Advanced gelatin-based vascularization bioinks for extrusion-based bioprinting of vascularized bone equivalents.

Bone tissue is highly vascularized. The crosstalk of vascular and osteogenic cells is not only responsible for the formation of the strongly divergent tissue types but also for their physiological maintenance and repair. Extrusion-based bioprinting presents a promising fabrication method for bone replacement. It allows for the production of large-volume constructs, which can be tailored to individual tissue defect geometries. In this study, we used the all-gelatin-based toolbox of methacryl-modified gelatin (GM), non-modified gelatin (G) and acetylated GM (GMA) to tailor both the properties of the bioink towards improved printability, and the properties of the crosslinked hydrogel towards enhanced support of vascular network formation by simple blending. The vasculogenic behavior of human dermal microvascular endothelial cells (HDMECs) and human adipose-derived stem cells (ASCs) was evaluated in the different hydrogel formulations for 14 days. Co-culture constructs including a vascular component and an osteogenic component (i.e. a bone bioink based on GM, hydroxyapatite and ASCs) were fabricated via extrusion-based bioprinting. Bioprinted co-culture constructs exhibited functional tissue-specific cells whose interplay positively affected the formation and maintenance of vascular-like structures. The setup further enabled the deposition of bone matrix associated proteins like collagen type I, fibronectin and alkaline phosphatase within the 30-day culture.

clickECM: Development of a cell-derived extracellular matrix with azide functionalities.

In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this "clickECM" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM. STATEMENT OF SIGNIFICANCE: The clickECM was produced by the incorporation of azide-functionalized sugar analogues into the extracellular glycans of fibroblast cell cultures by metabolic oligosaccharide engineering. By introducing these azide groups into the glycan structures, we enabled this cell-derived ECM for bioorthogonal click reactions. Click chemistry provides extremely specific reactions with high efficiency, high selectivity, and high reaction yields. We could show that the azide functionalities within the clickECM are chemically accessible. Based on our here described clickECM technique it will be possible to create and investigate new clickECM materials with tunable bioactive properties and additional functionalities, which offers a promising approach for basic and applied research in the field of biomaterial science, biomedical applications, and tissue engineering.

Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D.

Bone homeostasis is maintained by osteoblasts (bone formation) and osteoclasts (bone resorption). While there have been numerous studies investigating mesenchymal stem cells and their potential to differentiate into osteoblasts as well as their interaction with different bone substitute materials, there is only limited knowledge concerning in vitro generated osteoclasts. Due to the increasing development of degradable bone-grafting materials and the need of sophisticated in vitro test methods, it is essential to gain deeper insight into the process of osteoclastogenesis and the resorption functionality of human osteoclasts. Therefore, we focused on the comparison of osteoclastogenesis and resorption activity on tissue culture polystyrene (TCPS) and bovine extracellular bone matrices (BMs). Cortical bone slices were used as two-dimensional (2D) substrates, whereas a thermally treated cancellous bone matrix was used for three-dimensional (3D) experiments. We isolated primary human monocytes and induced osteoclastogenesis by medium supplementation. Subsequently, the expression of the vitronectin receptor (αVß3) and cathepsin K as well as the characteristic actin formation on TCPS and the two BMs were examined. The cell area of human osteoclasts was analyzed on TCPS and on BMs, whereas significantly larger osteoclasts could be detected on BMs. Additionally, we compared the diameter of the sealing zones with the measured diameter of the resorption pits on the BMs and revealed similar diameters of the sealing zones and the resorption pits. We conclude that using TCPS as culture substrate does not affect the expression of osteoclast-specific markers. The analysis of resorption activity can successfully be conducted on cortical as well as on cancellous bone matrices. For new in vitro test systems concerning bone resorption, we suggest the establishment of a 2D assay for high throughput screening of new degradable bone substitute materials with osteoclasts.

Basic research and clinical applications of non-hematopoietic stem cells, 4-5 April 2008, Tubingen, Germany.

