97. Gene expression changes with short- and long-term cannabinoid treatments in a spinal fusion model

BACKGROUND CONTEXT The rising opioid epidemic is a public health crisis at the crossroads of orthopedic care and pain management. Medical marijuana is a potential non-opioid analgesic yet to be studied in the surgical setting and its effects on fracture repair are not fully understood. In addition to the analgesic effects of cannabinoids, studies have demonstrated potentially osteo-inductive properties, albeit controversial at the molecular level. Osteoblasts, osteoclasts and bone peripheral nerve terminals have been shown to have endocannabinoid receptors (CB1, CB2), comparably expressed to that of brain tissue. PURPOSE This study aims to understand the molecular tissue and cell response to the administration of tetrahydrocannabinol (THC) and cannabidiol (CBD) compounds in a spinal fusion model. STUDY DESIGN/SETTING Basic science animal model. OUTCOME MEASURES Quantitative PCR (qPCR) analysis. METHODS Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). 40 adult Sprague-Dawley rats were used for this study. Posterolateral lumbar spinal fusion was performed at the L4-L5 level utilizing allogenic bone graft. Four rats were used as donors for allografts, while 36 rats were divided among the 4 treatment groups. Treatment groups consisted of Saline control, 5mg/kg THC, 5mg/kg CBD, and 10mg/kg CBD+THC. Treatment was delivered through weekly 0.1 ml intraperitoneal injections. Newly formed bone and callus tissue were harvested 2- and 8-weeks postsurgery for assessment. qPCR analysis was performed to quantify changes in expression of osteogenic and cannabis receptor genes. Shapiro-Wilk normality test was performed. Based on the normality, One-way ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's multiple comparisons tests were performed using GraphPad Prism. Two-tailed values of p<0.05 were considered statistically significant. RESULTS qPCR data at 2 weeks indicated downregulated RANKL/OPG ratios skewing towards osteogenesis in the CBD and CBD+THC groups, when compared to Saline, with the THC group demonstrating a downward trend, though not reaching significance. ALPL, BMP4, SOST, CTNNB1 were all significantly higher in the CBD group compared to Saline, with CTNNB1 also showing an upregulating trend in the THC and CBD+THC groups. Data at 8 weeks shows no change in expression of CTNNB1, MMP13, Col1A1. Cannabis receptor CB1 was unchanged, while CB2 and GPR55 were upregulated for all treatment groups. TRPV1 receptor was downregulated for CBD and CBD+THC groups. Osteoblast activity markers ALPL and RUNX2 at 8 weeks were downregulated for all treatment groups, while SOST was downregulated for CBD and THC compared to Saline. In the CBD+THC group RANK, RANKL and OPG are downregulated compared to Saline. OPG was generally downregulated, but only significant for the CBD+THC group. Interestingly, RANKL/OPG ratio showed upregulation in the CBD and CBD+THC groups. RANKL showed upregulation in the CBD and THC groups. CONCLUSIONS Osteogenesis factors were upregulated (ALPL, BMP4, B-catenin pathway) in the cannabinoid treated groups at 2 weeks, which indicates potential bone regeneration enhancement. The RANKL/OPG ratio as an indicator for the metabolic state of bone showed downregulation at 2 weeks and upregulation at 8 weeks. Coincidentally, in the physiological setting, the ratio is lower in the early phases and increases at the later remodeling phases of healing and, thus, cannabinoids show enhanced effects. Overall, cannabinoids did not indicate any negative side effects on bone healing. On the contrary, there is the potential to enhance physiologic healing. FDA Device/Drug Status This abstract does not discuss or include any applicable devices or drugs. The rising opioid epidemic is a public health crisis at the crossroads of orthopedic care and pain management. Medical marijuana is a potential non-opioid analgesic yet to be studied in the surgical setting and its effects on fracture repair are not fully understood. In addition to the analgesic effects of cannabinoids, studies have demonstrated potentially osteo-inductive properties, albeit controversial at the molecular level. Osteoblasts, osteoclasts and bone peripheral nerve terminals have been shown to have endocannabinoid receptors (CB1, CB2), comparably expressed to that of brain tissue. This study aims to understand the molecular tissue and cell response to the administration of tetrahydrocannabinol (THC) and cannabidiol (CBD) compounds in a spinal fusion model. Basic science animal model. Quantitative PCR (qPCR) analysis. Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). 40 adult Sprague-Dawley rats were used for this study. Posterolateral lumbar spinal fusion was performed at the L4-L5 level utilizing allogenic bone graft. Four rats were used as donors for allografts, while 36 rats were divided among the 4 treatment groups. Treatment groups consisted of Saline control, 5mg/kg THC, 5mg/kg CBD, and 10mg/kg CBD+THC. Treatment was delivered through weekly 0.1 ml intraperitoneal injections. Newly formed bone and callus tissue were harvested 2- and 8-weeks postsurgery for assessment. qPCR analysis was performed to quantify changes in expression of osteogenic and cannabis receptor genes. Shapiro-Wilk normality test was performed. Based on the normality, One-way ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's multiple comparisons tests were performed using GraphPad Prism. Two-tailed values of p<0.05 were considered statistically significant. qPCR data at 2 weeks indicated downregulated RANKL/OPG ratios skewing towards osteogenesis in the CBD and CBD+THC groups, when compared to Saline, with the THC group demonstrating a downward trend, though not reaching significance. ALPL, BMP4, SOST, CTNNB1 were all significantly higher in the CBD group compared to Saline, with CTNNB1 also showing an upregulating trend in the THC and CBD+THC groups. Data at 8 weeks shows no change in expression of CTNNB1, MMP13, Col1A1. Cannabis receptor CB1 was unchanged, while CB2 and GPR55 were upregulated for all treatment groups. TRPV1 receptor was downregulated for CBD and CBD+THC groups. Osteoblast activity markers ALPL and RUNX2 at 8 weeks were downregulated for all treatment groups, while SOST was downregulated for CBD and THC compared to Saline. In the CBD+THC group RANK, RANKL and OPG are downregulated compared to Saline. OPG was generally downregulated, but only significant for the CBD+THC group. Interestingly, RANKL/OPG ratio showed upregulation in the CBD and CBD+THC groups. RANKL showed upregulation in the CBD and THC groups. Osteogenesis factors were upregulated (ALPL, BMP4, B-catenin pathway) in the cannabinoid treated groups at 2 weeks, which indicates potential bone regeneration enhancement. The RANKL/OPG ratio as an indicator for the metabolic state of bone showed downregulation at 2 weeks and upregulation at 8 weeks. Coincidentally, in the physiological setting, the ratio is lower in the early phases and increases at the later remodeling phases of healing and, thus, cannabinoids show enhanced effects. Overall, cannabinoids did not indicate any negative side effects on bone healing. On the contrary, there is the potential to enhance physiologic healing.