Novel biomechanical quantification methodology for lumbar intraforaminal spinal nerve adhesion in a laminectomy and disc injury rat model.

Spinal nerve fibrosis following injury or surgical intervention may play an important role in the pathophysiology of chronic back pain. In this current study, we demonstrate the role of biomechanical quantification of lumbar intraforaminal spinal nerve adhesion and tethering in the analysis of the post-laminectomy condition and describe a direct methodology to make this measurement. Twenty age-matched Sprague-Dawley male rats were divided into operative and non-operative (control) groups. Operative animals underwent a bilateral L5-L6 laminectomy with right-side L5-6 disc injury, a post-laminectomy pain model previously published by this lab. At eight weeks, animals were sacrificed and the strength of adhesion of the L5 intraforaminal spinal nerve to surrounding structures was quantified using a novel biomechanical methodology. Operative animals were found to have a significantly greater load to displace the intact right L5 spinal nerve through the intervertebral foramen when compared to control animals. The findings show that the post-laminectomy condition creates quantifiable fibrosis of the spinal nerve to surrounding structures and supports the conclusion that this fibrosis may play a role in the post-laminectomy pain syndrome.

High-molecular-weight hyaluronan inhibits macrophage proliferation and cytokine release in the early wound of a preclinical postlaminectomy rat model.

BACKGROUND CONTEXT: Failed back syndrome, a condition that affects 3-14% of postoperative spine patients, is characterized by the recurrence of radicular pain after spinal decompression. The source of this pain in some patients is thought by many investigators to be the result of epidural scarring and nerve root tethering, but this is controversial. We have previously demonstrated that in a disc-injury model the untreated postlaminectomy rats develop a significant proliferative fibrous response at 8 weeks with spinal nerve scarring to the disc and adjacent pedicle, and increased sensitivity to tactile allodynia testing in the related sensory dermatome. Topical high-molecular-weight hyaluronan (HMW HA) moderates both the proliferative fibrosis and the behavioral pain response. PURPOSE: Our purpose is to study the time-related changes in the proinflammatory cytokine and monocyte/macrophage profiles in the epidural space in the early postlaminectomy untreated and HMW HA gel treated groups. STUDY DESIGN/SETTING: A modified rat laminectomy with disc injury model was employed to assess epidural fibrosis between and around the spinal nerves using a quantitative immunohistochemistry assessment approach along with correlative enzyme-linked immunosorbent assay analysis. METHODS: Lumbar laminectomies at L5 and L6 with a L5-L6 disc injury were performed on 120 adult male Sprague-Dawley rats. The rats were then randomized into one of two groups: untreated and treated. The treatment group received a one-time topical application of 0.1 cc of HMW HA gel directly to the laminectomy site just before wound closure. The rats were then randomly subdivided into survival periods of 24 hours, 72 hours, and 7 days. Immunohistochemistry was performed on fresh frozen sections and stained for interleukin-1 beta (IL-1beta) and monocytes/macrophages (ED-1) using monoclonal antibodies and 3, 3' diaminobenzidine (DAB) chromogen. The amount of stain in each specimen was then quantified using the National Institutes of Health computer imaging analysis system. RESULTS: The semiquantified data from the histological specimens demonstrated significant decreases in the IL-1beta and IL-6 infiltration observed at 24 hours in the epidural space and around the right nerve root (p=.0296 and 0.0195, respectively) in the HA gel treated group. Additionally, significant decreases in the monocyte/macrophage infiltration were observed at 72 hours in the epidural space around the left nerve root (p=.0039) and right nerve root (p=.0072) in the HA gel treated group. At 7 days, IL-1beta, IL-6, and macrophage infiltration of the wound had declined in both the HA gel and the untreated groups. The enzyme-linked immunosorbent assay data support the same pattern as seen in the histological results. CONCLUSION: These results demonstrate that treatment of postlaminectomy wounds with HMW HA gel decreases the number of monocytes and macrophages and the concentration of certain cytokines in the early inflammatory phase of healing. There are several plausible explanations for this effect. First, the HMW HA may block the interaction of short-chain low-molecular-weight HA with proinflammatory cell surface receptors. The interaction of these short-chain oligo-HA fragments, upon cell-surface receptor binding, induces changes in inflammatory cells that lead to increased cell motility and migration into the wound area. Second, the addition of exogenous HMW HA may cause a dilution effect in the wound, thereby decreasing the concentration of inflammatory cells in the extracellular matrix of the region of injury. Finally, the migration of inflammatory cells may be decreased in the viscous environment of the HMW HA. The first explanation is believed by the authors of this paper to be the more likely mechanism. HMW HA probably mutes the proinflammatory effects of the low-molecular weight fragments, leading to decreased inflammation, and thus decreased fibrosis and scar formation noted in the chronic model.

