The main obstacle for tissue engineering is the difficulty in producing structurally and functionally well-organized tissues from in vitro cultured cells. Thus, on one hand the research is focusing towards bioactive three-dimensional materials (scaffolds) able to stimulate specific cellular processes. In fact the problem exists that cells cultured in scaffolds have great difficulty to adhere and proliferate if they don’t recognize bioactive molecules. In this respect biological polymers are used in the preparation of synthetic matrices to be used as tissue engineering scaffolds. On the other hand biological research is focusing on morphological and functional properties of cells seeded onto bioactive materials to evaluate their viability, adherence and proliferation, fundamental steps for successful tissue engineering. Surgical specimens were treated with type Ia collagenase and cultured in FCS/EGF supplemented DMEM. Cellular characterization was carried out on 3rd passage cells. Fibroblasts were seeded on Matri-cell, a substrate rich in basal lamina constituents, or PVA-gelatin sponges. Pulmonar ovine fibroblasts were also employed to set up the experimental procedures of cell seeding on scaffolds and histological methods. Immunocytochemistry was carried out to evaluate the presence of cytokeratin, fibroblast antigen, S-100 protein, TGF-beta1, fibronectin, type I collagen. Cytochemistry allowed to examine the synthesis of glysosoaminoglycans (Alcian blu method) and glycoproteins (PAS reaction). A fibroblast-like morphology and phenotype were found in the human cells isolated and selected from yellow ligaments. An high expression of fibroblast Ag, fibronectin and type I collagen but low TGF-beta1 and no cytokeratin immunoreaction were observed. A different localization of the detected antigens was found in the isolated fibroblasts depending on the type of substrate: a strong immunoreactive network of collagen I fibres was observed around cells grown on Matri-cell compared to the granular immunoprecipitates observed in the cytoplasm of fibroblasts grown on non coated plastic. Fibronectin was detected mainly at the extracellular level in Matri-cell cultured fibroblasts. Fibroblasts seeded on PVA-gelatin are viable and adherents to the substrates. Alcian blue reaction demonstrated the active production of glysosoaminoglycans both in Matri-cell or PVA-gelatin cultured cells, suggesting that these substrates allow extracellular matrix molecule production
Based on the concepts of White and Panjabi of 1990 we have classified vertebral instabilities into congenital and acquired. The congenital instabilities are due to evident bone alterations that bring about mechanical instability, such as in spondylolisthesis and bone defects of formation and segmentation, or are caused by alteration of the elastic stability of the spinal column, such as in ligamental luxation of the Ehlers-Danlos syndrome or neuromuscular asthenia. Acquired vertebral instabilities include the extensive and much discussed issue of degenerative instabilities and secondary instabilities associated with rheumatoid arthritis, traumatic pathology, neoplastic, iatrogenic instabilities, etc. The spine is a complex structure in elastic equilibrium between functional demand and the physiological resistance of the motor segments, that is the articulations, capsules and ligaments, and the muscles. Like Aulisa and Vinciguerra (1994) we are inclined to refer to stable and unstable equilibrium of the spine and to distinguish “mechanical” stability from “biological” stability. There are authoritative cases of evident macro-instability where the functional units, even though affected by serious mechanical alteration, are able to conserve a totally asymptomatic vertebral column in a state of elastic compensation for a long time. We have classified our cases according to Christian Pfirrmann’s classification of lumbar intervertebral disc degeneration (2001), completing it with the three types of disc degeneration that Modic suggested in 1998. We present our case histories from 2001 to 2002 of macro-instabilities of lumbo-sacral spine treated with stabilisation, PLIF and fusion and of micro-instabilities treated with dynamic stabilisation in neutralisation without fusion. In instabilities when one or more motor segments do not respond to permanent stress and the discs begin to change structurally and demonstrate phenomena of fissuring and dehydration, to the point of assured degeneration and collapse, we have developed a two-fold method of treatment:
MICRO-INSTABILITY: when the degenerative phenomena are still in progress and TAC, RMN and functional radiographs can identify an early phase, we propose dynamic stabilisation in neutralisation in order to restore the height of the disc and cancel the disc-radicular conflict, thus maintaining the capacity of movement of the functional unit. MACRO-INSTABILITY: when the clinical examination and imaging study show late-stage degenerative instability with collapse of the disc space and insufficiency of level with evident somatic traction spurs and reactive sclerosis of vertebral plates, then we believe that today only fusion can relieve the painful symptoms.