A copper sulfate and hydroxylysine treatment regimen for enhancing collagen cross‐linking and biomechanical properties in engineered neocartilage

Abstract

The objective of this study was to improve the biomechanical properties of engineered neotissues through promoting the development of collagen cross-links. It was hypothesized that supplementing medium with copper sulfate and the amino acid hydroxylysine would enhance the activity of lysyl oxidase enzyme to form collagen cross-links, increasing the strength and integrity of the neotissue. Neocartilage constructs were generated using a scaffoldless, self-assembling process and treated with copper sulfate and hydroxylysine, either alone or in combination, following a 2-factor, full-factorial study design. Following a 6-wk culture period, the biomechanical and biochemical properties of the constructs were measured. Results found copper sulfate to significantly increase pyridinoline (PYR) cross-links in all copper sulfate-containing groups over controls. When copper sulfate and hydroxylysine were combined, the result was synergistic, with a 10-fold increase in PYR content over controls. This increase in PYR cross-links manifested in a 3.3-fold significant increase in the tensile properties of the copper sulfate + hydroxylysine group. In addition, an 123% increase over control values was detected in the copper sulfate group in terms of the aggregate modulus. These data elucidate the role of copper sulfate and hydroxylysine toward improving the biomechanical properties of neotissues through collagen cross-linking enhancement.

Topographic Variations in Biomechanical and Biochemical Properties in the Ankle Joint: An In Vitro Bovine Study Evaluating Native and Engineered Cartilage

“…Different mechanical and biochemical stimuli can be combined to enhance the functional properties of the engineered tissue on par with native tissue values. 23,27,40,41 In this study the native tibial plafond exhibited significantly better properties in comparison with all other locations of the ankle joint. The measured differences of tibial plafond articular cartilage were also mirrored in the biomechanical and biochemical properties of neocartilage constructs generated using tibial plafond cells.…”

“…Next, samples were suspended in 50 mL of 10-mmol/L pyridoxine and 2.4-mmol/L homoarginine and then diluted 5Â in 0.5% heptafluorobutyric acid in 10% acetonitrile. For highperformance liquid chromatography, 10 mL of each sample was used as previously described, 27 by use of pyridinoline standards (Quidel, San Diego, CA), to quantify the cross-link content.…”

Passive Strain-Induced Matrix Synthesis and Organization in Shape-Specific, Cartilaginous Neotissues

“…[46][47][48] Further, treatment of cell-seeded collagen gels with sodium azide, an ATP inhibitor, or cytochalasin D, a cytoskeletal inhibitor, has been found to inhibit such matrix reorganization following mechanical stimulation. 49,50 Previous work has also correlated increased biomechanical properties in engineered neotissues with increased crosslinks, 38,51 suggesting that the greater functional properties in the middle zone of 10-g-loaded constructs are likely related to the increased crosslink content in this region. Such work indicates that cells, in response to a mechanical input, generate intrinsic traction forces to align compact, and crosslink collagen fibers such that they counter the newly loaded environment.…”

“…55,56 Instead, it is now shown that a highly complex and integrated relationship appears to exist that connects not only overall matrix composition, but also its architecture with the functionality of both native and engineered cartilaginous tissues. 34,38,40,[57][58][59] Gaining a better understanding of the intricate relationship between structure and function in engineered tissues should be the focus of future studies to aid in advancing tissue engineering endeavors.…”

“…Pyridinoline (PYR) collagen crosslinks were analyzed and quantified via high-performance liquid chromatography (HPLC) using PYR standards (Quidel, San Diego, CA), as previously described. 38,39 Histology and immunohistochemistry HistoPrep (Fisher Chemical, Vernon Hills, IL) was used to freeze samples, which were sectioned at 12 mm. Slides were then fixed in formalin before being stained with Safranin O/Fast Green to examine GAG distribution and with Picrosirius Red for total collagen content.…”