Aldemar Andres Hegewald

Regeneration of intervertebral disc tissue by resorbable cell-free polyglycolic acid-based implants in a rabbit model of disc degeneration.

Study Design. Different biologic strategies exist to treat degenerative disc disease. Tissue engineering approaches favor autologous chondrocyte transplantation. In our one-step-approach, a resorbable cell-free polyglycolic acid (PGA)-based implant is immersed in serum from whole blood and implanted into the disc defect directly after discectomy. Objectives. The aim of our study was to investigate the capacity of a cell-free implant composed of a PGA felt, hyaluronic acid, and serum to recruit disc cells and stimulate repair tissue formation in vivo after microdiscectomy in a rabbit model. Summary of the Background Data. Disc tissue has a limited ability to regenerate after the degeneration process was once initiated. Therefore, we developed a cell-free resorbable implant that is able to attract local cells into the defect and induce proper repair tissue formation. Methods. The cell-free implant consisting of PGA and hyaluronic acid was immersed in allogenic serum and implanted into the disc defect after discectomy in New Zealand white rabbits. One week and 6 months after the operation, the disc height index and the T2-weighted signal intensity index were determined using plane radiographs and magnetic resonance imaging. Finally, discs were explanted and investigated histologically. Animals with discectomy only served as controls. Results. In our animal studies, we could demonstrate that the T2-weighted signal intensity of the operated discs decreased in both groups 1 week after surgery. However, after 6 months, the T2-weighted signal intensity index increased by 45% in the implanted group whereas the index decreased further by 11% in the sham group. This corresponded to changes in the disc height index. Furthermore, the histologic examinations indicated cell migration into the defect and showed tissue regeneration. Conclusion. The implantation of a cell-free PGA-hyaluronic acid implant immersed in serum after discectomy induces regeneration, resulting in improvement of the disc water content and preservation of the disc height 6 months after surgery.

Acute Hypoperfusion Immediately after Subarachnoid Hemorrhage: A Xenon Contrast-Enhanced CT Study

The acute neurological deficit present immediately after subarachnoid hemorrhage (SAH) correlates with overall outcome. Only limited data are available to quantify changes in cerebral perfusion in this acute phase, and this study sought to characterize those changes within the first 12 h post-SAH. Xenon contrast-enhanced CT scanning was performed in 17 patients (Hunt and Hess grade [HH] 1-3, n = 9; HH 4-5, n = 8) within 12 h after SAH. Cerebral blood flow (CBF) was analyzed in all cortical and central vascular regions of interest (ROI), as well as infratentorial ROI. Hemodynamic stress distribution (central/cortical ROI) was also calculated. Asymptomatic patients without perfusion deficits served as controls (n = 5), and Glasgow Outcome Scale score (GOS) was determined 3 months after the event. Intracranial pressure (ICP) and cerebral perfusion pressure (CPP) were within normal limits in all patients. CBF was significantly reduced in all patients with SAH (34 mL/100 g x min) compared to controls (67 mL/100 g x min; p < 0.001). Patients in better clinical condition (HH 1-3) presented with significantly less reduction of CBF (41 mL/100 g x min) compared to patients with more severe hemorrhage (HH 4-5: 24 mL/100 g x min; p < 0.001), and had better outcomes. Changes in perfusion were more pronounced in supratentorial than in infratentorial ROI. Hemodynamic stress distribution was most pronounced in patients with higher HH grade (p < 0.05). The first 12 h after SAH are characterized by persistent, severe reduction of CBF, which in turn correlates with HH grade, but is independent of ICP or CPP. Acute peripheral vasospasm of the microvasculature, not detectable by conventional angiography, may account for this early phase of prolonged hypoperfusion.

