John R. Matyas

The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering.

An understanding of developmental biology can provide useful insights into how different tissue-engineered repairs might be designed. During embryogenesis of the intervertebral disk, the cells of the notochord play a critical role in initiating tissue formation, and may be responsible for development of the nucleus pulposus. In some species, including humans, these notochordal cells may eventually be lost, either through apoptosis or terminal differentiation, and are replaced by chondrocyte-like cells. However, there is some evidence that the notochordal cells may persist in at least some humans. This review discusses some of the potential applications of notochordal cells in tissue engineering of the nucleus pulposus.

Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison

The nuclei pulposi of the intervertebral discs (IVDs) contain a mixed population of cell types at various stages of maturation. This tissue is formed either by or with the help of cells from the embryonic notochord, which appear to be replaced during development by a population of chondrocyte‐like cells of uncertain origin. However, this transition occurs at widely varying times, depending upon the species – or even breed – of the animal being examined. There is considerable debate among spine researchers as to whether the presence of these residual notochordal cells has a significant impact upon IVD degeneration models, and thus which models may best represent the human condition. The present study examines several different species commonly used in lumbar spine investigations to explore the variability of notochordal cells in the IVD.

Regional variations in the cellular matrix of the annulus fibrosus of the intervertebral disc

The three‐dimensional architecture of cells in the annulus fibrosus was studied by a systematic, histological examination using antibodies to cytoskeletal components, in conjunction with confocal microscopy. Variations in cell shape, arrangement of cellular processes and cytoskeletal architecture were found both within and between the defined zones of the outer and inner annulus. The morphology of three, novel annulus fibrosus cells is described: extended cordlike cells that form an interconnected network at the periphery of the disc; cells with extensive, sinuous processes in the inner region of the annulus fibrosus; and cells with broad, branching processes specific to the interlamellar septae of the outer annulus. The complex, yet seemingly deliberate arrangement of various cell shapes and their processes suggests multiple functional roles. Regional variations in the organization of the actin and vimentin cytoskeletal networks is reported across all regions of the annulus. Most notable is the continuous, strand arrangement of the actin label at the discs periphery in contrast to its punctate appearance in all other regions. The gap junction protein connexin 43 was found within cells from all regions of the annulus, including those which did not form physical connections with surrounding cells. These observations of the cellular matrix in the healthy intervertebral disc should contribute to a better understanding of site‐specific changes in tissue architecture, biochemistry and mechanical properties during degeneration, injury and healing.

Stress governs tissue phenotype at the femoral insertion of the rabbit MCL

The cells in the midsubstance portion of skeletal ligaments typically have elongated shapes, but where ligaments insert into bone the cells appear very rounded and the tissue phenotype is that of fibrocartilage. Between the midsubstance and the insertions there is a gradient in cell shape and tissue phenotype that has been hypothesized to reflect a gradient of mechanical stresses. To test this hypothesis, cell shapes (an index of tissue phenotype) were quantified in the central part of the femoral insertion of the rabbit medial collateral ligament by computer-assisted histomorphometry. Morphometric measurements were correlated with the mechanical stresses and strains in the central part of the insertion as predicted by finite element analysis. Throughout the ligament the direction of the predicted principal tensile stresses coincides with the direction of the collagen fibers which curve from the midsubstance to meet the femur at nearly right angles. Principal compressive stresses also occur within the ligament: the highest are localized near the bone; the lowest in the midsubstance. The areas with the roundest cells correspond to the areas with the highest principal compressive stresses in the model; the areas with the flattest cells correspond to the areas with the lowest compressive stresses in the model. A correlation between cell shape and mechanical stresses suggests that physiological loading of the MCL is important for the maintenance of tissue phenotype throughout this insertion. We theorize that the cells in ligament insertions adapt to the prevailing local mechanical environment.

