combined with implantation of a demineralised bone matrix for non-traumatic osteonecrosis of the femoral head.

INTRODUCTION: Core decompression is the standard surgical procedure in the treatment of early stage non-traumatic osteonecrosis of the femoral head (ONFH). However, there is still a debate whether decompression in combination with supplementary augmentation by bone grafts, growth factors, or cell implementation is superior to conventional decompression alone. This study evaluated patients after core decompression combined with an augmentation by a demineralised bone matrix, and particularly aimed to report long-term conversion rates to total hip replacement (THR). MATERIALS AND METHODS: 14 patients with 18 hips suffering from ONFH (Ficat stage I-IIB) underwent this surgical procedure. All patients underwent radiographic and MRI investigations at baseline and at follow-up periods of 12 and 24 months. The clinical follow-up was done using the Merle d'Aubigné-score for an average period of 9 years after surgery. RESULTS: 14 of the 18 subjects (77 %) achieved at least a good clinical result after 2 years. The Merle d'Aubigné-score improved significantly after 12 (p = 0.0001) and 24 months (p = 0.0002). However, the MRI volumetric analysis showed an increased necrotic bone volume from 3.16 ± 0.54 to 3.88 ± 0.62 cm(3) (p = 0.04). Within 9 years, 13 out of 18 cases (72 %) required further surgery by THR. Only 7 out of 18 subjects (39 %) reported an ongoing postoperative clinical benefit, and would retrospectively redo the same surgical approach again. The five patients that did not require THR were still satisfied after 9 years. CONCLUSIONS: In patients with early- stage femoral head osteonecrosis core decompression combined with the implantation of a demineralised bone matrix leads to a limited, temporary pain relief as seen in core decompression alone. However, long-term results were not encouraging with a high rate of conversion to arthroplasty. Therefore, core decompression with implantation of a demineralised bone matrix may be not appropriate to avoid THR in the long term.

[PHTLS team course: a pilot project. Structured student education in prehospital care of severely injured patients]. / Der PHTLS-TEAM-Kurs ­ ein Pilotprojekt. Strukturierte studentische Ausbildung in der präklinischen Versorgung schwerverletzter Patienten.

BACKGROUND: The training of medical school students at the University of Heidelberg seems to be improvable regarding prehospital trauma treatment compared to an established anaesthesiology-based training for medical emergencies. This study addresses the current situation and possibilities for advancing this training. MATERIAL AND METHODS: A baseline was set by interviews of the medical school students. Based on this the hypothesis was postulated that there is a deficit in the education of the medical school students concerning the training in prehospital trauma treatment. This was proved by questionnaires given to the students in the 7th and 8th semesters at the University of Heidelberg Medical School. The results were evaluated and a possible approach for improvement was developed. RESULTS: A total of 111 questionnaires could be evaluated. It could be shown that the existing education was not effectual and that there is a need for a praxis-orientated 1-day course in prehospital trauma treatment. CONCLUSION: Especially the treatment of multiply injured patients is a challenge for young medical professionals. However, there is a high motivation to learn and train in emergency medicine. The students long for a practical trauma course compared to the advanced medical CPR course provided by the Department of Anaesthesiology of the University of Heidelberg. Those algorithm-based trauma courses do exist with PHTLS® and ATLS®. Based on these courses we developed the PHTLS® TEAM course.

Autologous bone cylinder transplantation with cannulated screw re-stabilisation: a new treatment option for delayed fracture healing of the femoral neck.

AIM: Delayed fracture healing and non-unions of the femoral neck after lag screw osteosynthesis occur particularly in multiply injured young patients, and then surgical revision is often required. Currently no evidence-based treatment guidelines exist and therapeutic options include both hip arthroplasties and femoral head-maintaining operations. Here we report on young patients with delayed fracture healing of the femoral neck. Patients underwent revision surgery by autologous bone cylinder transplantation with mechanical re-stabilisation by cannulated lag screws. MATERIAL AND METHODS: We reviewed all patients after femoral neck screw osteosynthesis and identified eight patients at 7.3 [3-24] months after initial osteosynthesis with persisting, or reoccurring postoperative pain. Average patient age was 43 [35-57] years and patient Harris Hip Score (HHS) numbers were low (52 ± 19). Before revision surgery the preoperative CT scans showed a partial bone consolidation (anterior and/or posterior cortices) in the absence of a complete bone consolidation of all cortices. Seven patients were treated by bone cylinder transplantation from the patient's own iliac crest; one patient underwent an inverse bone cylinder procedure. Seven patients were additionally treated by re-insertion of 1-2 lag screws to increase mechanical stability. RESULTS: After revision surgery the average patient follow-up period was 42 [12-89] months. Five patients achieved favourable clinical and radiographic outcome with both complete bone union and return to work within 7.2 ± 2.75 months. One patient showed fracture healing but developed an aseptic femoral head osteonecrosis. Two patients failed to achieve complete bone consolidation. The postoperative HHS was 92 ± 4 in patients with favourable clinical outcome (n = 5) and 89 ± 2 after second revision surgery (2 hip arthroplasties; 1 valgus osteotomy). Both groups had significantly better HHS numbers compared with before surgical revision (p < 0.05). DISCUSSION: These data show that in this difficult-to-treat subset of young patients with delayed fracture healing of the femoral neck, autologous bone cylinder transplantation with mechanical re-stabilisation should be considered as a promising surgical revision strategy before hip arthroplasty.