Adequacy of herniated disc tissue as a cell source for nucleus pulposus regeneration

OBJECTnThe object of this study was to characterize the regenerative potential of cells isolated from herniated disc tissue obtained during microdiscectomy. The acquired data could help to evaluate the feasibility of these cells for autologous disc cell transplantation.nnnMETHODSnFrom each of 5 patients (mean age 45 years), tissue from the nucleus pulposus compartment as well as from herniated disc was obtained separately during microdiscectomy of symptomatic herniated lumbar discs. Cells were isolated, and in vitro cell expansion for cells from herniated disc tissue was accomplished using human serum and fibroblast growth factor-2. For 3D culture, expanded cells were loaded in a fibrin-hyaluronan solution on polyglycolic acid scaffolds for 2 weeks. The formation of disc tissue was documented by histological staining of the extracellular matrix as well as by gene expression analysis of typical disc marker genes.nnnRESULTSnCells isolated from herniated disc tissue showed significant signs of dedifferentiation and degeneration in comparison with cells from tissue of the nucleus compartment. With in vitro cell expansion, further dedifferentiation with distinct suppression of major matrix molecules, such as aggrecan and Type II collagen, was observed. Unlike in previous reports of cells from the nucleus compartment, the cells from herniated disc tissue showed only a weak redifferentiation process in 3D culture. However, propidium iodide/fluorescein diacetate staining documented that 3D assembly of these cells in polyglycolic acid scaffolds allows prolonged culture and high viability.nnnCONCLUSIONSnStudy results suggested a very limited regenerative potential for cells harvested from herniated disc tissue. Further research on 2 major aspects in patient selection is suggested before conducting reasonable clinical trials in this matter: 1) diagnostic strategies to predict the regenerative potential of harvested cells at a radiological or cell biology level, and 2) clinical assessment strategies to elucidate the metabolic state of the targeted disc.

Biomechanical testing of a polymer-based biomaterial for the restoration of spinal stability after nucleotomy.

BackgroundSurgery for disc herniations can be complicated by two major problems: painful degeneration of the spinal segment and re-herniation. Therefore, we examined an absorbable poly-glycolic acid (PGA) biomaterial, which was lyophilized with hyaluronic acid (HA), for its utility to (a) re-establish spinal stability and to (b) seal annulus fibrosus defects. The biomechanical properties range of motion (ROM), neutral zone (NZ) and a potential annulus sealing capacity were investigated.MethodsSeven bovine, lumbar spinal units were tested in vitro for ROM and NZ in three consecutive stages: (a) intact, (b) following nucleotomy and (c) after insertion of a PGA/HA nucleus-implant. For biomechanical testing, spinal units were mounted on a loading-simulator for spines. In three cycles, axial loading was applied in an excentric mode with 0.5 Nm steps until an applied moment of ± 7.5 Nm was achieved in flexion/extension. ROM and NZ were assessed. These tests were performed without and with annulus sealing by sewing a PGA/HA annulus-implant into the annulus defect.ResultsSpinal stability was significantly impaired after nucleotomy (p < 0.001). Intradiscal implantation of a PGA-HA nucleus-implant, however, restored spinal stability (p < 0.003). There was no statistical difference between the stability provided by the nucleus-implant and the intact stage regarding flexion/extension movements (p = 0.209). During the testing sequences, herniation of biomaterial through the annulus defect into the spinal canal regularly occurred, resulting in compression of neural elements. Sewing a PGA/HA annulus-implant into the annulus defect, however, effectively prevented herniation.ConclusionPGA/HA biomaterial seems to be well suited for cell-free and cell-based regenerative treatment strategies in spinal surgery. Its abilities to restore spinal stability and potentially close annulus defects open up new vistas for regenerative approaches to treat intervertebral disc degeneration and for preventing implant herniation.

Human intervertebral disc-derived cells are recruited by human serum and form nucleus pulposus-like tissue upon stimulation with TGF-β3 or hyaluronan in vitro

The aims of this work were to test whether human intervertebral disc-derived nucleus pulposus cells (hNP-cells) are attracted by human serum and to analyze if matrix generation from hNP-cells is promoted under the influence of transforming growth factor-beta3 (TGF-beta3) or hyaluronan (HA) in vitro. Using the multi-well chemotaxis assay to determine cell migration under the influence of different concentrations of human serum, it was demonstrated that dedifferentiated hNP-cells are able to migrate towards a serum fraction gradient in a concentration-dependent manner. Re-differentiation capacity of hNP-cells in 3D micro-masses under the influence of TGF-beta3 or hyaluronan was also tested. Gene expression analysis of types I, II, III and IX collagen, as well as aggrecan, COMP and LINK of hNP-cells in 3D-micro-mass cell-culture revealed a strong increase of these markers in TGF-beta3 treated cells. Furthermore, histochemical and immuno-histochemical staining after 28d showed proteoglycan and type II collagen-rich matrix for both, the TGF-beta3 and the hyaluronan treated cells. These findings show that TGF-beta3 or hyaluronan are able to induce the differentiation and that human serum stimulates the migration of hNP-cells in vitro. Therefore, hyaluronan and serum are suited for cell-free biomaterials as cell migration and differentiation inducing factors intended for biological treatment strategies of the intervertebral disc.

