Strain-induced strengthening of bioprosthetic tissue in physiologically super-saturated calcium phosphate solution
Hmiel, Corey Dean
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It is important to develop bioprosthetic devices that can replace damaged collagen-based connective tissues such as periodontal ligaments and joint tendons that show variable degrees of mineralization from their rigid hard-tissue insertions to their flexible central zones. Tanned bovine pericardium, when utilized in implanted bioprosthetic heart valves, has been found to slowly acquire such distributed bone-like calcium hydroxyapatite deposits often causing failure due to an increase in rigidity. Amassing predominantly at regions of maximum strain, it has been shown that this process can be significantly accelerated using super-saturated calcium phosphate solutions in laboratory apparatus. This investigation examined the potential strengthening of tanned pericardial tissue strips when strained in room-temperature calcium phosphate solution, at 30X physiological concentration, as a first step toward fabrication of bone-bonding bioprosthetic ligaments and tendons. Preliminary trials explored the mineral deposition utility of oscillating (1–50Hz) versus constant strain, over variable (1–100 hours) times, as well as differential stress conditions for dog bone-shaped versus rectangular samples. Application of 10% strain for 4 hour periods was known to yield suitable positive strength changes when applied to 2mm-wide tanned pericardium strips immersed in the calcium phosphate solution. For these selected conditions, twenty-five main experiments were done employing adjacent cut strips of tanned pericardium in sets of three for each trial: one strip as-received, prepared for surgical use; a second strip, placed into the solution container surrounding the tensile test grips, as a solution-exposure-only Control; and the third strip held at 10% strain for 4 hours at room temperature in the mineralization solution. Specimens of each type were examined by Multiple Attenuated Internal Reflection Infrared (MAIR-IR) Spectroscopy for evidence of phosphate mineral uptake, by Scanning Electron Microscopy and Energy Dispersive x-ray (SEM/EDS) analysis for presence and location of possible calcium and phosphate deposits, by confocal Infrared Microscopy (IM) to visualize tissue fibrillar orientation with regard to the tensile force direction, and by conventional x-ray imaging to assess possible increase in internal densification of the mineralization solution-incubated tissues. Conventional tensile testing of all specimens was performed, producing statistically insignificant results indicating only modest preferential strengthening of the strain-held samples over the as-received and static immersion controls: Ultimate stress values exhibited approximately a 10% increase following a JAW solution exposure couple with tensile loading. Average ultimate load-to-failure values were as follows; for as-received 2mm pericardium strips = 6.92N ±3.58N; for solution Control pericardium = 7.03N ±3.46N; for 10% strained, solution-exposed pericardium = 7.64 ±3.25N. MAIR-IR revealed small amounts of preferential phosphate uptake into the strain-held tissue specimens, making it unlikely that their strength increase was due to calcium ion cross-linking alone. SEM/EDS showed scattered superficial deposits of Calcium- and Phosphorus-rich nodules, with no evidence for periodicity or localization to regions of maximum strain. Microscopy showed disorganized fibrous lamellae not well-oriented into the tensile direction, suggesting that more completely oriented collagenous substructures might obtain even greater strength increases. Sample subgroups of possibly different orientations did show statistically significant strength increases. Standard x-ray imaging did not reveal preferable tissue densification in any case, indicating that added calcium phosphate deposits were mostly superficial.