Methods to monitor distribution and metabolic activity of mesenchymal stem cells following in vivo injection into nucleotomized porcine intervertebral discs.

Intervertebral disc (IVD) degeneration involves a series of biochemical and morphological changes leading to loss of spinal stability and flexibility. Cell therapy is promising to reconstitute IVDs with new cells, however, sustained metabolic activity seems crucial for an active contribution to regeneration. The aim of the present study was to establish methods for separate follow up of persistence and activity of autologous porcine mesenchymal stem cells (pMSC) after implantation into IVDs of Goettingen minipigs in vivo in order to conclude about the potential of such an intervention strategy. For quantitative follow up, the transfer matrix was supplemented with Al(2)O(3) particles and pMSC which were retrovirally labeled with firefly luciferase (pMSC-Luc). Six mature Goettingen minipigs underwent matrix based cell transfer after partial nucleotomy of lumbar IVDs (n = 24). Day 0 and day 3 segments were analyzed for retained volume of Al(2)O(3) particles by micro-computed-tomography (muCT) and for cell activity by luciferase enzyme assessment. Three days after injection a reduction of Al(2)O(3) particles (P = 0.028) to about 9% and of pMSC-Luc activity to about 7% of initial values (P = 0.003) was detected, which suggests loss of 90% of the implant material under in vivo conditions without evidence for reduced pMSC-Luc metabolic activity (P = 0.887). In conclusion, separate follow up of implant material and cell activity was possible and unravels problems with in vivo implant persistence after annular puncture rather than quick loss of cell activity. Therefore, IVD-regeneration-strategies should increasingly focus on annulus reconstruction in order to reduce implant loss due to annular failure.

[Controlled distraction as a therapeutic option in moderate degeneration of the intervertebral disc -- an in vivo study in the rabbit-spine model]. / Kontrollierte Distraktion als therapeutische Option bei mittelgradiger Bandscheibendegeneration -- Eine In-vivo-Studie am Kaninchen-Wirbelsäulenmodell.

AIM: The aim of this study was to investigate the effects of temporary distraction on a degenerated intervertebral disc to characterize regenerative changes associated with disc distraction. METHOD: New Zealand white rabbits (n = 32) were used for this experimental animal study. The rabbits were randomly assigned to one of five groups. 6 animals were loaded for 28 days using a custom-made external loading device to stimulate disc degeneration (G2). In 6 animals the discs were first loaded for 28 days and after 28 days loading time the discs in six animals were treated as dynamic distraction with an external distraction device (G1). In six animals the discs were distracted for 28 days without previous loading (G5) and in six animals the discs were loaded for 28 days and afterwards the loading device was removed for 28 days for recovery without distraction (G3). Six animals were sham operated (G4) without application of axial load. After 28 to 56 days loading and distraction time, the animals were sacrificed and the lumbar spine was harvested for histological and radiographic analysis. Histology was performed according to a degeneration score and disc height was calculated radiographically. For the cell viability examination, the number of apoptotic cells was determined. RESULTS: After 28 days of loading (G2), the discs showed a significant decrease in disc space of the treated segment. Histologically, a disorganization of the architecture of the annulus occurred. The number of dead cells increased significantly in the annulus and cartilage endplate. These changes were reversible after 28 days of distraction (G1). The disc thickness increased significantly to physiological levels as compared to the specimens from the 28 days loading group without distraction. Histologically, the discs showed signs of tissue regeneration after 28 days of distraction (G1). The number of apoptotic cells decreased significantly in comparison to the loaded discs without distraction (G2). CONCLUSION: The results of this study suggest that disc regeneration can be induced by axial dynamic distraction in the moderately degenerated rabbit intervertebral disc. The decompressed rabbit intervertebral discs showed signs of tissue recovery at the cellular and histological levels after temporary disc distraction.

[Necrotizing fasciitis following therapeutic injection in a shoulder joint]. / Nekrotisierende Fasziitis nach Injektionstherapie im Schultergelenk.

Necrotizing fasciitis is an inflammatory, rapidly progressive soft tissue infection usually caused by Streptococcus pyogenes or by a combination of aerobic and anaerobic microorganisms. Here we report the case of a patient who developed necrotizing fasciitis near the site of therapeutic injections. An orthopaedic surgeon in private practice had given the 74-year-old patient, who suffered from left shoulder pain, cortisone injections in his left shoulder joint. During the course of this therapy, the patient developed necrotizing fasciitis. Despite radical surgical debridement of the patient's back, left thorax and amputation of his left arm, the patient expired 15 h after arriving at our department. In cases such as these, patient survival depends upon an early diagnosis followed by immediate radical surgical intervention including complete opening of fascial compartments and excision of necrotic tissues.