Introduction: Adipose tissue has been known for some time to contain regenerative cells. These regenerative cells are able to differentiate into a nucleus pulposus-like phenotype when exposed to environmental factors similar to disc. In an effort to develop a clinical option for cell placement and assess the response of the cells to the post-surgical milieu, adipose-derived cells were collected, concentrated, and transplanted under fluoroscopic guidance directly into a surgically damaged disc in a dog model.

Methods: After IACUC approval 12 dogs, 2 years of age, were obtained. Adipose cells were harvested from the super-scapular region of the neck (scruff) and adherent cells separated, collected, and labeled with DAPI. Three lumbar intervertebral disc levels in each dog underwent a partial nucleotomy; other levels served as non-operated controls. Levels of intervention as well as the regimen of treatment were dually randomized. Three interventions were used in this study; adipose-derived cells in hyaluronic acid (HA) carrier, HA alone, or no intervention. All deliveries were guided by fluoroscopy. The dogs were radiographed, received MRI scans and then euthanized by 12 months. The disc tissue was harvested from the lumbar spine in each dog. Cells labeled with DAPI prior to implantation were evident in the tissue. Matrix composition was assessed for aggrecan, Types I and II collagen by both RT-PCR (Table I) and ELISA (Table II) to assess and compare matrix regeneration. mRNA and protein from each level are presented with respect to normal values defined as the 100 percent expression.

Results: The results can be summarized as:

the regenerative cells are viable following implantation.

Discussion: This study provides evidence that adipose derived cells might offer a reliable source of regenerative potential capable of bio-restitution. The span of this study was sufficient to show that freshly isolated adipose-derived cells can be transplanted percutaneously and will survive the trauma associated with post-surgical inflammation to remain viable and produce appropriate, tissue-specific matrix.

Study Design: Experimental study in dogs:

Objective: To assess the efficiency of disc chondrocyte transplantation in a canine model.

Summary of Background Data: Conventional clinical treatments of intervertebral disc herniation and degeneration are focused on excision of damaged tissue, stabilization, and spinal fusion. The development and refinement of cell-based therapeutics for tissue regeneration and repair have spawned a multitude of applications including autologous disc chondrocyte transplantation. For clinical application the efficiency of disc chondrocyte transplantation was assessed using a pre-clinical canine model to show the technical feasibility and biological relevance for disc repair and retardation of disc degeneration. This report examined the protein expression of transplanted disc chondrocytes and their role in the clinically observed disc repair following autologous disc chondrocyte transplantation.

Methods: The nucleus and inner annulus were sampled from four skeletally-mature dogs by micro-discectomy. Disc chondrocytes were isolated and propagated under GMP validated conditions including completely autologous serum conditions. Two months later, the cultured cells were transplanted through the contralateral side of experimental discs after testing complete healing of the annulus by measuring intradiscal pressure stability. After seven months the animals were humanely killed. One half of the vertically halved lumbar spines were embedded in paraffin and sections were analysed histologically and immunohistochemically.

Results: Histological examinations revealed large clusters of cells within the nucleus area of the treated discs. Cells within these cell clones were found to be viable and surrounded by de novo synthesized matrix as evidenced by a distinct histological staining and immunohistochemical expression pattern. A disc-specific expression of collagen type I and II and hyaline-specific proteoglycans was observed indicating the regenerative and reconstructive capacity of the transplanted disc chondrocytes.

Conclusions: These results indicate the contribution of transplanted disc chondrocytes to the observed clinical success of this cell-based therapy.

Diurnal changes in the loads acting on the spine affect the water content and height of the intervertebral discs. We have reviewed the effects of these changes on spinal mechanics, and their possible clinical significance. Cadaveric lumbar spines subjected to periods of creep loading show a disc height change similar to the physiological change. As a result intervertebral discs bulge more, become stiffer in compression and more flexible in bending. Disc tissue becomes more elastic as its water content falls, and its affinity for water increases. Disc prolapse becomes more difficult. The neural arch and associated ligaments resist an increasing proportion of the compressive and bending stresses acting on the spine. Observations on living people show that these changes are not fully compensated for by modified muscle activity. We conclude that different spinal structures are more heavily loaded at different times of the day. Therefore, the time of onset of symptoms and signs, and any diurnal variation in their severity, may help us understand more about the pathophysiology of low back pain and sciatica.