Topical high molecular weight hyaluronan reduces radicular pain post laminectomy in a rat model.

BACKGROUND CONTEXT: A controversy exists about the mechanism of causation of the post-laminectomy pain syndrome. Some believe that epidural scarring, and attendant spinal nerve and nerve root scarring and tethering to the disc or pedicle at the site of surgery contributes to post-laminectomy pain in such patients. However, clinical outcome studies on this question are inconclusive and the assertion remains controversial. Definitive studies to help resolve the question are needed. Previously our laboratory has reported on a preclinical post-laminectomy model that mimics the postoperative proliferative fibrotic response grossly, as well as by biochemical assessment of the collagen content within the spinal canal. The post-laminectomy fibrotic response was attenuated in that study by application of a topical antifibrotic (high molecular weight hyaluronan gel) or by insertion of an absorbable roofing barrier (0.2-mm-thick Macropore sheet material) over the laminectomy defect before wound closure. The question remains of relevance of the attenuation of the fibrotic response to post-laminectomy chronic pain syndromes. PURPOSE: The purpose of this study is to evaluate the effect of therapeutic attenuation of proliferative scar within the spinal canal post laminectomy on the pain-related behavioral response in a preclinical rat model. STUDY DESIGN/SETTING: An established L5-L6 rat laminectomy model with a unilateral L5-6 disc injury was employed to assess postoperative proliferative fibrosis of the L5 spinal nerves using quantitative biochemical hydroxyproline assessment of the collagen content in four experimental groups. These observations were correlated with gross descriptions of spinal nerve scarring or tethering. Associated manifestations of a sensory pain-related response in the L5 spinal nerve receptor area of the hind paws was studied using standard tactile allodynia assessment with the von Frey hair technique. The tactile allodynia findings were supplemented by weekly descriptors of behavioral pain manifestations. METHODS: Bilateral laminectomies at L5 and L6 and a unilateral right disc injury (L5-6) were performed on 35 male adult Sprague-Dawley rats, weighing 400+ grams (approved by the VA Institutional Animal Care Use Committee). The study consisted of four groups: 1) normal nonoperative control; 2) a sham-operated group; 3) an untreated laminectomy-disc injury group; and 4) a laminectomy-disc injury treatment group in which 0.1 cc topical high molecular weight hyaluronan (HMW HA) gel was layered over the dura and into the laminectomy canal before closure. Before animals were entered into the study, they were checked for the presence of abnormal response to the tactile testing procedure of the L5 sensory receptor area. Animals exhibiting anomalous responses were excluded from the study. Behavioral testing for tactile allodynia was performed at weekly intervals post laminectomy beginning at 3 weeks. Pain-related behavior was characterized at weekly intervals. A behavioral test cage with a wire mesh floor allowed for tactile allodynia testing. Graduated von Frey hairs whose stiffness increased logarithmically from 0.41 to 15 g were used for tactile allodynia tests. The animals were killed 8 weeks postoperatively for analysis. The dissected spinal nerve and nerve root specimens were studied biochemically for hydroxyproline content to estimate total collagen in and around the L5 neural structures. Statistical analyses were performed using analysis of variance and a Fisher comparison t test. RESULTS: The major observations on the untreated preclinical post-laminectomy rat model previously described by this laboratory were confirmed. All untreated animals developed a tail contracture concave toward the right (disc injury side) consistent with asymmetrical lumbar muscle spasm. Only one animal in the HA gel treatment group had a tail contracture. It was of mild degree and occurred in an animal that demonstrated slightly increased right L5 tactile sensitivity. Gross inspection of the dissected specimens demonstrated spinal nerve scarring and tethering to the disc and pedicle greater on the right than the left in untreated animals, findings that were markedly reduced in the treatment group. Collagen content of the L5 spinal nerve and nerve roots with attached scar were significantly lower in the HA gel treatment group than in the untreated laminectomy group (p=.0014). Pain behavioral testing of the L5 receptor area of the right hind paw in the untreated laminectomy group showed markedly increased sensitivity to tactile allodynia testing compared with the corresponding limb of the control group (p=.0001), to the corresponding limb of the sham group (p=.0001), and compared with the HMW HA gel treatment group (p=.0010). Comparisons of the pain behavioral data between the sham and the post-laminectomy HA gel treatment group and the control animals lacked statistical significance. CONCLUSION: This study supports the concept of a relationship between perineural fibrosis and radicular neuropathy in the model described, and emphasizes the role of disc injury and spinal nerve retraction in the post-laminectomy fibrotic process. Furthermore, it shows promise for preliminary assessment of interventions with other anti-inflammatory agents, for characterization of the neurochemical profile of the post-laminectomy pain state, and for exploration of newer pharmaceutical agents potentially useful in the prevention or management of the post-laminectomy syndrome. Post-laminectomy scar is but one of many potential causes of the post-laminectomy pain syndrome. Furthermore, a cautionary note must be emphasized as in all studies using preclinical models, conclusions drawn from the studies cannot be extended directly to patients without confirmatory clinical follow-up studies.

