Bone ingrowth

Bone ingrowth into a dry Ti implant shows a typical bone response to an implant that does not elicit an unwanted foreign body response over 6 weeks as seen in Figure 6.2a. At 2 weeks, there is a large spread of mineralising immature bone around the defect area that is discernable from µCT that can be seen to penetrate the open Ti structure. This is typical of the callus response to a bone defect, as the bone quickly aims to stabilise the defect area. By 4 weeks, local remodelling of the mineralising callus can be seen as the bone inside the Ti implant becomes visibly thicker and more coherent. This is reflected in the bone thickness distribution, seen in Figure 6.2b, where the bone thickness in 2

Figure 6.2 2D slices from KCT of tibiae after 2, 3, 4 and 6 weeks implantation of dry Ti samples. The VOI used for quantification is shown as a red outline in (a). The bone thickness distribution

inside the VOI is measured using accessible volume at each time point, as shown in (b). The yellow arrows indicate the evolution of immature bone into contiguous segments of bone over

week samples show a large proportion (98%) of bone trabeculae thickness less than 100 µm and modal value of 42 µm whilst by 6 weeks, only 60% of bone trabeculae are less than 100 µm thick and the modal value of 122 µm.

Similar bone responses were observed in the Ti+PRP (Figure 6.3) and Ti+blood cases (Figure 6.4), with local callus formation around the defect area followed by thickening and consolidation of the bony struts inside the Ti implant. In all three cases, there was good bone integration into the Ti implants, but bone was seen to line the Ti surface rather than completely filling the pores.

Figure 6.3 2D slices from KCT of tibiae after 2, 3, 4 and 6 weeks implantation of Ti+PRP samples. The VOI used for quantification is shown as a red outline in (a). The bone thickness distribution inside the VOI is measured using accessible volume at each time point, as shown in

The amount of direct contact between the ingrown bone and the Ti implant increases and bone is seen to grow along the surface of the Ti for all treatment types (Figure 6.5). Despite this, very little bone is seen to grow away from the original defect space and into the Ti. This is surprising as good bone0Ti contact area would suggest that the Ti is providing a good conduit for bone to grow along and being formed directly from the surface. This goes against previous literature that suggested that bone growth began at the centre of the channel mouth and towards the channel in a cone shape (Frosch et al., 2003). This may be due to the complex pore structure of the Ti implants, rather than the single channel experiments carried out by Frosch et al. that causes bone growth from multiple entry points and becomes gradually preferential to form along the surface. The increase in BIC over time in the Ti dry samples, increasing from 18% to 65% of the Ti surface (Ti dry) in the VOI

Figure 6.4 2D slices from KCT of tibiae after 2, 3, 4 and 6 weeks implantation of Ti+blood samples. The VOI used for quantification is shown as a red outline in (a). The bone thickness distribution inside the VOI is measured using accessible volume at each time point, as shown in

becoming covered could be put forward as evidence towards this. The increase in BIC could be attributed to firstly the increasing volume of bone inside the implant but also secondly that as the bone inside the implant is remodelling over time. Bone grew on the Ti surface in order to close the defect area. The low values of BIC at very early stages also suggest that bone does not form on the surface initially. This behaviour was seen in both the PRP and blood cases, thus these coatings did not seem to make the surfaces any more osteoconductive than the dry case. The dry case showed significant differences between the time points (2 and 4 weeks, 2 and 6 weeks, 3 and 6 weeks and 4 and 6 weeks, all p = 0.0247) and similarly in the blood group (2 and 3 weeks, 2 and 4 weeks, 2 and 6 weeks, all p = 0.0247). In the dry case, no statistical difference was seen in the 2 and 3 weeks (p = 0.0633), and between 3 and 4 weeks (p = 0.2563), which suggest that local remodelling began sometime after 4 weeks implantation causing significant increases in BIC. This remodelling stage may have been brought forward by the addition of blood, where a significant difference was seen already by the 2nd and 3rd weeks, suggesting remodelling began occurring as early as 3 weeks into implantation. The PRP cases showed no significant differences between time points (p = 0.0606 – 0.5), but these analyses were of limited use due to the small sample size (n =2 in some cases).

As seen in Figure 6.5, there was very little difference between the different treatment types which would suggest that neither PRP nor blood has any significant effect on improving bone attachment to Ti surfaces (dry vs PRP, p = 0.0606 – 0.5; dry vs blood, p = 0.1376 – 0.4136 at all time0points). This can be attributed to several factors. First of all, these surgical sites are already bloody, which could render any additional growth factors or cells provided by the blood treatment less meaningful. This is true also for PRP as it is a derivative of the blood such that all the proteins provided by PRP may already be present in abundance in a bloody site. Secondly, the implants were treated as a surgeon would before surgery; i.e. no steps were taken to ensure the homogenous coating of the inner Ti surface with PRP or blood. This could be done, for example, by putting the implant and PRP/blood solution in a vacuum to coat the surface. The implants were visibly covered in blood or PRP before implantation. However this is not considered a serious flaw as no real preference of bone growth along the outside of the implant was observed. Thirdly, the use of PRP is thought to require the presence of thrombin and CaCl2 in sufficient concentrations (142.8 U/ml and 14.3 mg/ml

Figure 6.5 The bone4implant contact area, BIC, over time for the different treatments. (p < 0.05, † dry, * blood)

respectively) in order to be ‘biologically active’ (Lacoste et al., 2003). In a bloody site however, it can be assumed that this threshold would have been reached.

One prior study found that osteoblast bone ingrowth into channels was restricted to 300 – 500 µm from the channel opening in vitro (Frosch et al., 2002). This study has shown that in vivo, this is not the case as connected segments of bone are seen to traverse across the width of the Ti implant through the pores. This can be explained by the fluid flow present in in vivo experiments that allow the transport and removal of cells inside the porous structure. The interconnectedness of the Ti pore channels also provide multiple ways of supplying an advancing bone front, rather than having the tissue stagnate due to the slower rate of vascularisation. Bone ingrowth, , can be seen to increase over time for all of the samples in the 6 week period although the nature of the bone changes throughout this time (Figure 6.6).

In the dry cases, a significant difference in was seen only at 6 weeks compared to 4 weeks (p = 0.0633 beween 2 and 3 weeks, p = 0.4136 between 3 and 4 weeks and p = 0.0247 between 4 and 6 weeks). This coincides with the timing of significantly increased BIC, as bone inside the defect and implant pore space becomes more consolidated. In the blood group, there were significant increases in bone ingrowth at 2 weeks (p = 0.0247) but none beyond 2 weeks. Again this correlates with the BIC values, which suggested that bone ingrowth and subsequent remodelling was aided by the addition of blood inside the pores of the Ti implant. Ultimately however, there were no significant difference between the conditions (dry, PRP and blood, p = 0.2563 – 0.4136), and thus no real effect of the addition of PRP and blood could be inferred in terms of the early bone ingrowth.

Comparing the values does not distinguish whether the increase in is driven by thickening of the bone trabeculae or the invasion of bone growth from the two ends of the defect. A decrease in SSA of the bone inside the VOI indicates a thickening of the bony struts (Figure 6.7).

Figure 6.6 The bone ingrowth percentage, is plotted for the different treatments. Error bars show the standard deviation between 3 samples. (p < 0.05, † dry, * blood)