Edward N. Hanley

Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls.

Study Design. A prospective analysis of 33 control and 39 surgical human lumbar disc specimens from the anulus was undertaken to assess disc cell extracellular matrix production and cell function. The authors of this study analyzed immunohistochemical distributions of Types I, II, III and VI collagen, in situ localization of apoptotic disc cells, and tartrate‐resistant acid phosphatase localization. Objectives. To quantify the incidence of apoptotic cell death in the anulus; examine the collagen distribution in the pericellular, territorial, and interterritorial matrix; examine matrix cell degeneration; and compare diseased tissue with normal tissue from control individuals. Summary of the Background Data. Previous studies of disc histopathology have focused on extracellular matrix morphology and on biochemical synthetic and degenerative changes, but little is understood about the cell populations within the disc that are responsible for these changes. Methods. In this study light microscopy, immunohistochemistry, enzyme histochemistry, and in situ hybridization were used to examine 33 patient and 39 control specimens of human anulus obtained either via surgical procedures or from donors to the Cooperative Human Tissue Network. Results. The high incidence of apoptotic cell death was significantly greater in the control group (73.1 ± 5.1% [mean ± SEM]; n = 20) than among surgical specimens (53.5 ± 5.6%; n = 20; P < 0.001); this was probably a result of the significantly greater average age in the control population (57.2 ± 3.1 years) compared with that in the patient population (44.3 ± 3.2 years; P < 0.001). Immunohistochemistry yielded findings that led to an expanded appreciation of the greatly modified extracellular domains that surrounded disc cells during aging and degeneration in both study groups. Enzyme histochemistry revealed the presence of tartrate‐resistant acid phosphatase activity in human disc cells. Conclusions. These findings reveal that there is a high incidence of apoptosis in the intervertebral disc. Surviving cells are not synthetically inactive but are, rather, producing inappropriate matrix products during aging and degeneration. In certain instances it appears that the matrix surrounding the cell may form an isolation barrier, which may influence individual cell activity and intercellular communication. These results point to the need to 1) more fully understand the cause of disc cell death via apoptosis and to determine whether this programmed cell death can be reversed or halted, and 2) more fully understand the dynamic relation between disc cells and the surrounding extracellular matrix, which they produce and remodel. The factors regulating extracellular matrix‐disc cell homeostasis in the disc are unknown, as is the relation between extracellular matrix and disc cell functional modulation. The morphologic findings of this study suggest that these issues are important considerations in disc cell biology. The identification of tartrate‐resistant acid phosphatase activity in disc cells allows for a new area of study of disc extracellular matrix remodelling. In summary, these new perspectives provide new parameters with which to assess disc cell health and function.

A comparison of radiographic findings in fusion and nonfusion patients ten or more years following lumbar disc surgery.

Ninety-six patients who had undergone disc excision and midline spinal fusion and 36 patients who had had simple disc excision had spinal radiographs made 10 or more years postoperatively. Claw spurs were found most commonly at the L2-3 and L3-4 levels in fusion patients, particularly male laborers. Traction spurs with segmental hypermobility were found more commonly at the L4-5 level in patients whose spines were not fused, particularly women. Total lumbar flexion-extension was greater in nonfusion than in fusion patients, but the L1 - 3 mobility was greater in those who had undergone fusion, suggesting a compensatory increase in the range of lumbar motion. Segmental mobility at levels of surgery in nonfusion patients was similar in those with good and those with poor clinical results. Disc space narrowing was common at levels of operation, but did not correspond to the clinical result. Pseudarthrosis was demonstrated in 26% of fusion patients, but was of no clinical significance. Although complex radiographic changes follow lumbar disc surgery, with or without failure, it is concluded that the plane radiograph is of little aid in determining the source of postoperative pain. The sole exception is that of acquired spondylolysis, which was found in 2.5% of this group of fusion patients, and was clearly associated with a poor clinical outcome. Symptomatic degenerative disc disease at levels above lumbar spinal fusions appears to be an uncommon clinical problem.

Anti-Apoptotic Effects of IGF-1 and PDGF on Human Intervertebral Disc Cells In Vitro

