Human Hypertrophic Cartilage Matrix for bone regeneration: evaluation using Micro-CT

Objectives: The osteogenic potential of soluble factors derived from human hypertrophic cartilage matrix (HCCM) was evaluated by means of micro-radiographic computerised tomography (µ -CT) and a newly developed bone algorithm. Methods: HHCM was derived from immortalized human hypertrophic chondrocytes grown in culture; conditioned growth medium was clarified by centrifugation and concentrated via 3,000 MWCO PES membranes. Three groups of ten adult male Sprague Dawley rats were anaesthetised (Isoflurane; N₂O/O₂) and a full thickness skin flap raised to expose the skull; using an 8mm trephine a defect was created across the midline of the calvarium. Defects were filled with 25µl HCCM on an 8mm collagen* disc (Gp1); 25µl of growth medium on a collagen disc (Gp2), or left untreated (control group). Five animals per group were sacrificed at 14 and 28 days using a schedule one method, specimens were dissected and fixed in formalin. Volume of new bone formation was assessed using µ-CT** and image processing software 3-D Calc*** (Bone specific algorithm). Results: Mean bone volume within defects at two weeks was Gp1: 6.46mm³; Gp2 3.74mm³ Control: 4.38 mm³ and at 4 weeks Gp1: 11.03 mm³; Gp2: 8.78 mm³; Control: 6.77 mm³. There was an increase in bone volume with time and more bone formed in the HCCM treated group than in group2 or untreated controls; however the differences were not statistically significant at either time interval (Kruskal-Wallis one-way ANOVA). Conclusions: Treatment of critical sized calvarial defects with HHCM derived factors resulted in a trend of increased bone formation at both time points when compared with controls. The use of µ-CT is an efficient means of assessing bone volumes in small experimental animal models of bone healing. *Helistat, Integra Lifesciences, Plainsboro, New Jersey US **SkyScan, Aartselaar, Belgium ***Collaborative project: SkyScan, Erasmus University Rotterdam and RUCA University Antwerp