Goodwin, Christian John; (1996) The use of the ESTA bioassay system to optimise the incorporation of recombinant human growth hormon into biomaterials for use in orthopaedic surgery. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The incorporation of a protein such as human growth hormone (hGH) into biomaterials may adversely effect its structural integrity. In this Thesis the delivery of bioactive hGH from a range of biomaterials has been investigated. The bioactivity was measured using the uniquely sensitive and precise MTT-ESTA bioassay system. Hormone damage was further assessed by determining the ratios of bio- to immunoactive hGH released. Only [tilde] 1 [percent] of the total hormone incorporated into a range of methacrylate based bone cements was released in vitro. However, because high hormone loading doses were possible, even these low percentage recoveries provided physiologically sufficient concentrations in the eluates. Release occurred in two phases, with more than 90[percent] being delivered in the first 24hrs followed by a prolonged slow release. Recovery and B:I ratios depended upon the formulation of the bone cements; recoveries were enhanced if combinations of monomers were used; no direct relationship with the water uptake properties of the cements was found. Greater recoveries (e.g. [greater-than]20[percent]) were obtained from a biodegradable material (poly(e- caprolactone)) and systems where the hGH was surface loaded such as porous polymeric scaffolds and hydroxy apatite coatings. The bioassay was adapted to assess the in vitro cytotoxicities of the biomaterials. All of the methacrylate bone cements, but not the biodegradable materials, proved to be cytotoxic. Monomer composition of the former greatly influenced the cytotoxicity. The colorimetric bioassay was also further developed. Use of the newer tetrazolium salts (XTT and MTS) combined with one of several intermediate electron acceptors yielded improved microculture tetrazolium assays (MTAs). Observations made during this optimization challenged common assumptions regarding links between changes in bioreduction of a tetrazolium salt and cell proliferation. In addition, the new MTAs were shown to depend exclusively upon the DT-diaphorase mediated electron-transfer pathway.

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