Study Design. Human cells from the anulus were grown in tissue culture in an experimental design to study the anti-apoptotic effect of two selected cytokines. Objectives. To determine whether two selected cytokines, insulin-like growth factor-1 and platelet-derived growth factor, were effective in decreasing apoptosis in human cells from the anulus grown in culture for 10 days. Summary of the Background Data. Previous studies have shown that there is a small cell population in the aging human intervertebral disc. Earlier work from the authors’ laboratory suggested that apoptosis (programmed cell death) may be a major contributing factor to the decrease in cell number. A wide variety of inhibitors of apoptosis have now been identified; the present report presents findings on the actions of insulin-like growth factor-1 and platelet-derived growth factor in retarding or preventing apoptosis. Methods. Using previously published culture methods, cells from the anulus of 25 subjects (mean age, 41.7 years) were grown in monolayer culture for 10 days and tested under the following conditions: 1) control growth in the presence of 20% fetal bovine serum; 2) positive control conditions promoting the development of apoptosis in the absence of serum; or 3) in dose–response regimes where insulin-like growth factor-1 or platelet-derived growth factor were added in the presence of only 1% fetal bovine serum (necessary for basal cell maintenance). Specimens were derived from 18 lumbar, 9 cervical, and 1 thoracic sites; the average Thompson score was III. Cells were grown on chambered slides and evaluated in situ using the TdT in situ apoptosis detection reaction to identify apoptotic cells. An average of 300 cells were counted in replicate cultures at each dose to determine the incidence of apoptosis; results were analyzed with standard statistical techniques. Cultured cells also were examined with transmission electron microscopy. Results. Serum withdrawal to a 1% level was used as a positive apoptosis control in vitro and resulted in a significantly greater percentage of apoptosis compared with the 20% serum negative control (1.02% ± 0.34 (28) versus 0.14% ± 0.04 (27; mean ± SEM (n)), P < 0.0001). Exposure to 50 ng/mL insulin-like growth factor-1 significantly reduced the percentage of apoptosis (vs.— 1% serum) to 0.49% ± 0.26 (P = 0.005); 500 ng/mL was also significantly effective (% apoptosis = 0.09% ± 0.04 (P = 0.0001). Platelet-derived growth factor at a dose of 100 ng/mL also significantly reduced apoptosis (0.18 ± 0.11, P = 0.0001). Conclusions. Data demonstrate a significant reduction in the percentage of apoptotic disc cells after exposure to 50–500 ng/mL insulin-like growth factor-1 or exposure to 100 ng/mL platelet-derived growth factor. These findings expand the understanding of the cell biology of the disc cell and show that selected cytokines can retard or prevent programmed cell death in vitro. The administration of these cytokines may have future therapeutic potential in the treatment of disc degeneration.

Autologous intervertebral disc cell implantation: a model using Psammomys obesus, the sand rat.

Study Design. Work presented here used a small animal model to illustrate the feasibility of autologous disc cell implantation. Objectives. To develop a small animal model for autologous disc cell implantation. Summary of the Background Data. The use of autologous disc cells in the potential treatment of disc degeneration offers attractive possibilities for novel therapies. Results are presented on the use of the sand rat (Psammomys obesus), a small rodent that spontaneously develops disc degeneration during aging, in experimental studies in which cells were harvested from a lumbar intervertebral disc, expanded in monolayer tissue culture, labeled with agents that allow subsequent immunolocalization of these cells, and implanted in a second disc site of the donor animal. Methods. Tissue culture, disc surgery, histology, and immunocytochemistry were used. Cells were either engrafted in a bioresorbable carrier tested for cell compatibility or injected into the recipient disc. Results were assessed with radiographic examination of the implantation site and with histology and immunocytochemistry. Conclusion. Data from 15 animals were obtained with engraftment resident in the animal for up to 33 weeks. Immunocytologic identification of engrafted cells showed that they integrated into the disc and were surrounded by normal matrix at time points up to 8 months postengraftment. Engrafted cells exhibited either a spindle-shaped morphology in the annulus or a rounded chondrocyte-like morphology in the nucleus. Although technically challenging, the authors’ experience showed that autologous disc cell implantation can be successful and that the sand rat is a valuable model for autologous disc cell studies.

Adipose-Derived Stem Cells: Characterization and Current Application in Orthopaedic Tissue Repair

Orthopaedic tissues, such as bone, cartilage, intervertebral disc and tendon, contain cells that are difficult to culture and stimulate in vitro for repair of damaged tissue. Stem cells have the ability to self-renew and differentiate into many tissue types. Recent progress in stem cell research has led to an enthusiastic effort to utilize stem cells for orthopaedic tissue regeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal cells (ASC) are an attractive, readily available adult stem cell that has become increasingly popular for use in many stem cell applications. Recent progress has been made in characterizing ASC and looking mechanistically at gene expression and cellular pathways involved in differentiation. This review focuses on (i) the characterization of ASC through expression of appropriate surface markers; (ii) modulation of in vitro differentiation of ASC through different scaffolds, growth factors, and media; and (iii) the use of ASC in orthopaedic tissue repair. Strategies for repair involve the use of differentiated or undifferentiated, fresh or passaged ASC, in conjunction with appropriate choice of media, growth factors and scaffolds. Recent in vivo studies utilizing ASC are discussed giving results on defect repair and potential for clinical orthopaedic tissue regeneration.

