Statistical evaluation of data was performed using GraphPad software package. Data are reported as mean ± standard deviation (SD) at a significance level of P < 0.05. One-way ANOVA with the Bonferroni test was carried out to compare the groups.

Electrospun fibres closely mimic the extracellular matrix of bone [²¹–²³], the fibres obtained during this study had an average diameter of 269 ± 102 nm with most fibres distributed between 100 and 300 nm (Figure 3).

The stability of the PVPA coating was examined using EDX analysis (Figure 4). The presence of phosphorous was monitored over a 21-day period after immersion of the scaffolds in DMEM. Scaffolds that had not undergone heat treatment were tested as a control. There was a sharp decrease in phosphorous wt% from day 0 to day 2 on both scaffold types, decreasing from 3.3 ± 0.08 wt% to 1.24 ± 0.13 wt% on heat-treated scaffolds and to 0.12 ± 0.02 wt% on untreated scaffolds. The initial release of phosphorous in the first two days of immersion could be advantageous in vivo as the apoptosis of osteoclasts immediately after implantation would allow osteoblasts to secrete matrix.

After day 2, there was no significant change in phosphorous concentration over the remaining culture period as shown in Figure 5; however, there was a significant difference in concentration between the two scaffold types; scaffolds which had undergone a heat treatment had a significantly higher (P < 0.001) concentration of phosphorous compared to untreated scaffolds. This result suggests that after the initial dissolution of PVPA, the coating becomes stable upon heat treatment.

It is hypothesised that PVPA is bound to PCL at the interface as a monomolecular layer, and there are subsequent interactions between the unbound PVPA molecules. It is suggested that up till day 2, the unbound PVPA are gradually released. This is followed by stabilisation of the coating as all unbound PVPA are released and only the monolayer of PVPA exists on the surface.

EDX revealed that the heat treatment of PCL/PVPA prevents the PVPA from being removed from the scaffold. During heat treatment the scaffold is taken above its crystallisation temperature. This leads to movement of the polymer chains and cooling leads to subsequent crystallisation of the polymer chains. The PVPA could become “trapped” during the heating and cooling of the polymer chains, which subsequently provides a stable PCL/PVPA scaffold.

The wettability of PCL/PVPA and PCL scaffolds was determined through water contact angle testing (Figure 5). There is a significant difference in the contact angle of PCL scaffolds once treated with PVPA, the contact angle is reduced from 123.3 ± 10.8° to 43.3 ± 1.0° (Table 2). The use of PCL without any pretreatment can be limited due to the hydrophobic nature of the polymer. The wettability testing demonstrates that the treatment of PCL with PVPA significantly improves the wetting nature of the polymer and no further treatment would be needed before cell seeding.

PVPA consists of negatively charged –COOH and –OH functional groups. The oxygen atoms exert a greater “pull” on the hydrogen atoms and consequently one of the atoms become electronegative. These groups are said to be polar and are therefore attracted to water as water is also polar, this property will make PVPA hydrophilic and consequently when PCL is treated with PVPA, the surface becomes hydrophilic.

FTIR analysis was employed to identify any changes in the chemical structure that could have occurred during the electrospinning process. When we compare electrospun PCL and a PCL pellet, the spectra reveals that there is no difference in the functional groups present after-electrospinning (Figure 6(a)). The change in the spectra upon the addition of PVPA to PCL reveals a –OH peak at 3450 cm⁻¹ (Figure 6(b)). This peak can be owed to the PVPA polymer, which contains many –OH functional groups. The C–H peaks present at 1750 cm⁻¹ and 2850 cm⁻¹ in electrospun PCL remained unchanged in the presence of PVPA. FTIR analysis has demonstrated that the electrospinning process has no effect on the functional groups present in the resulting structure.

The thermal properties of the polymer scaffolds were determined through DSC; the results are outlined in Table 3. The treatment of PCL with PVPA has had no significant difference on the thermal properties of the scaffolds. This is most likely due to there being only a low concentration of PVPA in comparison to the PCL substrate therefore having no significant effect on the thermal properties.

