Influence Of Osmotic Loading On Organ Culture Model For Rabbit Intervertebral Disc

Objective To develop an organ culture model of rabbit intervertebral discs in vitro and investigate effects of osmotic loading on cell viability and metabolism of intervertebral discs.Methods Thoracolumbar and lumbar intervertebral discs were harvested after sacrifice from 10 New Zealand white rabbits (4～6 months old, n =9/animal) which were divided into two groups. The intervertebral discs were maintained in organ culture with iso-osmotic (300 mOsm/kg, iso-osmotic group) or hyperosmotic(410 mOsm/kg, hyperosmotic group) media. Cell viability, structural integrity and proteoglycan content were assessed using Mitotracker Green probe, histochemical and biochemical methods before culture and on 7,14,21,28 days after culture.Result The fluorescence intensity of fresh intervertebral disc was the maximum(11503±402). During organ culture in vitro,there was a significant decrease in fluorescence in the hyperosmotic group on 14 days(9202±907)(P<0.05), the fluorescence intensity of hyperosmotic group on 7,21and 28 days were 10504±710,10860±711 and 10713±953. The fluorescence yield on 7,14,21and 28 days of iso-osmotic group were 11350±351,11207±385,10914±300 and 10862±229, and there was no significant difference compared to fresh intervertebral disc(P>0.05). There was no significant change in fluorescence yield on 7,21and 28 days between two groups(P>0.05). During organ culture, the nucleus pulposus and anulus fibrosus maintained the structural characteristics of the intact tissue on the whole. There was a significant decrease of proteoglycans in both hyperosmotic hyperosmotic group(3.33±.028) and iso-osmotic group (2.83±0.25) after 7 days culture time compared to fresh intervertebral disc (5.03±0.37) (P<0.01). There was no additional loss of proteoglycans was detected on 14,21 and 28 days than on day 7(P>0.05). No significant difference in proteoglycan content was found between tow groups on the same period (P>0.05).Conclusion Organ culture model of intervertebral discs could preserve organ integrity and cell viability at least 4 weeks under iso-osmotic or hyperosmotic loading.