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.

Neurological outcome in surgically treated patients with incomplete closed traumatic cervical spinal cord injury.

STUDY DESIGN: Retrospective study based on a reference paper. Neurological outcome in patients who were managed surgically with closed traumatic cervical spine injury was evaluated using the ASIA motor scoring system and Frankel grading. OBJECTIVES: To assess the accuracy of motor charting and Frankel grading as tools to evaluate neurological outcome in closed traumatic cervical spine injury, and also to evaluate how the surgically treated patients fared in their neurological recovery by measurement tools as in the reference paper. SETTING: National Spinal Injuries Centre, Stoke Mandeville Hospital, Aylesbury, UK. METHOD: Fifty-seven patients were admitted within 2 days of the injury with closed traumatic cervical spine injuries (1997-2004). Thirty-seven (65%) met the inclusion criteria as per the referenced paper, that is, were treated surgically, were Frankel grade B and above and had at least 12 months follow up. The remaining 20 patients were not included as they did not meet the inclusion criteria. The breakdown of the 20 patients is given in Table 1. The mean recovery percentage (MRP) and mean deficit percentage (MDP) were calculated as per the referenced paper. RESULTS: An evaluation of 37 patients surgically treated, who had follow up of at least 12 months, showed that preservation of pin prick below the level of lesion, and preservation of anal tone and perianal sensation were good prognostic indicators. There was no correlation between degree of encroachment of canal or the degree of kyphosis to MDP or MRP. The mean time from injury to mobilization was 7.6 days in 25 out of 37 patients. Twelve of the 37 patients had prolonged immobilization because of ITU stay or because they were initially treated conservatively. Three out of the 37 patients developed DVT/PE. Mean hospital stay was 6.4 months. CONCLUSION: The neurological outcome in surgically treated patients is comparable to the conservatively treated patients. The Frankel grading and ASIA motor charting combined is a powerful tool in assessing the neurological outcome in closed traumatic cervical spinal injured patients. Until now there has been no evidence to suggest that the obvious advantages of surgical management of closed cervical spine injuries (better alignment, easier manual handling and early mobilization) is traded for poorer neurological outcome.

Management of degenerative changes and stenosis of the lumbar spinal canal secondary to cervical spinal cord injury.

We describe the case of a 47-year-old female who sustained a C5/6 fracture with C6 complete spinal cord injury 26 years ago. She presented with increased spasticity of the lower extremities, the abdominal wall and episodes of autonomic dysreflexia. Imaging of the spine revealed post-traumatic kyphosis at the level of the injury and degenerative changes of the lumbar spine with marked facet joint hypertrophy at the level of L4/5 causing severe spinal canal stenosis. Discussants of this case comment on the possible pathophysiological mechanisms causing autonomic dysreflexia, especially the development of degenerative changes, Charcot arthropathy and the role of tethering mechanisms. The diagnostic options and management approaches are also discussed.

Quality of life in patients with spinal cord injury--basic issues, assessment, and recommendations.

INTRODUCTION: Spinal cord injury (SCI) often causes severe disabilities. The degree of functional impairment strongly depends on the level and completeness of lesion (tetraplegic, paraplegic). But evaluation of outcomes also needs to consider the broader concept of health-related quality of the life (HRQL) for SCI patients. A multinational group of clinicians and researchers assessed this concept and reviewed the available instruments for measurement of quality of life in this group of patients. TIME POINTS: Phase I is in the acute clinic; phase II during rehabilitation; phase III after discharge home. Annual follow-up investigations should be maintained. The phase of initial care (phase 0) is important for prognosis and should, therefore, be part of the documentation. INSTRUMENTS: Criteria used to evaluate current QoL measures: reliability, validity, responsiveness, availability of translations, application in SCI patients, existing population norms. Several specific instruments or subscales exist for the following domains: physical and psychological functioning, pain, and handicap. Well-known generic measures of HRQL also have been applied to SCI patients, and a disease-specific instrument has been developed (SCIQL-23). A variety of subjective quality of life measures were evaluated as well. GROUP CONSENSUS/GUIDELINE: Prior to discharge from rehabilitation, the group suggested the use of the Functional Independence Measure, the Hospital Anxiety and Depression Scale and a Visual Analogue Scale for pain. Following discharge from the acute clinic, the SF-36, the Craig Handicap Assessment and Reporting Technique, the Quality of Well-being Scale, or the Life Satisfaction questionnaire were proposed. However, the evidence supporting the use of these instruments is sparse.

