Joon Y. Lee

Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice

Background: Cells that express bone morphogenetic protein-2 (BMP-2) can now be prepared by transduction with adenovirus containing BMP-2 cDNA. Skeletal muscle tissue contains cells that differentiate into osteoblasts on stimulation with BMP-2. The objectives of this study were to prepare BMP-2-expressing muscle-derived cells by transduction of these cells with an adenovirus containing BMP-2 cDNA and to determine whether the BMP-2-expressing muscle-derived cells would elicit the healing of critical-sized bone defects in mice. Methods: Primary cultures of muscle-derived cells from a normal male mouse were transduced with adenovirus encoding the recombinant human BMP-2 gene (adBMP-2). These cells (5 ¥ 105) were implanted into a 5-mm-diameter critical-sized skull defect in female SCID (severe combined immunodeficiency strain) mice with use of a collagen sponge as a scaffold. Healing in the treatment and control groups was examined grossly and histologically at two and four weeks. Implanted cells were identified in vivo with use of the Y-chromosome-specific fluorescent in situ hybridization (FISH) technique, and their differentiation into osteogenic cells was demonstrated by osteocalcin immunohistochemistry. Results: Skull defects treated with muscle cells that had been genetically engineered to express BMP-2 had >85% closure within two weeks and 95% to 100% closure within four weeks. Control groups in which the defect was not treated (group 1), treated with collagen only (group 2), or treated with collagen and muscle cells without adBMP-2 (group 3) showed at most 30% to 40% closure of the defect by four weeks, and the majority of the skull defects in those groups showed no healing. Analysis of injected cells in group 4, with the Y-chromosome-specific FISH technique showed that the majority of the transplanted cells were located on the surfaces of the newly formed bone, but a small fraction (approximately 5%) was identified within the osteocyte lacunae of the new bone. Implanted cells found in the new bone stained immunohistochemically for osteocalcin, indicating that they had differentiated in vivo into osteogenic cells. Conclusions: This study demonstrates that cells derived from muscle tissue that have been genetically engineered to express BMP-2 elicit the healing of critical-sized skull defects in mice. The cells derived from muscle tissue appear to enhance bone-healing by differentiating into osteoblasts in vivo. Clinical Relevance: Ex vivo gene therapy with muscle-derived cells that have been genetically engineered to express BMP-2 may be used to treat nonhealing bone defects. In addition, muscle-derived cells appear to include stem cells, which are easily obtained with muscle biopsy and could be used in gene therapy to deliver BMP-2.

Evidence for skeletal progenitor cells in the degenerate human intervertebral disc.

Study Design. To identify and characterize endogenous progenitor cell population from intervertebral disc. Objective. To determine if progenitor cells exist in degenerate human discs. Summary of Background Data. Back pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disc. Because disc disease is accompanied by a loss of cellularity, there is considerable interest in regeneration of cells of both the anulus fibrosus (AF) and nucleus pulposus (NP). Methods. To determine if skeletal progenitor cells are present in the disc, samples were obtained from the degenerate AF and NP of 5 patients (Thompson grade 2 and 3, mean age 34 ± 7.6 years) undergoing anterior cervical discectomy and fusion procedures as well as adult rat lumbar spine. Results. Cells isolated from degenerate human tissues expressed CD105, CD166, CD63, CD49a, CD90, CD73, p75 low affinity nerve growth factor receptor, and CD133/1, proteins that are characteristic of marrow mesenchymal stem cells. In osteogenic media, there was an induction of alkaline phosphatase activity and expression of alkaline phosphatase, osteocalcin, and Runx-2 mRNA. When maintained in adipogenic media, a small percentage of cells displayed evidence of adipogenic differentiation: accumulation of cytosolic lipid droplets and increased expression of peroxisome proliferator-activated receptor-&ggr;2 and lipoporotein lipase mRNA. AF- and NP-derived cells also evidenced chondrogenic differentiation. CD133 (+) cells in the AF were able to commit to either the chondrogenic or adipogenic lineages. The results of the human disc studies were confirmed using cell derived from the NP and AF tissue of the mature rat disc. Conclusion. The analytical data indicated that the pathologically degenerate human disc contained populations of skeletal progenitor cells. These findings suggest that these endogenous progenitors may be used to orchestrate the repair of the intervertebral disc.

Postoperative management protocol for incidental dural tears during degenerative lumbar spine surgery: a review of 3,183 consecutive degenerative lumbar cases.

