Sustaining Composition in Nucleus Pulposus Allograft during Allograft Tissue Processing

INTRODUCTION
Current treatments for disc degeneration focus on attaining symptomatic relief without addressing underlying disc pathophysiology such as tissue loss. As a strategy for replacing tissue lost due to degenerative change, a process for morselizing nucleus pulposus allograft (NP) was developed with a goal of supplementing tissue loss associated with chronic disc disruption linked with discogenic back pain.
AIMS/OBJECTIVES
These data characterize and compare NP allograft with native nucleus pulposus tissue following mechanical processing. To achieve a potential for percutaneous delivery in clinical care, it is critical to reduce the native tissue to a particulate without compromising essential facets of the composition such as proteoglycans, collagens, or water sorption.
METHODS
Intervertebral discs were sourced from thoracic (T)12 to lumbar (L)5 spine segments of cadaveric donors. Native tissue samples were excised using a scalpel or scissors, and then cut into ~1.5 cm3 pieces. Processed NP tissue samples were lyophilized and processed via cryomill to particulate to attain particles less than 250µm prior to terminal sterilization. Processed NP tissue particulate was obtained from 40 donors (n=20 females and n=20 males) and divided equally into each of the following four age groups: 15-25, 26-35, 36-45, 46-55 years old GAG content and swelling capacity were assessed. Alcian blue and Safranin O staining was used to assess proteoglycan distribution within tissue. Proteoglycan content was assessed in ug/mg dry weight of tissue, and water sorption calculated by weight following rehydration according to Kohn, et al. 2015. GAG content was assessed with a commercial dimethylmethylene blue assay per manufacturer’s instructions (Chondrex, 6022). Water sorption calculated by weight following rehydration as noted. Histochemical assessment was made by light microscopy. All quantitative data were analyzed using GraphPad Prism (Version 9). Data from the same donor were paired. Differences between groups were determined using a one-way ANOVA with Tukey post hoc analysis. Statistical significance was indicated by p-value < 0.05 between groups..
RESULTS
Both native NP tissue and NP particulate stained positively for Alcian blue and Safranin-O indicating proteoglycan-rich tissue. There were no significant differences between native NP tissue and NP particulate for GAG content. The average GAG content of 40 donors was 666.1±156.9 µg/mg tissue and was similar between females (663.7±139.0 µg/mg) and males (668.6±176.6 µg/mg). Data were analyzed to determine if sex and age had significant effects on GAG content. There was no significant effect of sex on GAG content (p = 0.9062), but as the average GAG content was lower as donor age increased, post hoc tests revealed that statistical significance could be observed for male donors (Figure 1). There was no significant interaction between sex and age (p = 0.4324) and water sorption followed GAG content.
CONCLUSIONS
Accepting that the loss of nucleus pulposus tissue has been linked to progressive degeneration, these data demonstrate that processing NP allograft tissue does not diminish any contribution attending the tissue with regard to proteoglycan content or water sorption. As it retains its ability to swell, tissue supplementation might contribute to an effective tissue supplement for disc tissue loss.