Biomechanical evaluation of a new modular rod-screw implant system for posterior instrumentation of the occipito-cervical spine: in-vitro comparison with two established implant systems.

Posterior instrumentation of the occipito-cervical spine has become an established procedure in a variety of indications. The use of rod-screw systems improved posterior instrumentation as it allows optimal screw positioning adapted to the individual anatomic situation. However, there are still some drawbacks concerning the different implant designs. Therefore, a new modular rod-screw implant system has been developed to overcome some of the drawbacks of established systems. The aim of this study was to evaluate whether posterior internal fixation of the occipito-cervical spine with the new implant system improves primary biomechanical stability. Three different internal fixation systems were compared in this study: the CerviFix System, the Olerud Cervical Rod Spinal System and the newly developed Neon Occipito Cervical System. Eight human cervical spine CO/C5 specimens were instrumented from C0 to C4 with occipital fixation, transarticular screws in C1/C2 and lateral mass or pedicle screws in C3 and C4. The specimens were tested in flexion/extension, axial rotation, and lateral bending using pure moments of +/- 2.5 Nm without axial preload. After testing the intact spine, the different instrumentations were tested after destabilising C0/C2 and C3/C4. Primary stability was significantly increased, in all load cases, with the new modular implant system compared to the other implant systems. Pedicle screw instrumentation tended to be more stable compared to lateral mass screws; nevertheless, significant differences were observed only for lateral bending. As the experimental design precluded any cyclic testing, the data represent only the primary stability of the implants. In summary, this study showed that posterior instrumentation of the cervical spine using the new Neon Occipito Cervical System improves primary biomechanical stability compared to the CerviFix System and the Olerud Cervical Rod Spinal System.

Load-displacement properties of the normal and injured lower cervical spine in vitro.

The objective of this study was to determine which discoligamentous structures of the lower cervical spine provide significant stability with regard to different loading conditions. Accordingly, the load-displacement properties of the normal and injured lower cervical spine were tested in vitro. Four artificially created stages of increasing discoligamentous instability of the segment C5/6 were compared to the normal C5/6 segment. Six fresh human cadaver spine segments C4-C7 were tested in flexion/extension, axial rotation, and lateral bending using pure moments of +/- 2.5 Nm without axial preload. Five conditions were investigated consecutively: (1) the intact functional spinal unit (FSU) C5/6; (2) the FSU C5/6 with the anterior longitudinal ligament and the intertransverse ligaments sectioned; (3) the FSU C5/6 with an additional 10-mm-deep incision of the anterior half of the anulus fibrosus and the disc; (4) the FSU C5/6 with additionally sectioned ligamenta flava as well as interspinous and supraspinous ligaments; (5) the FSU C5/6 with additional capsulotomy of the facet joints. In flexion/extension, significant differences were observed concerning range of motion (ROM) and neutral zone (NZ) for all four stages of instability compared to the intact FSU. In axial rotation, only the stage 4 instability showed a significantly increased ROM and NZ compared to the intact FSU. For lateral bending, no significant differences were observed. Based on these data, we conclude that flexion/extension is the most sensitive load-direction for the tested discoligamentous instabilities.

Computer-assisted surgery in posterior instrumentation of the cervical spine: an in-vitro feasibility study.