The three-dimensional architecture of the notochordal nucleus pulposus: novel observations on cell structures in the canine intervertebral disc

Cells from the nucleus pulposus of young (< 2 years) and old (> 5 years) non‐chondrodystrophoid dogs were studied using routine histology, confocal laser scanning microscopy and transmission electron microscopy. The architecture of cell structures – from the tissue scale down to subcellular scale – was reported. Clusters of notochordal cells were observed in young nuclei pulposi, ranging from 10 to 426 cells each. These clusters resisted mechanical disruption and showed evidence of cell–cell signalling via gap junctions. Cells (30–40 µm in diameter) within the clusters had a physaliferous appearance, containing numerous large inclusions which ranged from 1 to 20 µm in diameter. The inclusions were surrounded by a dense actin cortex but were not contained by a lipid bilayer. The contents of the inclusions were determined not to be predominantly carbohydrate or neutral lipid as assessed by histochemical staining, but the exact composition of the contents remained uncertain. There were striking differences in the cell architecture of young vs. old nuclei pulposi, with a loss of both cell clusters and physaliferous cells during ageing. These observations demonstrate unique cell structures, which may influence our understanding of the differences between notochordal and chondrocytic cells in the nucleus pulposus. Such differences could have substantial impact upon how we think about development, degeneration and repair of the intervertebral disc.

The effects of increased tension on healing medial collateral ligaments

The effects of motion and increased levels of stress on the biomechanical, biochemical, and morphological properties of healing medial collateral ligaments were assessed in a rabbit model. In one group, the medial collateral ligament of the left hindlimb was transected and allowed to heal with cage activity for either 6 or 12 weeks. In another group, the transected ligaments were permitted to heal for 4 weeks and then were placed under increased stress by inserting a stainless steel pin perpendicularly underneath the healing medial collateral ligament. The animals were allowed cage activity for an additional 2 or 8 weeks. The varus-valgus joint laxity and the stress-strain properties of the medial collateral ligament substance were obtained. Further, the quan tity of total collagen, amount and ratio of the collagen cross-links, dihydroxylysinonorleucine and hydroxyly sinonorleucine, and the histologic appearance of the healing medial collateral ligaments were evaluated for all groups. At 6 weeks, knees with a transected medial collateral ligament were twice as lax as the controls. However, joints with the stainless steel tension pin had varus-valgus values approximately 1.5 times those of the controls. At 12 weeks, joints with increased stress were not statistically different from the controls. The group that had healing with increased stress for 12 weeks produced the highest stress for a given strain compared to any other group. Also, the total collagen levels and the ratio of dihydroxylysinonorleucine/hy droxylysinonorleucine were the closest to normal of any transected group. Finally, qualitative histologic improve ments were seen, including a more longitudinal arrange ment of collagen fibers and decreased cellularity.

Recommendations for the use of preclinical models in the study and treatment of osteoarthritis

yCanadian Arthritis Network, Canada zDepartment of Surgery, McGill University, Montreal, Quebec, Canada xCentocor Pharmaceuticals, Malvern, PA, USA k Ecole Polytechnique, Montreal, Canada {Harvard University, Boston, MA, USA #Universite de Montreal, Montreal, Canada yyConsumer Advisory Council, Canadian Arthritis Network, Canada zzUniversity of Calgary, Calgary, Canada xxMount Sinai Hospital, Toronto, Canada kk Sanofi-Aventis, Frankfurt, FRG, Germany {{University Hospital Nijmegen, Nijmegen, The Netherlands ##University of California, San Diego, USA

ISSLS prize winner: Collagen fibril sliding governs cell mechanics in the anulus fibrosus: an in situ confocal microscopy study of bovine discs.