Enhancing tissue repair in annulus fibrosus defects of the intervertebral disc: analysis of a bio‐integrative annulus implant in an in‐vivo ovine model

Annulus fibrosus repair techniques for the intervertebral disc (IVD) address the unsolved problem of reherniation after IVD herniation and might facilitate the development of nucleus pulposus replacement techniques for IVD diseases. This study investigates the suitability of a bio‐integrative annulus implant.Standardized box defects were applied to the annulus L3/4 and L4/5 of 16 sheep, followed by randomized insertion of the textile polyglycolic acid/polyvinylidene fluoride annulus implant in one of the defects. Explantation was conducted after 2, 6 and 12 weeks, followed by provocative pressure testing and histological analysis. At 2 weeks’ follow‐up, all specimens of the control defect group demonstrated uncontained herniated nucleus pulposus tissue in the annulus defects. For the treated specimens, the annulus implant consistently provided an effective barrier for herniating nucleus pulposus tissue, with no implant dislocation at all time‐points. After 2 weeks, a homogeneous cell infiltration of the annulus implant was observed, leading to a progressive directional matrix build‐up. Repair tissue thickness was significantly stronger with the annulus implant at all follow‐ups (p < 0.01). No pronounced foreign body reaction and no difference in the amount of supra‐annular scar tissue over the defect sites were observed. The implantation procedure inflicted annulus damage adjacent to the defect. At later time‐points, however, no difference in comparison with the control defect group was evident. The investigated biointegrative annulus implant showed promising results with regard to biointegration, enhancement of repair tissue and function as a mechanical barrier in an ovine model.

The chemokines CXCL10 and XCL1 recruit human annulus fibrosus cells.

Study Design. Human annulus fibrosus tissue and cells were analyzed for the presence of chemokine receptors and the migratory effect of selected chemokines. Objective. To investigate spontaneous repair mechanisms and underlying cell recruitment in response to annular tears and degenerative defects. Summary of Background Data. Resorption of herniated disc tissue and the attempt to close annulus tears with repair tissue occur spontaneously. Although chemokines are suggested to play a role in resorption of herniated disc tissue, the role of chemokines in annulus fibrosus homeostasis and repair remains unclear. Methods. Cells were isolated from annulus fibrosus tissue and expanded in the presence of human serum. Multiwell chemotaxis assays were used to analyze the migratory effect of human serum and 0 to 1000 nM concentrations of the chemokines CXCL7, CXCL10, CXCL12, CCL25, and XCL1 on annulus fibrosus cells (AFCs) (n = 9 per chemokine and dose). Presence of corresponding chemokine receptors in AFCs was determined by real-time polymerase chain reaction analysis and immunohistochemistry. Results. Serum (0.1%–10%) significantly (P < 0.01) stimulates the migration of AFCs. Compared with untreated cells, the migration of cells was significantly (P < 0.01) enhanced upon stimulation with 100 to 1000 nM CXCL10 and 1000 nM XCL1. Chemokine receptors showed low expression levels in expanded AFCs as assessed by polymerase chain reaction. Immunohistochemical staining of the CXCL10 receptor CXCR3 and the XCL1 receptor XCR1 showed that the presence of the particular receptors in AFCs expanded under conventional cell culture conditions. In native annulus fibrosus tissue, CXCR3 was evident, whereas XCR1 could not be detected. Conclusion. The findings suggest that chemokines, in particular CXCL10, effectively recruit isolated AFCs. This suggests that chemokines are involved in annulus fibrosus homeostasis and potentially in spontaneous annulus repair attempts. This might have important implications for biological annulus-sealing strategies.