Transarticular C1/2 screws are widely used in posterior cervical spine instrumentation. The use of pedicle screws in the cervical spine remains uncommon. Due to superior biomechanical stability compared to lateral mass screws, pedicle screws can be used, especially for patients with poor bone quality or defects in the anterior column. Nevertheless there are potential risks of iatrogenic damage to the spinal cord, nerve roots or the vertebral artery associated with both posterior cervical spine instrumentation techniques. Therefore, the aim of this study was to evaluate whether C1/2 transarticular screws as well as transpedicular screws in C3 and C4 can be applied safely and with high accuracy using a computer-assisted surgery (CAS) system. We used 13 human cadaver C0-C5 spine segments. We installed 1.4-mm Kirschner wires transarticular in C 1/2, using a specially designed guide, and drilled 2.5-mm pedicle holes in C3 and C4 with the assistance of the CAS system. Hole positions were evaluated by palpation, CT and dissection. Forty-eight (92%) of the 52 drilled pedicles were correctly positioned after palpation, imaging and dissection. The vertebral artery was not injured in any specimen. All of the 26 C1/2 Kirschner wires were placed properly after imaging and dissection evaluations. No injury to vascular or bony structures was observed. C /2 transarticular screws as well as transpedicular screws in the cervical spine can be applied safely and with high accuracy using a CAS system in vitro. Therefore, this technique may be used in a clinical setting, as it offers improved accuracy and reduced radiation dose for the patient and the medical staff. Nevertheless, users should take note of known sources of possible faults causing inaccuracies in order to prevent iatrogenic damage. Small pedicles, with a diameter of less than 4.0 mm, may not be suitable for pedicle screws.

Epidural blood patch under fluoroscopic control: non-surgical treatment of lumbar cerebrospinal fluid fistula following implantation of an intrathecal pump system.

The treatment of lumbar cerebrospinal fluid fistula in the presence of an intrathecal catheter is known to be difficult. Open revision surgery is recommended in the literature, although the rate of recurrence is high. The epidural blood patch technique is well established as a successful treatment for post-dural-puncture headaches. Recent work about the distribution of the injected blood and theoretical considerations about the mechanism of action make this method suitable for the occlusion of spinal leakage even in the presence of an intrathecal catheter. In this note technical details are given for a successful therapy of lumbar cerebrospinal fluid fistula including the right positioning of the opening of the needle (cerebrospinal fluid can be expected intrathecally and epidurally) by injection of contrast medium first for myelography then for epidurography. In this procedure the (epidural) distribution of autologous blood can be indirectly controlled by compression of the dural sac. The method is easy to perform, and the possible risks are small.

Characteristics of an extended internal fixation system for polysegmental transpedicular reduction and stabilization of the thoracic, lumbar, and lumbosacral spine.

The Kluger internal fixator, with its artificial fulcrum outside the operative site, had to be extended for multisegmental use. Three different prototypes, called Central Bar (CB), Double Bar I (DB I) and Double Bar II (DB II) were designed, which were fully compatible with the existing reduction system. To evaluate the ability of these newly developed systems to provide primary stability in a destabilized spine, their stiffness characteristics and stabilizing effects were investigated in multidirectional biomechanical stability tests and compared with those of the clinically well-known Cotrel-Dubousset (CD) system. The investigations were performed on a spine tester using freshly prepared calf spines. The model tested was that of an intact straight spine followed by a defined three-column lesion simulating the most destabilizing type of injury. Pure moments of up to 7.5 Nm were continuously applied to the top of each specimen in flexion/extension, left/right axial rotation, and left/right lateral bending. Segmental motion was measured using a three-dimensional goniometric linkage system. Range of motion and stiffness within the neutral zone were calculated from obtained load-displacement curves. The DB II attained 112.5% (P = 0.26) of the absolute stiffness of the CD system in flexion and enhanced its stability in extension by up to 144.3% (P = 0.004). In axial rotation of the completely destabilized spine, this system achieved 183.3% of the stiffness of the CD system (P < 0.001), and in lateral bending no motion was measured in the most injured specimens stabilized by the DB II. The DB I, which was the first to be designed and was considered to provide high biomechanical stability, did not attain the stiffness standard set by the CD system in either flexion/extension or axial rotation of the most injured spine. The study confirms that it is worthwhile to evaluate in vitro the biomechanical properties of a newly developed implant before its use in patients, in order to refine weak construction points and help to reduce device-related complications and to better evaluate its efficacy in stabilizing the spine.