Topical high-molecular-weight hyaluronan and a roofing barrier sheet equally inhibit postlaminectomy fibrosis.

BACKGROUND CONTEXT: The relevance of epidural fibrosis to failed back surgical outcomes remains controversial. Previous studies on the correlation between epidural fibrosis and clinical outcome after laminectomy are inconclusive, and clinical approaches applied to reduce postlaminectomy spinal canal scarring have produced mixed outcomes. PURPOSE: Improved preclinical models are required to address the fundamental question of the relationship between postlaminectomy fibrosis and chronic pain. This study is directed at establishing small animal postlaminectomy models characterized by significantly reduced scar within the spinal canal postoperatively. Such preclinical models are offered as a platform for future studies to explore the potential relationship between postlaminectomy epidural fibrosis and persistent neuropathy with its potential for altered spinal mechanisms for pain processing, so-called spinal facilitation. Such experiments could be constructed in these models for comparison of pain behavior and its underlying neurochemistry both in the presence and absence of extensive postlaminectomy epidural scar. STUDY DESIGN/SETTING: A modified rat laminectomy model was employed to assess epidural fibrosis using a quantitative biochemical collagen assessment approach along with correlative histology. This group served as the control for comparison with groups in which antifibrotic measures were employed. We compared antifibrotic efficacy of a bioabsorbable roofing barrier sheet placed over the laminectomy defect with topical high-molecular-weight hyaluronan (HMW HA) gel, each applied postoperatively to prevent proliferative epidural scarring. Routine biomechanical tensile strength testing was employed to assess wound-healing strength. METHODS: A bilateral laminectomy (L5 and L6) with associated unilateral disc injury (L5-L6) was performed in 98 male Harlan Sprague-Dawley rats. The laminectomy models described incorporated a unilateral disc injury at L5-L6 because herniated disc material has been shown to contribute proinflammatory cytokines in the postoperative wound. Five groups were employed for the study: 1) normal controls without surgery; 2) a laminectomy-disc injury group without treatment; 3) a laminectomy-disc injury group treated with topical HMW HA gel; 4) a laminectomy-disc injury group treated with 0.2-mm thick bioabsorbable roofing barrier sheet in which a protected space was maintained between overlying paraspinous muscles and the dura and 5) a 0.02-mm thin barrier sheet treatment group in which the sheet was placed directly on the dura. The animals were sacrificed at 3- and 8-week postoperative intervals for analysis. The dissected specimens were studied biochemically for hydroxyproline content to estimate total collagen within the canal and on the dura between L4 and L7. Additional specimens were prepared histologically and stained with Masson-Goldner Trichrome stain to confirm presence of proliferative collagen and to describe the presence or absence of wound-healing scar adherence to the dura. The surgical incisions were studied biomechanically by uniaxial tensile testing to determine ultimate force, strain and prefailure stiffness. Statistics were performed using analysis of variance. RESULTS: Gross appearance and histology studies showed that the untreated laminectomy group demonstrated postoperative scar formation that is adherent between the wound and the dorsum of the dura mater in both 3- and 8-week groups. Proliferative scar was substantially increased grossly between the 3- and 8-week intervals. By gross observation there was adherence of the L5 spinal nerve to the underlying disc and adjacent pedicle on the disc injury side. Gross observation of treatment groups, in contrast, disclosed that both the 0.2-mm thick roofing barrier sheet and topical HMW HA gel each prevented scar attachment to the dural sleeve at both the 3- and 8-week postoperative