The mechanical properties of a material to be used in bone tissue engineering have an important role in the scaffold design. Mechanical testing was carried out to determine the compressive strength and Young's modulus of the polymer scaffolds. The two properties were determined using previously reported equations and the load-displacement curve obtained (Figure 7). The load-displacement curve displays a significant difference between the load withstood by PCL and PCL/PVPA. The compressive strength of PCL/PVPA scaffolds has been calculated to be 72 MPa, this is significantly higher than that of PCL scaffolds (14 MPa) (Table 4). A similar trend was determined for the Young's modulus, the Young's modulus of PCL/PVPA was determined to be 3.9 GPa and 0.9 GPa for PCL scaffolds. The compressive strength of PCL/PVPA is ten-times greater than that of trabecular bone (7–10 MPa), and the Young's modulus is eight times greater (0.05–0.5 GPa).

The significant increase in mechanical strength in the presence of PVPA could be owed to the presence of intermolecular interactions between the PVPA-coated PCL scaffolds. The highly negatively charged oxygen atoms within PVPA are attracted to hydrogen atoms present within the scaffolds structure, this interaction provides strength to the resulting structure and hence an increase in the compressive strength.

In the area of bone tissue engineering it is important to closely mimic the natural strength of bone as possible. This can be problematic due to the high compressive strength of bone. The mismatch of mechanical properties between natural bone and synthetic materials can lead to failure of an implanted material. The mechanical properties suggest that the scaffolds have sufficient mechanical strength to be used as a packing substance in trabecular bone voids. The increase in the mechanical properties of PCL/PVPA scaffolds is owed to the properties of PVPA. The solidification of PVPA gives rise to a strong material that is hypothesised to be stronger than PCL. The tensile and compressive properties of PVPA have not been tested. It is hypothesised that the presence of PVPA throughout the PCL structure would provide strength throughout the structure and hence sufficient mechanical support in bone tissue. The mechanical properties of the scaffold can be tailored by changing the amount of PVPA present within the structure. An optimum concentration will have to be determined to allow for an advantageous effect on cells growth as well as on the mechanical properties.

Preliminary studies have shown that osteoblast cells are able to favourably attach and proliferate on the PCL/PVPA scaffold (Figure 8). After 14 days in culture, a more pronounced cytoskeleton was observed in osteoblasts cultured on PCL/PVPA scaffolds, this is most likely to be due to superior spreading of the cells due to the significantly lower water contact angle of the PCL/PVPA scaffold when compared to PCL scaffolds [²³, ²⁴]. Further studies investigating the effects of the scaffold on osteoblast activity will be carried out.

Scanning electron microscopy (SEM) was used to visualise osteoclasts cultured on PCL, PCL/PVPA, and glass substrates over 7 days under standard conditions. At day 1, there were viable cells present on all scaffold types (Figure 9). A high number of cell colonies formed on PCL/PVPA scaffolds. It is not clear whether this is due to the hydrophilic nature of the substrate which encourages rapid attachment of cells or whether it is due to the PVPA polymer causing cells to group together and a possible indicator of the initiation of apoptosis. Cells present on both PCL and PCL/PVPA scaffolds displayed superior spreading when compared to the glass substrate. This can be owed to the morphology of the electrospun fibres; the fibrous structure provides a greater surface area for cell attachment, proliferation and integrin binding. At day 7, there was a marked difference in the morphology of cells present on the substrate types. Large, multinucleated cells formed on PCL substrates, which had an elongated, rounded morphology. There were very few cells present on PCL/PVPA substrates; there was evidence of cell debris on the surface. In contrast to cells present on PCL substrates, cells on the glass surface were multinucleated, however, with a flattened morphology.

It is hypothesised that the presence of PVPA in culture leads to the formation of nonhydrolysable analogues of ATP, AppCp. The accumulation of these analogues within the osteoclast cells leads to loss of its function and eventual cell death [²⁵, ²⁶]. From SEM analysis, it has been shown that cells are present at day 1, suggesting that cells are not apoptosing immediately upon treatment with PVPA.