Study Design. Retrospective review. Objectives. To review the experience of a single institution with degenerative lumbar surgery and to identify cases of incidental dural tears (DTs); and to report on the efficacy of our postoperative management protocol for DT, which relies on early mobilization. Summary of Background Data. DTs are a common complication of degenerative lumbar spine surgery. However, the management strategies for this complication vary from one surgeon to another. Methods. A total of 3,183 degenerative lumbar spine cases (decompression and/or fusion) were reviewed. Cases complicated by an incidental DT were identified. Patients who required a return to the operating room for a second procedure were identified and the operative findings were reviewed. Results. The incidence of DT during primary lumbar surgeries was 7.6% (153 of 2,024 cases), which compares to an incidence of 15.9% for revision cases (185 of 1,159 cases). If recognized during the index procedure, all DTs were repaired using a 4-0 silk suture. Six patients (4 primary, 2 revisions) who did not improve despite our postoperative management protocol were taken to the operating room for irrigation and debridement, repair of the defect, and placement of a subfascial drain to closed suction. All 6 patients went on to do well and did not have any further complications. Conclusions. DTs are common during degenerative lumbar spine surgery. Revision surgery is twice as likely as primary surgery to result in this complication. Our postoperative early mobilization protocol appears to be an effective and safe management strategy for treating this complication (98.2% success rate). Very few patients (6 of 338, or 1.8%) needed a reoperation.

Enhancement of Bone Healing Based on Ex Vivo Gene Therapy Using Human Muscle-Derived Cells Expressing Bone Morphogenetic Protein 2

Molecular biological advances have allowed the use of gene therapy in a clinical setting. In addition, numerous reports have indicated the existence of inducible osteoprogenitor cells in skeletal muscle. Because of this, we hypothesized that skeletal muscle cells might be ideal vehicles for delivery of bone-inductive factors. Using ex vivo gene transfer methods, we genetically engineered freshly isolated human skeletal muscle cells with adenovirus and retrovirus to express human bone morphogenetic protein 2 (BMP-2). These cells were then implanted into nonhealing bone defects (skull defects) in severe combined immune deficiency (SCID) mice. The closure of the defect was monitored grossly and histologically. Mice that received BMP-2-producing human muscle-derived cells experienced a full closure of the defect by 4 to 8 weeks posttransplantation. Remodeling of the newly formed bone was evident histologically during the 4- to 8-week period. When analyzed by fluorescence in situ hybridization, a small fraction of the transplanted human muscle-derived cells was found within the newly formed bone, where osteocytes normally reside. These results indicate that genetically engineered human muscle-derived cells enhance bone healing primarily by delivering BMP-2, while a small fraction of the cells seems to differentiate into osteogenic cells.

Nucleus pulposus replacement: basic science and indications for clinical use.

Study Design. A critical review of available and emerging nucleus pulposus replacement implants. Objectives. To review the biomechanics, design, and clinical data of currently available and developing nucleus pulposus replacement technologies. Summary of Background Data. The interest in minimally invasive treatment of degenerative disc disease has grown as the technology for intervertebral motion-sparing devices continues to improve. Replacement of nucleus pulposus without anular obliteration represents a tempting alternative to spinal fusion procedures. The aim in nucleus pulposus replacement is to slow adjacent level degeneration, restore normal loads to the diseased level, and restore segmental spinal biomechanics. Methods. A literature review of currently available biomaterials, biomechanics, and available preclinical and clinical data on nucleus pulposus replacement implants. Results. New synthetic biomaterials have recently been developed to closely mimic native biomechanics during compressive loading cycles of the intervertebral disc. This, in conjunction with improved understanding of global spine biomechanics, has allowed the development of novel nucleus replacement implants. These implants are currently at different stages of preclinical and clinical investigations. Conclusions. Although some of the newly designed prosthesis have shown some promising results in preclinical studies, rigorous short- and long-term clinical evaluations will be critical in evaluating their true efficacy.

New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering.

Study Design. Human intervertebral disc cells were cultured in a new three-dimensional “pellet culture” system as an alternative to conventional alginate bead microspheres. Histologic, biochemical, and immunohistologic assays were performed to characterize this new culturing method for disc cells. The feasibility of using the pellet culture system to study effects of gene therapy was also assessed. Objectives. To characterize a new and simpler, three-dimensional culture system for human intervertebral disc cells and to assess the feasibility of its use for gene therapy and tissue engineering studies. Summary of Background Data. The alginate microsphere three-dimensional culture system has been the most utilized culture method for disc cells, but it is technically difficult and has some disadvantages. Recently, the “pellet culture” method, a simpler three-dimensional culture system, was described for bone marrow stromal cells. This simpler method might be useful for in vitro and in vivo studies of disc cells as well as for delivery of exogenous genes. Methods. Isolated human intervertebral disc cells were centrifuged at low speeds to form aggregates and allowed to grow as pellets for up to 3 weeks. At various times these pellet cultures were analyzed grossly, histologically, and immunohistologically. Their ability to incorporate [35S]sulfate in their response to TGF-&bgr;1 was also analyzed. The ability of these pellets to deliver and express exogenous genes in vivo was analyzed by implantation of pellet cultures in muscles of SCID mice. Results. Within several days the intervertebral disc cells were able to form mature three-dimensional aggregates that were each well encapsulated by a fibrous capsule. These pellets successfully synthesized proteoglycan and collagen Type II matrix as determined by histology and immunohistochemistry. In response to TGF-&bgr;1, the pellets increased synthesis of proteoglycan and collagen Type II. When implanted into thigh muscles of SCID mice, the pellets remained aggregated and expressed the &bgr;-galactosidase marker gene in vivo for up to 2 weeks. Conclusions. The pellet culture system is a new and technically simple method to culture human intervertebral disc cells. The majority of the human intervertebraldisc cells retained their native phenotype in this threedimensional system and expressed a marker gene both in vitro and in vivo. Thus, this pellet system might be useful for in vitro and in vivo biochemical studies as well as for studies involving gene therapy and tissue engineering.

Ex Vivo Gene Therapy to Produce Bone Using Different Cell Types

Gene therapy and tissue engineering promise to revolutionize orthopaedic surgery. This study comprehensively compares five different cell types in ex vivo gene therapy to produce bone. The cell types include a bone marrow stromal cell line, primary muscle derived cells, primary bone marrow stromal cells, primary articular chondrocytes, and primary fibroblasts. After transduction by an adenovirus encoding for bone morphogenetic protein-2, all of the cell types were capable of secreting bone morphogenetic protein-2. However, the bone marrow stromal cell line and muscle derived cells showed more responsiveness to recombinant human bone morphogenetic protein-2 than did the other cell types. In vivo injection of each of the cell populations transduced to secrete bone morphogenetic protein-2 resulted in bone formation. Radiographic and histologic analyses corroborated the in vitro data regarding bone morphogenetic protein-2 secretion and cellular osteocompetence. This study showed the feasibility of using primary bone marrow stromal cells, primary muscle derived cells, primary articular chondrocytes, primary fibroblasts, and an osteogenesis imperfecta stromal cell line in ex vivo gene therapy to produce bone. The study also showed the advantages and disadvantages inherent in using each cell type.

Reliability of a novel classification system for thoracolumbar injuries: the Thoracolumbar Injury Severity Score.

Study Design. Prospective study of 5 spine surgeons rating 71 clinical cases of thoracolumbar spinal injuries using the Thoracolumbar Injury Severity Score (TLISS) and then re-rating the cases in a different order 1 month later. Objective. To determine the reliability of the TLISS system. Summary of Background Data. The TLISS is a recently introduced classification system for thoracolumbar spinal column injures designed to simplify injury classification and facilitate treatment decision making. Before being widely adopted, the reliability of the TLISS must be studied. Methods. A total of 71 cases of thoracolumbar spinal trauma were distributed on CD-ROM to 5 attending spine surgeons, including clinical/radiographic data, details of the TLISS, and a scoring sheet in which cases would be scored using the system. The surgeons were later assigned the task with the cases reordered. Intraobserver and interobserver reliability was calculated for TLISS components, total score, and surgeons treatment decision using the Cohen unweighted kappa coefficients and Spearman rank-order correlation. Results. Interrater reliability assessed by generalized kappa coefficients was 0.33 ± 0.03 for injury mechanism, 0.91 ± 0.02 for neurologic status, 0.35 ± 0.03 for posterior ligamentous complex status, 0.29 ± 0.02 for TLISS total, and 0.52 ± 0.03 for treatment recommendation. Respective results using the Spearman correlation were 0.35 ± 0.04, 0.94 ± 0.01, 0.48 ± 0.04, 0.65 ± 0.03, and 0.51 ± 0.04. Surgeons agreed with the TLISS recommendation 96.4% of the time. Intrarater kappa coefficients were 0.57 ± 0.04 for injury mechanism, 0.93 ± 0.02 for neurologic status, 0.48 ± 0.04 for posterior ligamentous complex status, 0.46 ± 0.03 for TLISS total, and 0.62 ± 0.04 for treatment recommendation. Respective results using the Spearman correlation were 0.70 ± 0.04, 0.95 ± 0.02, 0.59 ± 0.05, 0.77 ± 0.04, and 0.59 ± 0.05. Conclusions. The TLISS has good reliability and compares favorably to other contemporary thoracolumbar fracture classification systems.

Infection after the surgical treatment of adolescent idiopathic scoliosis: evaluation of the diagnosis, treatment, and impact on clinical outcomes.

Study Design. Retrospective case-control. Objective. The purpose of this study was to evaluate a single surgeons experience with infection after surgical treatment of adolescent idiopathic scoliosis, with a focus on the diagnosis, treatment, and impact on radiographic and patient-reported outcomes. Summary of Background Data. Although previous studies have evaluated this postoperative complication, no studies to date have looked at the impact of this complication on both radiographic and patient-reported outcomes. Methods. From 1986 to 2004, 236 patients were identified who underwent surgical treatment of adolescent idiopathic scoliosis and had at least 2-year follow-up. The medical records of patients who developed infection were retrospectively reviewed in detail. Preoperative and most recent postoperative radiographic parameters and Scoliosis Research Society 24 outcomes of both infected and noninfected patients were compared. Results. Of 236 patients 7 (3%) developed an infection. One was acute (17 days postoperative), and 6 were delayed (average 34.2 months postoperative). The most common presenting complaints included back pain (5 of 7) and localized swelling (4 of 7). All patients with delayed infection were treated with 1 surgery (irrigation and debridement, instrumentation removal) and 6 weeks of intravenous antibiotics. Of 6 patients 3 had pseudarthrosis. Culture results were: Staphylococcus epidermidis (n = 2), Propionibacterium acnes (n = 1), Enteroccocus faecalis (n = 1), Group A Streptococcus (n = 1), no growth (n = 1). The patient with acute infection required 6 surgical procedures and 16 weeks of antibiotics. Cultures were positive for methicillin resistant Staphylococcus aureus and Serratia marscesens. Revision fusion surgery was performed 5 months after the infection was treated. Compared with the noninfected patients, those with infection had lower percent thoracic (P = 0.01) and lumbar (P = 0.06) curve correction. There was no difference in the pain, function, self-image, satisfaction, or total Scoliosis Research Society 24 scores. Conclusion. Postoperative infection after the surgical treatment of idiopathic scoliosis can successfully be treated with irrigation and debridement, instrumentation removal, and a course of antibiotics. Although less curve correction was achieved in the infected group, there were no differences in patient-reported outcomes when compared with the noninfected group.

Surgical Decision Making for Unstable Thoracolumbar Spine Injuries: Results of a Consensus Panel Review by the Spine Trauma Study Group

Objectives: The optimal surgical approach and treatment of unstable thoracolumbar spine injuries are poorly defined owing to a lack of widely accepted level I clinical literature. This lack of evidence-based standards has led to varied practice patterns based on individual surgeon preferences. The purpose of this study was to survey the leaders in the field of spine trauma to define the major characteristics of thoracolumbar injuries that influence their surgical decision making. In the absence of good scientific data, expert consensus opinions may provide surgeons with a practical framework to guide therapy and to conduct future research. Methods: A panel of 22 leading spinal surgeons from 20 level I trauma centers in seven countries met to discuss the indications for surgical approach selection in unstable thoracolumbar injuries. Injuries were presented to the surgeons in a case scenario survey format. Preferred surgical approaches to the clinical scenarios were tabulated and comments weighed. Results: All members of the panel agreed that three independent characteristics of thoracolumbar injuries carry primary importance in surgical decision making: the injury morphology, the neurologic status of the patient, and the integrity of the posterior ligaments. Six clinical scenarios based on the neurologic status of the patient (intact, incomplete, or complete) and on the status of the posterior ligamentous complex (intact or disrupted) were created, and consensus treatment approaches were described. Additional circumstances capable of altering the treatments were acknowledged. Conclusions: Decision making for the surgical treatment of thoracolumbar injuries is largely dependent on three patient characteristics: injury morphology, neurologic status, and posterior ligament integrity. A logical and practical decision-making process based on these characteristics may guide treatment even for the most complicated fracture patterns.