Engineering of polymer-based grafts with cells derived from human nucleus pulposus tissue of the lumbar spine

Intervertebral disc degeneration is considered a major source of low back pain. We therefore examined an absorbable polyglycolic acid (PGA) biomaterial for its utility to support disc tissue regeneration. Microdiscectomy for lumbar disc herniation was performed in six patients. Intervertebral disc cells were isolated and in vitro cell expansion was accomplished using human serum and FGF2. In a fibrin–hyaluronan solution, disc cells were loaded on PGA scaffolds and cultured for 2 weeks. Formation of disc tissue was documented by histological staining of the extracellular matrix as well as gene expression analysis of typical disc marker genes. The use of human serum and FGF2 ensures efficient isolation and expansion of human disc cells. During this phase, dedifferentiation of the disc cells was observed. Subsequent 3D tissue culture of disc cells in PGA scaffolds, however, is accompanied by the induction of typical disc marker genes, resulting in tissue containing glycosaminoglycans and collagens. Propidium iodide/fluorescein diacetate (PI/FDA) staining documented that 3D assembly of disc cells in PGA scaffolds allows prolonged culture and high viability of disc cells. Disc cells from tissue of the nucleus compartment can be reliably isolated and expanded in vitro with FGF. In combination with a fibrin–hyaluronan solution and loaded on a PGA scaffold, disc cells from expansion culture commence a redifferentiation process. PGA‐based scaffolds could be useful as temporal matrices for regenerative disc repair approaches. Copyright

Hypoperfusion in the Acute Phase of Subarachnoid Hemorrhage

PURPOSEnAcute disruption of cerebral perfusion and metabolism is a well-established hallmark of the immediate phase after subarachnoid hemorrhage (SAH). It is thought to contribute significantly to acute brain injury, but despite its prognostic importance, the exact mechanism and time course is largely unknown and remains to be characterized.nnnMETHODSnWe investigated changes in cerebral perfusion after SAH in both an experimental and clinical setting. Using an animal model of massive, experimental SAH (n=91), we employed Laser-Doppler flowmetry (LDF), parenchymal microdialysis (MD; n=61), Diffusion-weighted imaging (DWI) and MR spectroscopy (MRS; n=30) to characterize the first hours after SAH in greater detail. The effect of prophylactic treatment with hypothermia (HT; 32°C) and an endothelin-A (ET-A) receptor antagonist (Clazosentan) was also studied. In a group of patients presenting with acute SAH (n=17) we were able to determine cerebral blood flow (CBF) via Xenon-enhanced computed tomography (XeCT) within 12 h after the ictus.nnnRESULTSnThe acute phase after SAH is characterized both experimentally and clinically by profound and prolonged hypoperfusion independent from current intracranial pressure (ICP), indicating acute vasospasm. Experimentally, when treated with hypothermia or a ET-A receptor antagonist prophylactically, acute hypoperfusion improved rapidly. DWI showed a generalized, significant decline of the apparent diffusion coefficient (ADC) after SAH, indicating cytotoxic edema which was not present under hypothermia. SAH causes a highly significant reduction in glucose, as well as accumulation of lactate, glutmate and aspartate (MD and MRS). HT significantly ameliorated these metabolic disturbances.nnnCONCLUSIONnAcute vasospasm, cytotoxic edema and a general metabolic stress response occur immediately after experimental SAH. Prophylactic treatment with hypothermia or ET-A antagonists can correct these disturbances in the experimental setting. Clinically, prolonged and ICP-independent hypoperfusion was also confirmed. As the initial phase is of particular importance regarding the neurological outcome and is amenable to beneficial intervention, the acute stage after SAH demands further investigation and warrants the exploration of measures to improve the immediate management of SAH patients.

A combinatorial relative mass value evaluation of endogenous bioactive proteins in three-dimensional cultured nucleus pulposus cells of herniated intervertebral discs: identification of potential target proteins for gene therapeutic approaches.

Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56 / range 29 - 84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for four weeks. Subsequently cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Independent from gender, age and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1β and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6 and 7, insulin-like growth factor 1, transforming growth factor beta 1 and 3, however, remained below the minimal detectable quantities. These findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases.