Timothy M. Ganey

Disc chondrocyte transplantation in a canine model: a treatment for degenerated or damaged intervertebral disc.

Study Design. Disc degeneration and osteoarthritis are diseases of the matrix. Chondrocytes that have been removed from damaged cartilaginous tissues maintain a capacity to proliferate, produce, and secrete matrix components, and respond to physical stimuli such as dynamic loading. A dog model was used to investigate the hypothesis that autologous disc chondrocytes can be used to repair damaged intervertebral disc. Objectives. Given the capacity for the cells in vitro to produce matrix molecules that would be appropriate for disc chondrocytes, the focus of the experiment was to investigate whether the cells would continue to sustain metabolic function after transplantation. Summary of the Background Data. No evidence for long-term integration exists for cell transplantation in species other than rats and rabbits. Furthermore, no controlled studies of 1-year duration have been published. Materials and Methods. Disc chondrocytes were harvested and expanded in culture under controlled and defined conditions, returned to the same animals from which they had been sampled (autologous transplantation) via percutaneous delivery. The animals were analyzed at specific times after transplantation by several methods to examine whether disc chondrocytes integrated with the surrounding tissue, produced the appropriate intervertebral disc extracellular matrix, and might provide a formative solution to disc repair. Results. In the context of degenerative changes in an injury model: (1) autologous disc chondrocytes were expanded in culture and returned to the disc by a minimally invasive procedure after 12 weeks; (2) disc chondrocytes remained viable after transplantation as shown by Bromodeoxyuridine incorporation and maintained a capacity for proliferation after transplantation as depicted by histology; (3) transplanted disc chondrocytes produced an extracellular matrix that displayed composition similar to normal intervertebral disc tissue. Positive evidence of proteoglycan content was supported by accepted histochemical staining techniques such as Safranin O-Fast Green; (4) both type II and type I collagens were demonstrated in the regenerated intervertebral disc matrix by immunohistochemistry after chondrocyte transplantation; and (5) when the disc heights were analyzed for variance according to treatment, a statistically significantcorrelation between transplanting cells and retention of disc height was achieved. Conclusions. Autologous chondrocyte transplantation is technically feasible and biologically relevant to repairing disc damage and retarding disc degeneration.

Intervertebral Disc Repair Using Adipose Tissue-Derived Stem and Regenerative Cells: Experiments in a Canine Model

Study Design. Therapeutic treatment of intervertebral disc repair using cells. Objective. The goal of the study was to test the hypothesis that repair of a damaged disc is possible using autologous adipose tissue derived stem and regenerative cells (ADRCs). Summary of Background Data. Degradation resulting from either acute or chronic repetitive disc injury leads to disc degeneration. However, if a damaged disc could be repaired in the early stages, before the onslaught of degradation, then the disc degeneration process may be slowed down. Methods. Twelve dogs underwent a partial nucleotomy at 3 lumbar levels (L3–L4, L4–L5, and L5–L6); adjacent levels served as nonoperated controls. The animals (or discs) were allowed to recover from the surgery for 6 weeks. At that time subcutaneous adipose tissue was harvested and ADRCs were isolated. The 3 experimental discs that had undergone a partial nucleotomy were randomized to receive: (1) ADRCs in hyaluronic acid carrier (Cells/HA); (2) HA only; or (3) No Intervention. Assessments of the 3 experimental discs plus the 2 adjacent untouched discs were made using MRI, radiography, histology, and biochemistry. The animals were killed at 6 months and at 12 months. Results. Repair in this study was specifically demonstrated through histology and biochemical analysis. Disc levels receiving ADRCs more closely resembled the healthy controls as evidenced in matrix translucency, compartmentalization of the anulus, and in cell density within the nucleus pulposus. Matrix analysis for Type-II collagen and aggrecan demonstrated evidence of a statistically better regenerative stimulation to the disc provided by ADRCs when compared to either the HA only or no intervention treatments. Conclusion. Autologous adipose tissue derived stem and regenerative cells, as used in this disc injury model, were effective in promoting disc regeneration, as evidenced by disc matrix production and overall disc morphology.

Does long-term compressive loading on the intervertebral disc cause degeneration?

Study Design. Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs of dogs for up to 53 weeks. Objective. To test the hypothesis that compressive forces applied to the intervertebral disc for a long period of time cause disc degeneration in vivo in a dog model. Summary of Background Data. It is a commonly held belief that high forces applied to the intervertebral disc, and to joints in general, play a role in causing degeneration. Methods. Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs (L3/L4) of 12 dogs. After up to a year, the dogs were killed, and their lumbar spines were removed and radiographed. The L3/L4 disc and the controls (T13/L1 and L4/L5) were excised and examined for visible signs of degeneration. The discs then were assessed using immunohistochemical analysis and enzyme-linked immunosorbent assay. Disc chondrocytes also were assayed for apoptosis. Results. No obvious signs of degeneration in the discs (L3/L4) that had been under compression for up to a year could be observed. There was no disc bulging, anular fissures, or disc space narrowing. Some changes were observed at the microscopic level, although no thickening of the endplate was apparent. The enzyme-linked immunosorbent assay analysis provided significant data for all three regions of the disc (nucleus, inner anulus, and outer anulus). When comparing the compressed disc (L3/L4) with either of the control discs (T13/L1 and L4/L5), in the compressed disc: 1) the nucleus contained less proteoglycan and more collagen I and II; 2) the inner anulus contained less proteoglycan and collagen I; and 3) the outer anulus contained more proteoglycan and less collagen I. The collagen II differences for the inner and outer anulus were not significant. Conclusion. Compression applied to the lumbar intervertebral discs of dogs for up to a year does not produce degeneration in any visible form. It does produce microscopic changes and numerical changes, however, in the amounts of proteoglycan and collagen in the nucleus, inner anulus, and outer anulus. The present results add no credence to the commonly held belief that high compressive forces play a causative role in disc degeneration.

Sever's injury: a stress fracture of the immature calcaneal metaphysis.

Magnetic resonance imaging (MRI) in children with a presumptive diagnosis of Sever’s apophysitis and with continuing pain after conservative treatment demonstrated bone bruising within the trabecular bone of the metaphyseal region adjacent to the calcaneal apophysis. Limited portions of the apophyseal secondary ossification center showed similar increased signal changes. MRI studies following treatment with immobilization showed subsidence or disappearance of the metaphyseal but not any apophyseal signal changes commensurate with improvement in symptoms. Accordingly, the disorder commonly referred to as Sever’s “apophysitis” may be a metaphyseal trabecular stress fracture, similar to the toddler’s calcaneal stress fracture that has minimal or no involvement of the apophyseal ossification center, and thus should not be referred to as an apophysitis. Rather, it appears to be an overuse injury causing microinjury within the developing metaphyseal “equivalent” trabecular bone that has not completely adapted to the changing biologic (biomechanical) requirements of the growing, athletically active child.

Contiguous discitis and osteomyelitis in children.

Magnetic resonance imaging of 16 patients with contiguous discitis and osteomyelitis provided a specific diagnosis and defined the anatomic extent of vertebral and soft-tissue involvement. Altered signal changes were evident in the disc, adjacent vertebra in the end plate and metaphyseal equivalent regions, and the anterior prevertebral tissues. Significant posterior spread and disc herniation were not evident. Fourteen patients had lumbar involvement; two had cervical involvement. The patients were followed-up for an average of 4 years 5 months. Scoliosis has developed in one patient, and four continue to have a loss of lumbar lordosis. By comparing serial roentgenograms, the mean decrease of disc-space height after the acute episode was 43% (range, 51-61%). There was no restitution of normal disc-space height at the latest follow-up roentgenogram in any of the patients. A 14% (average) narrowing of the vertebral foramina was evident in seven cases. In one patient, a fusion progressively developed 7 years after the acute episode (before full skeletal maturity). However, several patients appear to be progressing toward fusion of adjacent vertebra. A study of histologic specimens elucidated vascular anatomy of the immature vertebra that further explain the disease characteristics.

Assessment of bone formation during osteoneogenesis: A canine model☆☆☆★

Distraction osteoneogenesis, callotasis, has been demonstrated to be an effective means of lengthening long bones. A variation of Ilizarovs technique produces a transport disk from one cut surface of bone within a defect and advances the disk to the opposite surface to close the defect. This process, previously described by Costantino et al. (Arch Otolaryngol Head Neck Surg 1990; 116:535-45), demonstrated bone formation within the distraction site. The precise mechanism of bone formation has not yet been described for the mandible. Four conditioned beagles were studied, with one control dog maintained in neutral fixation and three dogs distracted at 0.25 mm every 8 hours. A two-cm defect was closed, and dogs were kept in fixation for 1 week after closure, after which they were killed. Three sites were evaluated: (1) the distraction seam, (2) the interface of the cortical and distracted bone, and (3) the cortexes at the closed defect. Each site was bisected, and one half was decalcified for immunohistochemical and hematoxylin and eosin pathologic evaluation. The vascular basement membrane was labeled for laminin and type IV collagen. Both of these substances demonstrate the differentiation of the vascular matrix component predisposing primary bone formation. Labels were intense at the distraction seam where intense angiogenesis occurred. No hyalin cartilage was observed at the distraction site, which indicates that the fixation was stable and that ossification occurred primarily without intermediate callous formation. This model demonstrated that osteoclasts within the canine model produce bone through primary bone formation within an angiogenic matrix rich in basement membrane laminin and type IV collagen. Likewise, bone is species specific in mineral composition for dog mandible. Understanding the formation and composition of distracted bone is essential for understanding application of this technique within the clinical setting.

Basement membrane of blood vessels during distraction osteogenesis.

A canine model of distraction osteogenesis has recently been developed that permitted the evaluation of bone formation and its vascularization during bifocal callotasis. In this model, the authors examined the composition of the blood vessels during distraction osteogenesis of the mandible for laminin and for Type IV collagen, both constituents of the vascular basement membrane. At the fibrous distraction site, at the juncture of the free cortical surface and the regenerated bone, and at the abutting cortical surfaces at the distal margin of the defect, laminin and Type IV collagen were present in all vessels.

Postnatal development of the human sternum.

Postnatal development and maturation of the human sternum are highly variable. Endochondral ossification centers (sternebrae) form within each cartilaginous segment of the sternum, with each center enveloped by a spherical growth plate. Within a cartilaginous center there may be either one or two ossification centers, those with two centers retaining and reflecting features of their bilateral embryonic origin. Malaligned bifid centers are clearly associated with rib articulation asymmetry as well. Expansion of individual ossification centers progresses within the peripheral cartilaginous domains of the sternum. With respect to the rostrocaudal axis, sternebrae form between the costosternal articulations. Consistent with the biology of endochondral transition, cartilage canals are evident throughout unossified regions of the hyaline matrix. Expanding ossification of adjacent sternebrae results in depletion of the common area of cartilage between the two sternebrae, and eventually in physiologic epiphysiodesis. Fusion of the mesosternebrae reciprocates the initial pattern of sternebral ossification site appearance, proceeding in a caudal-to-cranial direction. Union of adjacent sternebrae, initiated through a central osseous bridge, progresses through anterior, lateral, cephalocaudal, and posterior domains to achieve synostosis. Accessory and bifid centers of ossification within the same intercostal space coalesce prior to adjoining adjacent sternebrae. Manubriosternal fusion is rare due to the presence of a fibrocartilaginous joint restricting ossification. The xiphoid process remains connected to the most caudal mesosternum via a common zone of hyaline cartilage that ossifies by middle to late adulthood. A single pattern of development does not appear fundamental to successful growth of the sternum, as morphological variants were common.

Osseous overgrowth after amputation in adolescents and children.

We retrospectively studied the incidence of primary surgical revision for stump overgrowth in a population of childhood and adolescent amputees. The anatomic location and the etiology of amputation are critical to the occurrence of overgrowth needing revision. Metaphyseal-level amputations are the most likely to develop overgrowth requiring revision (50%), whereas diaphyseal amputations are slightly less likely (45%). Joint disarticulations never develop overgrowth. Traumatic amputations are the most frequent mode of injury requiring revision of overgrowth (43%), followed by congenital or intrauterine amputations (30%) and elective amputations (20%). Radiographic classification of the osseous overgrowth helps define its severity and degree of ossific progression. Surgical revisions are usually performed when overgrowth reaches a grade 3 classification. The majority of skeletally immature diaphyseal- or metaphyseal-level amputees, including those with certain preexisting orthopaedic conditions, retain the ability to develop osseous overgrowth at the apex of the stump skeleton.

Pathologic morphology of the dislocated proximal femur in children with cerebral palsy.

We describe the gross and microscopic anatomic changes in the hip that result from the deforming forces in children with neuromuscular imbalance. Twelve dislocated proximal femora that had been resected from children with spastic diplegia or tetraplegia were evaluated with respect to their gross, microscopic, and radiographic structure. The epiphyses were wedge shaped with deformation of the femoral head apparent in all cases. In addition to a severe loss of articular cartilage, a furrowed erosion of epiphyseal bone suggested a sustained, blunt, band-like force across the surface of the hip where it opposed the acetabular labrum. The underlying physis of the capital femur was irregular with aberrant histologic structure, whereas that of the lesser trochanter was hypertrophic and angulated in a superior and anterior direction. A significant degree of valgus was not noticeable in most specimens. In summary, the spastic adductor and iliopsoas, responsible for the changes in the lesser trochanter, work in conjunction with the hip flexor and internal rotator muscles to subluxate the proximal femur. In the process, the superior rim of the acetabulum and capsule causes focal deformation of the superolateral femoral head, creating a fulcrum upon which the hip then progressively subluxates. The indentation locks the femoral head at the lateral acetabular margin, preventing complete dislocation, but leading to bone pain consequent to cartilage erosion.