Microporosity Affects Bioactivity of Macroporous Hydroxyapatite Bone Graft Substitutes

Abstract

This paper describes an investigation into the influence of microporosity on early osseointegration within porous hydroxyapatite scaffolds. Two batches of phase pure porous hydroxyapatite were produced with total porosities of approximately 80%, but with varied levels of microporosity such that the strut porosity of the two batches were 10 and 20%. Cylindrical specimens 4.5mm in diameter were implanted in the distal femur of 6 month New Zealand White rabbits and retrieved for histological and histomorphometric analysis at 1 and 3 weeks. Optical microscopy demonstrated variation in the degree of capillary penetration and bone morphology within scaffolds at 1 and 3 weeks, respectively. Moreover, histomorphometry demonstrated that there was significantly more bone ingrowth within HA80-2 scaffolds and that the rate of bone formation within these scaffolds was significantly faster. These results indicate that the bioactivity of porous hydroxyapatite scaffolds may be improved by increasing the level of microporosity within the ceramic struts. Introduction The combined affects of an ageing population and greater expectations in quality of life have resulted in an increasing global demand for orthopaedic implants for the replacement or augmentation of damaged bones and joints. In bone grafting current ‘gold standards’ include the use of autograft (living bone from the patient) or allograft (dead, sterilised bone from bone banks) but these methods are increasingly recognised as non-ideal due to limitations in supply and consistency [1]. Porous ceramics have been considered for use as bone graft substitutes in the treatment of bone defects for over 30 years [2]. In particular, calcium phosphates such as hydroxyapatite (HA) have been promoted as a result of their osteoconductive properties. However, while it is well recognised that both the rate of integration and the final volume of regenerated bone may be primarily dependent on various features of the macro-porosity, such as volume fraction, pore size and pore connectivity [3], recent in vitro studies have demonstrated bone cell sensitivity to the level of microporosity within the ceramic struts [4]. The aim of this study was to investigate the influence of microporosity on early osseointegration within porous HA (PHA) scaffolds. Materials and Methods Two batches of phase pure PHA were produced using a novel slip foaming technique [5], both had total porosities of approximately 80%, but varied in the volume fraction of porosity distributed 2 Title of Publication (to be inserted by the publisher) Figure 1 – (a) Porosity distribution within batches HA80-1 and HA80-2. Micropore morphology within the struts of batches (b) HA80-1 and (c) HA80-2. Bar = 50 μm between their macropore (>50μm) and micropore (<20μm) populations (Fig. 1). This variation was such that the strut porosity of batches HA80-1 and HA80-2 were 10 and 20%, respectively. Morphological characterisation of both the macroand microporosity was performed through a combination of immersion densitometry and image analysis of serial sections using a Zeiss Axioskop optical microscope linked to a KS300 image analyser. Macropore size, interconnection size and connectivity index, a measure of inter-pore connectivity, were all measured. Both the open and closed % of microporosity within the struts was quantified. Cylindrical specimens 4.5mm in diameter were implanted in the distal femur of 6 month New Zealand White rabbits and retrieved for histological and histomorphometric analysis at 1 and 3 weeks. The volume of new bone ingrowth was calculated using a Weibel grid and the mineral apposition rate (MAR) was determined through the administration of fluorochrome labels at 1 and 2 weeks and measurement of the inter-label distance using a Zeiss Axioskop optical microscope with a UV light source, linked to KS300 image analyser. Figure 2 – (a) Macropore interconnection data and (b) porosity distribution of open and closed microporosity within batches HA80-1 and HA80-2. 0 1 2 3 4 5 6 7 8 HA80-1 HA80-2 M ic ro po ro si ty (% ) Open Closed (b) (a) 0 10 20 30 40 50 60 70 80 90 HA80-1 HA80-2 P or os ity (% ) Microporosity Macroporosity (a) (b) (c) 0 100 200 300 400 500 600 Interconnection Size Connectivity Index HA80-1 HA80-2 Figure 3 – Variation in ingrowth morphology within batches (a) HA80-1 (Goldner’s trichrome) and (b) HA80-2 (Toluidine blue). Bar = 100 μm