The pressure distribution between the cartilaginous surfaces in the human hip joint was measured using pressure-sensitive film. Five cadaveric hip joints (aged between 58 and 87 years) were measured at three positions and loads, representing three instants in the stance phase of the walking cycle. The pressure distribution was not uniform, indicating that the cartilage did not, to any great extent, distribute the applied load. The maximum pressures recorded were about 10 MN/m2. The anterosuperior surface of the cartilage was identified as an area of high pressure.

Cadaveric lumbar discs were injected with chymopapain and subjected to a series of mechanical tests over a period of up to 19 hours. Discs from the same spine injected with saline were used as controls. The results showed that chymopapain had no measurable effect on the mechanical properties of the disc apart from the increased height and stiffening caused by fluid injection. Another series of tests on isolated pieces of disc material showed that chymopapain could reduce the size of prolapsed nuclear material by 24% in one hour and by 80% in 48 hours. It is concluded that, in the short-term, chymopapain has a negligible effect on the mechanics of a disc but it can reduce the size of any prolapsed nuclear material with which it comes in contact.

One hundred and thirty-nine discs from cadaveric lumbar spines were injected with a mixture of radio-opaque fluid and dye. Discograms were taken and the discs were then sectioned in the sagittal plane. Examination of the sections revealed that injected fluid did not at first mix with the disc matrix but pushed it aside to form pools of injected fluid. The location of these pools, and hence the appearance of a discogram, depended on the stage of degeneration of the disc. It is concluded that useful clinical information can be obtained from discograms.

A series of experiments showing how posture affects the lumbar spine is reviewed. Postures which flatten (that is, flex) the lumbar spine are compared with those that preserve the lumbar lordosis. Our review shows that flexed postures have several advantages: flexion improves the transport of metabolites in the intervertebral discs, reduces the stresses on the apophyseal joints and on the posterior half of the annulus fibrosus, and gives the spine a high compressive strength. Flexion also has disadvantages: it increases the stress on the anterior annulus and increases the hydrostatic pressure in the nucleus pulposus at low load levels. The disadvantages are not of much significance and we conclude that it is mechanically and nutritionally advantageous to flatten the lumbar spine when sitting and when lifting heavy weights.

Cadaveric lumbar spine specimens of "motion segments", each including two vertebrae and the linking disc and facet joints, were compressed. The pressure across the facet joints was measured using interposed pressure-recording paper. This was repeated for 12 pairs of facet joints at four angles of posture and with three different disc heights. The results were that pressure between the facets increased significantly with narrowing of the disc space and with increasing angles of extension. Extra-articular impingement was found to be caused, or worsened, by disc space narrowing. Increased pressure or impingement may be a source of pain in patients with reduced disc spaces.

Forty-one cadaveric lumbar intervertebral joints from 18 spines were flexed and fatigue loaded to simulate a vigorous day's activity. The joints were then bisected and the discs examined. Twenty-three out of 41 of the discs showed distortions in the lamellae of the annulus fibrosus and, in a few of these, complete radial fissures were found in the posterior annulus.

Specimens of femoral cortical bone from normal subjects and from patients with osteoporosis were mechanically tested in tension to destruction. The osteoporotic bone showed less strength and less stiffness than the normal bone; these reductions are related to the increased cavity area in osteoporosis. Further, the osteoporotic bone is not able to absorb as much energy before fracture as the normal bone; but this difference is not related to changes in cavity area.

A casting method for measuring the intra-articular space in the loaded hip is described. The results of tests on 22 hips from 22 cadavers show that the joint space is variable both in location and size and can disappear under light loads. It seems likely that the size and shape of the space influence how much access synovial fluid has for lubrication and nutrition.

Cadaveric lumbar intervertebral joints were loaded to simulate the erect standing posture (lordosis), and the erect sitting posture (slightly flexed). The results show that, after the intervertebral disc has been reduced in height by a period of sustained loading, the apophysial joints resist about 16 per cent of the intervertebral compressive forces in the erect standing posture, whereas in the erect sitting posture they resist none. The implications of this in relationship to degenerative changes and to low backache are discussed.

A method is described whereby fractures of the neural arch similar to those in spondylolysis are produced experimentally. The forces, bending moments and displacements required to initiate the fractures are given; The mechanical aspects in the aetiology of spondylolysis are explained by a simplified two-dimensional force analysis.