intervals. Furthermore, both the HMW HA gel and 0.2-mm thick roofing barrier sheet treatment groups had significant reduction of total collagen content in the laminectomy specimens measured biochemically at the two time periods compared with the untreated controls. Histologically, the HMW HA gel and the 0.2-mm thick barrier sheet findings were consistent with the gross observations concerning lack of adherence between scar of the overlying wound and the dura. Notably, both the 0.2- and the 0.02-mm barrier sheets became enveloped by a fibrotic envelope consistent with a foreign body reaction. In the group in which the 0.02-mm thin sheet was placed within the canal on top of the dura, there was an increase of fibrosis around the sheet within the canal leading to a space-occupying mass within the canal. Although the 0.2-mm thick roofing barrier placed external to the canal became enveloped by scar, it appeared to attract proliferative scar away from the epidural space, leaving the dura relatively free of scarring or adherence to overlying tissues. The mechanical properties of the incisional wound increased significantly between 3 and 8 weeks. The ultimate strength, stress, strain and stiffness of the several groups were similar at each time point. CONCLUSION: These results provide two preclinical rat laminectomy models of potential usefulness for the future study of the relevance of epidural fibrosis to behaviorally defined pain states, and for the study of the potential of an altered neurochemical signature in postlaminectomy pain conditions. Such preclinical models have become standard in studies of pain behavior and its neurochemistry in preclinical sciatic nerve and spinal nerve injury models, and should be of utility in the studies of postlaminectomy fibrosis. There was progressive scar proliferation and maturation in the untreated postlaminectomy group in the postoperative interval between 3 and 8 weeks. HMW HA gel applied topically and a 0.2-mm thick bioabsorbable Macropore sheet used as a roofing barrier each significantly reduced postlaminectomy proliferative scar without affecting the integrity of incisional wound healing. However, if the 0.02-mm thin barrier sheet used in this study is placed within the canal in contact with the dura and adjacent to the pedicles, the process of reabsorption results in a fibrotic mass within the canal. The preferred barrier sheet placement for this model is clearly in a roofing position bridging over the open epidural space. It must be placed in a manner to block off the paraspinous muscle healing response and still leave a gap between the sheet and the dura.

A preclinical post laminectomy rat model mimics the human post laminectomy syndrome.

Chronic low back pain with sciatica complicating post laminectomy surgery is poorly understood. It is likely that some aspects of persistent pain of the syndrome results from spinal facilitation in which there is lowering of pain excitation levels. A small animal preclinical model is needed that mimics the clinical condition to permit detailed studies of the underlying altered neurochemistry of the sensory pathways. We propose herein a rat laminectomy model containing the elements required for study of the neurobiology of the condition. The model consists of a surgical laminectomy that includes L5 spinal nerve manipulation and disc injury, elements necessarily employed in human disc herniation surgery. At 8 weeks post laminectomy the proposed model demonstrates paraspinous muscle spasm, tail contracture, behavioral pain behavior, tactile allodynia, epidural and nerve root scarring, and nerve root adherence by scar to the underlying disc and adjacent pedicle. Two underlying pain facilitation states are invoked in the clinical condition: (1) an inflammatory state required to achieve wound healing; and (2) a nerve injury state resulting from nerve manipulation and subsequent epidural scarring, spinal nerve scarring, and spinal nerve tethering to the adjacent disc and pedicle. Both pain facilitation states are active in the model.

