564 THE JOURNAL OF BONE AND JOINT SURGERY  RESEARCH The development of fibronectin-functionalised hydroxyapatite coatings to improve dermal fibroblast attachment in vitro C. J. Pendegrass, M. El-Husseiny, G. W. Blunn From University College London, London, United Kingdom  C. J. Pendegrass, PhD, Lecturer  M. El-Husseiny, MD, Student  G. W. Blunn, PhD, Professor The Centre for Biomedical Engineering, University College London, The Royal National Orthopaedic Hospital, Brockley Hill, Stanmore, Middlesex HA7 4LP, UK. Correspondence should be sent to Dr C. Pendegrass; e-mail: c.pendegrass@ucl.ac.uk ©2012 British Editorial Society of Bone and Joint Surgery doi:10.1302/0301-620X.94B4. 27698 $2.00 J Bone Joint Surg Br 2012;94-B:564–9. Received 2 June 2011; Accepted after revision 5 January 2012 The success of long-term transcutaneous implants depends on dermal attachment to prevent downgrowth of the epithelium and infection. Hydroxyapatite (HA) coatings and fibronectin (Fn) have independently been shown to regulate fibroblast activity and improve attachment. In an attempt to enhance this phenomenon we adsorbed Fn onto HA-coated substrates. Our study was designed to test the hypothesis that adsorption of Fn onto HA produces a surface that will increase the attachment of dermal fibroblasts better than HA alone or titanium alloy controls. Iodinated Fn was used to investigate the durability of the protein coating and a bioassay using human dermal fibroblasts was performed to assess the effects of the coating on cell attachment. Cell attachment data were compared with those for HA alone and titanium alloy controls at one, four and 24 hours. Protein attachment peaked within one hour of incubation and the maximum binding efficiency was achieved with an initial droplet of 1000 ng. We showed that after 24 hours one-fifth of the initial Fn coating remained on the substrates, and this resulted in a significant, three-, four-, and sevenfold increase in dermal fibroblast attachment strength compared to uncoated controls at one, four and 24 hours, respectively. Conventional stump–socket prostheses are the treatment of choice for most above-knee ampu- tees; however, a poorly fitting socket will fre- quently cause pressure sores, infection and an abnormal gait. 1 An intraosseous transcutaneous amputation prosthesis (ITAP) can overcome these problems 2 as well as providing improved proprioception. 3 During normal wound heal- ing, epithelial cells at the margin of the wound migrate to re-establish the protective barrier function of the skin. Around an ITAP this results in downgrowth of the epithelium and pocket formation, which in turn provides a route whereby pathogens can enter and cause infection. For an ITAP to be successful, a tight seal at the skin–implant interface is essential. 4,5 Deer antlers have been studied as biomimetic models of ITAP 6 and have emphasised the cru- cial role of dermal tissue adhesion in preventing downgrowth. Surface modifications and syn- thetic hydroxyapatite (HA) coatings have been incorporated into the design of ITAPs to enhance dermal adhesion, 4-7 and biological coatings have been shown to improve dermal fibroblast attachment both in vitro and in vivo. 8,9 Cell adhesion is modulated by the extra- cellular matrix (ECM). The glycoprotein fibronectin (Fn) is a principal component of ECM, and it contains cell integrin-binding sequences; these include the amino acid sequence arginine-glycine-aspartic acid (RGD in one-letter amino acid code), through which it promotes cell-matrix adhesion. 10 Fn is readily adsorbed onto biomaterials, and enhances fibroblast activity 11 and attachment by upregu- lation of focal adhesion expression in vitro. 8,12 Focal adhesions are specialised electron-dense regions of the plasma membrane which create intimate, discrete contacts of 10 nm to 15 nm with the substratum of cells. 13 Quantification of the number of focal adhesions per unit cell area has been shown to be an accurate way of meas- uring the direct biophysical strength of dermal fibroblast attachment in vitro. 14 HA is a naturally occurring mineral apatite that comprises 70% of bone. 15 Synthetic HA coating of endoprostheses promotes osseo- integration, 16 and Fn functionalisation of HA- coated titanium alloy has been shown to increase dermal tissue attachment in vivo. 9 Despite extensive investigations into the inter- action of ECM proteins with HA, 17-20 the pre- cise nature of the interaction is not clear. This study investigated the loading and release kinetics of Fn from HA in the hope of provid- ing new data that may enable us to establish an optimal coating regime capable of enhancing dermal fibroblast attachment. This could then be applied to ITAPs in order to enhance the interface between implant and skin. The aim of