Posterolateral spinal fusion (PSLSF) in rabbits is a challenging model for bone substitutes because the transverse processes are extremely thin and the space to be filled with bone is greater than critical and meiopragic in terms of vascularity.

Several investigators have shown beneficial effects of PRP in bone and soft-tissue healing processes. However, controversial results have been reported in clinical setting analysing the effectiveness of PRP. Aim of the present study was to test the effectiveness of PRP in experimental model of PLSF in rabbits.

Aims: Aims of this study was to perform a quantitatively evaluation of newly formed bone, vascular density (VD) and their correlation in animal model of posterolateral spinal fusion based on skeletal stem cells (SSCs) combined with a coral.

Methods: 15 rabbits received cell-material constructs, 15 rabbits were sham-operated (decortication of transverse apophyses), 15 rabbits received material alone. After 6 months the animals were sacrified. We performed a semi-quantitative and quantitative histologycal analysis of the fusion mass. To assess the VD, sections of the fusion mass were immunolabelled for alpha-smooth muscle actin as a vascular marker.

Results: No complete fusion was observed in all groups and no bone was formed in the interapophyseal region. Aboundant newly formed bone was observed in the peri-apophyseal regions in 60% of cases. The quantitative analysis showed a significantly higher amount of bone and VD in animals treated with cells and/or biomaterial alone compared to sham (p< 0.05). Periapophyseal VD and new bone formation was significantly higher compared to interapophyseal region in all groups (p< 0.05). Positive correlation exist between newly formed bone and vascolar density (p = 0,0009).

Conclusions: Interapophyseal region is scarcely vascolarized. The study shows a positive correlation between VD and osteogenesis. The inadequacy of staminal cells could be related with the poor survival after the implant. For the use of stam cells in the APL are necessary more studies in order to clarify the survival and in situ differentiation of the grafted cells in short and mid term.

Posterolateral spinal fusion is considered one of the most challenging condition for bone graft substitutes since using autogenous bone graft pseudarthrosis have been reported in 30% of cases.

MATERIALS AND METHODS.We develop a model of posterolateral spinal fusion in the rabbit based on skeletal stem cells (SSCs) loaded into a coral-hydroxyapatite material (Pro-Osteon 500RTM). 15 rabbits received cell-material constructs, 15 rabbits were sham-operated (decortication of transverse apophyses), 15 rabbits received material alone. The animals were housed for 6 months and radiographically monitored. At sacrifice, the explanted spine was analyzed by conventional and high resolution (Faxitron) radiography, and the outcome judged, blind of histology results, by two orthopedic surgeons.

RESULTS: radiographic evaluation showed a fusion rate rate of 90% in animals treated with cell constructs or biomaterial alone, and no fusion in the sham controls. Histology revealed abundant new bone formation directly on the scaffold in the cell construct and biomaterial alone groups, but no evidence of bone formation in the midregion of the interapophyseal space, where poorly vascular, dense fibrous tissue was observed.

CONCLUSIONS: The study shows that:

1) the cell-biomaterial constructs which per se were highly efficient in previous animal studies, used in different absolute quantities but identical ratios were not efficient in the direct preclinical model.

After the embryonic period, notochord remnants persist inside the intervertebral disc (IVD), where they give rise to the nucleus pulposus. Notochordal cells (NTCs) gradually disappear during maturation. This phenomenon is correlated with onset of disc degeneration. The objective of this study was to design a protocol for the isolation of NTCs to study his role in IVD regeneration.

Lumbar IVDs from immature rats were either enzymatically dissociated or mechanically taken out or cells isolation. Cells RNA extraction for PCR analysis was performed to assay Sonic and Indian Hedgehog (Ihh and Shh) and his receptor Patched (Ptc) expression.

NTCs were readily detectable in culture as large vacuolated “physalipherous” cells, with the enzymatic method. The cells isolated mechanically were enable to grow in monolayer while grown 2 weeks in a 3-D pellet culture. Ihh and Ptc was expressed in the cells isolated with both method, while Shh was expressed only in the cells isolated through the mechanical method.

Our findings show that the better way to isolate a pure population of NTCs is a mechanical extraction from a immature IVD. This is a first step in order to study his role for the regeneration of IVD.