Senescence in cells of the aging and degenerating intervertebral disc: Immunolocalization of senescence-associated β-galactosidase in Human and sand rat discs

Study Design. Human intervertebral disc anulus tissue was obtained in a prospective study of cell senescence. Localization of the senescence biomarker &bgr;-galactosidase (senescence associated &bgr;-galactosidase, SA-&bgr;-gal) was used for quantitative determination of the % senescent cells. Discs were obtained from surgical specimens or control donors. Discs were also studied from the lumbar spine of the sand rat. Experimental studies were approved by the authors’ Human Subjects Institutional Review Board and animal use committee. Objectives. To determine the incidence of cell senescence in human discs with Thompson Grades I through V using immunocytochemistry to quantify the percentage of cells positive for the senescence biomarker SA-&bgr;-gal. Summary of Background Data. Cell senescence has been recognized as a potential factor playing a role age-related disc degeneration. Senescent cells are viable but have lost the ability to divide. Senescence cells, however, are metabolically active. Methods. Fifty-seven discs specimens from 54 subjects were examined with immunocytochemistry for anti-SA-&bgr;-gal immunocytochemical localization to identify senescent cells. The fraction of positive cells was determined with quantitative histomorphometry. Results. Quantitative histomorphometry of human discs show an overall incidence of SA-&bgr;-gal-positive cells of 29.9% (±24.8, SD), with a range from 0 to 92.01%. Analysis by ANOVA of the % senescent cells grouped by Thompson grade showed significant increases in senescence with increasing disc degeneration (P < 0.0001). Further analysis with Tukey’s test showed significant differences between the % senescent cells in Grades I/II versus IV, and versus V. SA-&bgr;-gal-positive cells were also present in discs of the aging sand rat spine. Conclusions. Quantitative analysis of immunohistochemical localization of SA-&bgr;-gal identified a sizeable population of senescent cells in the aging/degenerating disc. It is important to discover more about the senescent disc cell population because these cells persist and accumulate over time within the disc. Since senescent cells cannot divide, senescence may reduce the disc’s ability to generate new cells to replace existing ones lost to necrosis or apoptosis.

Disc Excision and Spine Fusion in the Management of Lumbar Disc Disease: A Minimum Ten-Year Followup

Seventy-nine percent of 312 patients who underwent lumbar disc surgery were evaluated at least 10 years postoperatively (mean=13.7 years). Residual back and nerve root symptoms and functional impairment were equally as common among the 143 patients who underwent fusion as they were among the 64 patients who did not. Thirty percent of the patients whose spines were fused and 37.7% of those patients whose spines were not fused were considered longterm failures because of persistent symptoms or the need for reoperation. Thirty-seven percent of the fusion patients had persistent graft donor site symptoms. Examined patients showed a high percentage of residual neurologic defects. An unexplained positive Trendelenburg sign was present in 14.8% of the fusion patients and in 18.2% of the patients whose spines were not fused. Although retrospective studies often have problems of accuracy, this analysis confirms other observations that midline spinal fusion offers few benefits in the management of lumbar disc disease.

Surgical treatment of isthmic lumbosacral spondylolisthesis. Analysis of variables influencing results.

Fifty consecutive patients underwent standardized surgical treatment for isthmic lumbosacral spondylolisthesis. Twenty-two (44%) had mechanical symptoms only and were treated with in situ fusion. Twenty-eight (56%) had back and radicular symptoms and underwent decompression and fusion. Follow-up averaged 40.4 months. Satisfactory results were achieved in 30 (60%). Patients under 30 and over 50 appeared to do better. Success rate was not related to degree of slippage. Success rate in compensation cases was 39%, versus 83% in non-compensation cases (P < 0.001); males, 53%, versus females, 78% (0.05 < P < 0.1); back pain only, 73%, versus radiculopathy, 50% (0.05 < P < 0.1); smokers, 48%, versus nonsmokers, 74% (0.05 < P< 0.1). Pseudarthrosis rate was 12%, and this correlated with failure (P<0.002). Thus, a trend towards an unsatisfactory outcome was seen in males, middle-aged individuals, those with a smoking habit, and patients with radicular symptoms. A compensable work situation and pseudoarthrosis had a profoundly negative influence on outcome.

The Indications for Lumbar Spinal Fusion With and Without Instrumentation

Study Design Literature review. Objectives A review, analysis, and discussion of the extensive literature on lumbar spinal fusion were done to attempt to place in perspective the indications and success rates fur lumbar spinal fusion with and without instrumentation. Summary of Background Data A wide variety of lumbar spinal conditions have been managed by spina! fusion. Results appear better when the diagnosis is very specific and related to definable instability or deformity in patienta with a stable psychologic state. Methods Search of literature. Results Success rates are higher in isthmic spondylolisthesis, unstable spinal stenosis syndromes (degenerative spondylolisthesis, degenerative scoliosis), and in patients with objective segmental instability. Variable success rates are reported for disc-related low back pain conditions and in patients with failed previous surgery. Instrumentation appears to be beneficial in situations where complex deformities or obvious instability is present. When applied for other diagnoses (e.g., internal disc disruption), results appear no better than with traditional surgical techniques. Conclusions The outcome of lumbar spinal fusion depends on careful assessment of the anatomic cause of pain and of the patients functional state and expectations.

Epidemiology Introduction: 1995 Focus Issue Meeting on Fusion

Jeffrey N. Katz, Kevin F. Spratt, Gunnar B. J. Andersson, Scott D. Boden, Robert D. Fraser, Scott R. Garfin, Vijay K. Goel, Edward N. Hanley Jr., Malcolm H. Pope, Volker K. H. Sonntag, Dale R. Sumner and Thomas A. Zdeblick