Biomechanical evaluation of a newly developed monocortical expansion screw for use in anterior internal fixation of the cervical spine. In vitro comparison with two established internal fixation systems.

STUDY DESIGN: The primary biomechanical stability of anterior internal fixation of the cervical spine obtained with a new monocortical expansion screw in vitro was evaluated. OBJECTIVES: To determine whether the anterior internal fixation of the spine obtained with the new monocortical expansion screw provides biomechanical stability comparable with that obtained with bicortical fixation. SUMMARY OF BACKGROUND DATA: The anterior plate instrumentation used with bicortical screw fixation in the cervical spine provides a primary stability superior to that associated with monocortical screw fixation. However, bicortical screws have the potential to perforate the posterior cortex. Therefore, monocortical instrumentation systems were developed, but without the biomechanical stability associated with bicortical systems. A new expansion screw for monocortical fixation was developed to improve biomechanical stability of monocortical systems. METHODS: Three different internal fixation systems were compared in this study: 1) H-plate with AO 3.5-mm bicortical screws, 2) cervical spine locking plate with monocortical screws, and 3) H-plate with the new monocortical expansion screws. Eight fresh human cadaver spine segments from C4 to C7 were tested in flexion-extension, axial rotation, and lateral bending using pure moments of +/- 2.5 Nm without axial preload. Five conditions were investigated consecutively: 1) intact spine; 2) uninstrumented spine with the segment C5-C6 destabilized; 3-5) instrumentation of the segment C5-C6 with the three implants mentioned above after removal of the disc and insertion of an interbody spacer. RESULTS: Between bicortical and monocortical expansion screw H-plate fixation, no significant differences were observed in all load cases concerning range of motion and neutral zone. The neutral zone and range of motion were significantly larger for the cervical spine locking plate than for bicortical and monocortical expansion screw fixation in all load cases, except neutral zone for axial rotation versus bicortical screw fixation. The instrumented cases only had a significantly lower range of motion and neutral zone than the intact cases in extension-flexion, whereas for lateral bending and axial rotation no significant differences could be observed. Because the experimental design precluded any cyclic testing, the data represent only the primary stability of the implants. CONCLUSIONS: In anterior instrumentation of the cervical spine using a H-plate, the new monocortical expansion screw provides the same biomechanical stability as the bicortical 3.5-mm AO screw and a significantly better biomechanical stability than the cervical spine locking plate. Therefore, the expansion screw may be an alternative to the bicortical fixation and does not involve the risk of penetration of the posterior vertebral body cortex.

[Diagnosis and therapy of spinal stenosis in the elderly]. / Diagnostik und Therapie der Spinalstenose beim älteren Menschen.

PROBLEM: Spinal stenosis is a common and increasing problem in the elderly population and a diagnostic and therapeutic challenge. METHODS: An overview of etiology, epidemiology, diagnostics and therapy is given based on a literature review of the years 1978-1998 and experiences since 1985. RESULTS: Dealing with spinal stenosis we have to distinguish concerning diagnostics and therapy between cervical and lumbar spinal stenosis. The cardinal symptom of the lumbar spinal stenosis is the claudicatio spinalis and of the cervical spinal stenosis the cervical myelopathy occasionally combined with radiculopathy. The first therapeutic step should be the conservative therapy in nearly all cases, the only exception is a severe cervical myelopathy where an operation is indicated. In case of persistent or progressive symptoms under a sufficient conservative therapy, operative therapy is indicated. The different possible therapy decisions will be based on flow-charts. CONCLUSION: Whereas in lumbar spinal stenosis the indication for operative therapy should be considered with reservation, in cervical spinal stenosis with myelopathy operative therapy should be considered at an early stage.

Strategy for the treatment of patients with spinal neoplasms.

Progress in oncological therapy within the last decade has enhanced the survival time of patients suffering from tumorous osteolyses of the spine. While the necessity of surgical intervention is often settled by acute clinical symptoms, the extent of surgery is certainly co-determined by the patient's expectations and the time of survival to be expected. We therefore developed a specific tumor algorithm for operations on the spine with special emphasis on prognosis and the attainable quality of life. The results of 154 patients with tumorous osteolyses of the thoracic and lumbar spine, who were treated according to our algorithm, underline the unequivocal advantages of initially posterior procedures.

Autotransfusion--bacterial contamination during hip arthroplasty and efficacy of cefuroxime prophylaxis. A randomized controlled study of 40 patients.

40 patients undergoing primary hip arthroplasty, given autologous processed blood transfusion, were randomized a receive no antibiotic prophylaxis (group A, n 20) or cefuroxime (1.5 g single injection; group B, n 20). Bacterial contamination at various steps in the autotransfusion procedure was assessed in liquid and solid culture media. The operation field and the wound drainage blood were never contaminated either of the groups but some of the suction tips were. Parts of the Vacufix blood collection bags of group A contained bacteria, but none in group B. Processed red blood cell concentrates in both groups showed bacterial growth. Greater blood loss did not increase the contamination rate in general. Isolated bacteria included the species Staphylococcus epidermidis, coagulase-negative staphylococci and Propionibacteria in both groups, but with different cell counts. In addition, Corynebacterium bovis et minutissimum and Moraxelle were identified in group A. In conclusion, autologous blood transfusion was a safe procedure. If contamination occurred, the bacterial count was low, and the bacteria of low pathogenicity. Antibiotic prophylaxis with cefuroxime reduced this contamination of suction tips and collection bags and limited the transfer of autologous blood products.

Biomechanical evaluation of a bioresorbable PLA dowel for arthroscopic surgery of the shoulder.

Metallic implants in the reconstructive surgery of the shoulder joint can lead to certain complications. Therefore, a small bioresorbable dowel made of PLA has been developed for arthroscopic refixation of the glenoid labrum. Biomechanical tests employing in vitro degradation should confirm the implant's viability before starting the clinical application. Pull-out experiments of sutures fixed with these dowels were performed. The bioresorbable implant fulfilled the mechanical expectation in its time-dependent stability under realistic conditions by far. A reliable fixation of sutures by the implant in clinical situations can be widely guaranteed with these experimental results.

The treatment of spondylolistheses with segmental reduction and interbody fusion by means of an internal fixator.

In spondylolisthesis with an indication for fusion and with a slipping of more than 50 % at least a partial reposition should be reached in general because the incidence of pseudarthrosis would increase with a fusion in situ and a large disturbance of the spinal statics would persist. Hereby with almost all methods an enlarged operative morbidity and often a longer fusion range has to be taken in account compared to the fusion in situ. Therefore, in smaller slippages the fusion in situ will be favoured because the disturbance of the statics is not so important, that such an effort combined with such methods is necessary. If the operation method with small spondylolisthesis and pseudospondylolisthesis allows the reposition without much effort and if the operative morbidity in comparison with the fusion in situ is not higher, then it is reasonable to fuse the cases with a spondylolisthesis Meyerding grade 1 and 2 in the anatomic corrected position too. Because the spinal fixator we use fills out these criteria we combine the correction of the position with the fusion also in cases of small spondylolisthesis. The incidence of neurologic complications correlates with the amount of the reposition distance and can be caused by preforaminal or extraforaminal lesions. The reduction of small malpositions could only produce preforaminal lesions. Using the spine fixator with its reposition instruments linked outside the wound and with it's uninhibited access to the segment and to the preforaminal neural structures during the whole repositioning these lesions can be avoided.