Outcomes in open tibia fractures: relationship between delay in treatment and infection.

BACKGROUND: Emergent irrigation and debridement has been accepted as a mainstay of open fracture treatment. The purpose of this study was to evaluate the infectious outcome of open tibia fractures relative to the time from injury to operative irrigation and debridement. METHODS: One hundred seventy-eight patients with 191 consecutive fractures were retrospectively reviewed. Of these, 103 patients with 106 fractures were available for this study, with an average follow-up of 10.23 months. RESULTS: Results revealed 21.7% type I fractures, 43.4% type II fractures, 16.0% type IIIa fractures, 11.3% type IIIb fractures, and 7.5% type IIIc fractures. Of all fracture types, 22.6% became infected and 5.7% went on to have osteomyelitis. The average time to treatment was not significantly different in infected versus noninfected fractures across fracture types. No infection occurred when the time to surgery was within 2 hours; however, no significant increase in infection was discovered with respect to patients treated after 6 hours compared with those treated within 6 hours. CONCLUSION: The results support the Gustilo grading system of open fractures as a significant prognostic indicator for infectious complication. We continue to support the emergent treatment of open tibia fractures.

Adhesion of perichondrial cells to a polylactic acid scaffold.

The number of chondrogenic cells available locally is an important factor in the repair process for cartilage defects. Previous studies demonstrated that the number of transplanted rabbit perichondrial cells (PC) remaining in a cartilage defect in vivo, after being carried into the site in a polylactic acid (PLA) scaffold, declined markedly within two days. This study examined the ability of in vitro culture of PC/PLA constructs to enhance subsequent biomechanical stability of the cells and the matrix content in an in vitro screening assay. PC/PLA constructs were analyzed after 1 h, 1 and 2 weeks of culture. The biomechanical adherence of PC to the PLA scaffold was tested by subjecting the PC/PLA constructs to a range of flow velocities (0.25-25 mm/s), spanning the range estimated to occur under conditions of construct insertion in vivo. The adhesion of PC to the PLA carrier was increased significantly by 1 and 2 weeks of incubation, with 25 mm/s flow causing a 57% detachment of cells after 1 h of seeding, but only 7% and 16% after 1 and 2 weeks of culture, respectively (p<0.001). This adherence was associated with marked deposition of glycosaminoglycan and collagen. These findings suggest that pre-incubation of PC-laden PLA scaffolds markedly enhances the stability of the indwelling cells.

Development-associated differences in integrative cartilage repair: roles of biosynthesis and matrix.

A recurring problem in tissue transplantation therapies for articular cartilage defects is the lack of integration between the implant and the host cartilage. Previous studies have shown that in vitro integration between explants of calf cartilage is markedly higher than that between fetal cartilage, despite similarly high levels of deposition of newly synthesized collagen. The aim of this study was to determine if cellular biosynthesis and extracellular matrix each contribute to these development-associated differences in integrative repair in vitro. The approach taken was to examine integration between specific combinations of cartilage explants that were apposed for two weeks. The cartilage matrix showed different propensities for repair, as integration of calf live cartilage to calf devitalized cartilage was greater than that of calf live cartilage to fetal devitalized cartilage. An inhibiting factor appeared to be present in fetal cartilage matrix since guanidine treatment of fetal devitalized cartilage was able to enhance its integration. The difference between integration to living cartilage and integration to devitalized cartilage, for calf and fetal tissue, indicated that the biosynthetic contribution to integration by calf cartilage was greater than the biosynthetic contribution by fetal cartilage. Thus, the increasing level of integration between fetal and fetal cartilage, fetal and calf cartilage, and calf and calf cartilage appeared to reflect both biosynthetic and matrix differences. Therapeutic strategies to enhance integration to cartilage may thus target both the extracellular components and the cellular biosynthetic activities of implants and host cartilage.