Study Design. In situ investigation of collagen and cell mechanics in bovine caudal discs using novel techniques of confocal microscopy. Objective. To measure simultaneously the in situ intercellular and collagen matrix mechanics in the inner and outer anulus fibrosus of the intervertebral disc subjected to flexion. Summary of Background Data. Mechanobiology studies, both in vivo and in vitro, clearly demonstrate that mechanical factors can influence the metabolic activity of disc cells, altering the expression of key extracellular matrix molecules. Essential to elucidating the mechanotransduction mechanisms is a detailed understanding of the in situ mechanical environment of disc cells in response to whole-body mechanical loads. Methods. Confocal microscopy was used to simultaneously track and capture in situ images of fluorescently labeled cells and matrix during an applied flexion. The position of the nuclear centroids was calculated before and after applied flexion to quantify the in situ intercellular mechanics of both lamellar and interlamellar cells. The deflection patterns of lines photobleached into the extracellular matrix were used to quantify collagen fibril sliding and collagen fibril strains in situ. Results. The extracellular matrix was observed to deflect nonuniformly due to the relative sliding of the collagen fibrils. Intercellular displacements within the lamellar layers were also nonuniform, both along a cell row andbetween adjacent rows. Within a cell row, the intercellular displacements were small (<1%). Conclusions. The in situ cell mechanics of anular cells was found to be strongly influenced by collagen fibril sliding in the extracellular matrix and could not be inferred directly from applied tissue loads.

The early molecular natural history of experimental osteoarthritis: I. Progressive discoordinate expression of aggrecan and type II procollagen messenger RNA in the articular cartilage of adult animals

OBJECTIVE To quantify changes in the chondrocyte metabolism of aggrecan core protein and type II procollagen messenger RNA (mRNA) during the early and middle phases of experimental osteoarthritis (OA) in animals. METHODS Experimental OA was induced by transecting the cranial cruciate ligament of the stifle joint in adult animals; articular cartilage was harvested and analyzed after 4, 10, and 32 weeks. RESULTS Northern blot analysis revealed no change in aggrecan mRNA 4 weeks after surgery compared with aggrecan mRNA in the unoperated contralateral control joints; aggrecan mRNA levels became significantly elevated by 10 and 32 weeks after surgery. In OA cartilage, type II procollagen mRNA was dramatically and progressively elevated at all times after surgery. The relative increases in type II procollagen mRNA exceeded the relative increases in aggrecan mRNA at all times after surgery, and these differences increased progressively over time. Articular chondrocytes became activated globally (total RNA increases) and specifically (mRNA increase) early after joint injury and remained activated throughout the early and middle phases of this experimental OA. CONCLUSION The early natural history of experimental OA is characterized by a progressive imbalance in the mRNA expression of aggrecan and type II procollagen in articular chondrocytes. These results suggest that the stimuli for the transcription of these 2 genes are fundamentally different in this animal model.

Hindlimb loading, morphology and biochemistry of articular cartilage in the ACL-deficient cat knee

The cat hindlimb is the best studied animal model of neuromuscular control, muscle mechanics/muscle morphology, and locomotor kinematics. Therefore, this model offers itself for intervention studies, where a musculoskeletal parameter is perturbed and the effects of this perturbation are compared with normal function. The objective of this study was to describe the effects of anterior cruciate ligament (ACL) transection in the cat knee and to correlate hindlimb loading with morphological and biochemical changes of articular cartilage. A distinct unloading of the deficient hindlimb was found when compared with the nonoperated hindlimb immediately after ACL transection, and persisted for about 16-18 weeks. Beyond about 18 weeks post-ACL transection, hindlimb loading returned to the symmetric pattern observed before surgery. In accordance with the expectations from the force-platform results, a decrease in muscle mass was found from muscles of the experimental hindlimb when compared to the mass of muscles from the contralateral hindlimb. This decrease of muscle mass was largest at 4 weeks and smallest at 35 weeks post-ACL transection. At 12 and 35 weeks post-ACL transection, cell density was increased and absolute amounts of hexuronic acid were elevated in the articular cartilage of the experimental knee compared with the corresponding values of the contralateral knee. Progressive changes of the articular cartilage towards osteoarthritis (OA) were not observed in the time frame of this experiment. The results suggest that anterior cruciate ligament transection in the cat produces initial changes in the knee that are similar to those observed previously in the dog and rabbit.