## 4 Discussion  
The IR spectra were assessed for all three LPA derivatives. To enable this, only the characteristic functional groups were measured, and no interference occurred. Fatty acid-free HSA was used; thus it did not contain any complex-forming molecules. The LPA was solubilized in methanol and water to avoid any secondary binding and to ensure the purity of the reagents for spectroscopic purposes. The stretching of the CH\({}_{2}\) groups is visible in the 2916-2926 cm\({}^{-1}\) range and around 2850 cm\({}^{-1}\). The deformation vibrations occur at 1467 cm\({}^{-1}\)[30], and at 1736 cm\({}^{-1}\) C=O stretching is visible [31]. The absorbance at 1266 cm\({}^{-1}\) has been described as the peak of the PO\({}_{2}\) asymmetric stretching vibration. However, this absorption is more profound in the spectrum of 18:2 LPA but should also appear in the spectra of 18:1 and 16:0 derivatives. Alternatively, this band can result from the CH\(\alpha\) rocking, which is visible in the case of double bonds, and which is likely to be more intense in the case of 18:2 LPA, due to the two double bonds [32]. The purity of LPA 18:1 and 18:2 is in the 90 % range; thus, it may also be an acyl isomer impurity. The P-O-C and C-O bonds appear at 1062 cm\({}^{-1}\) and 1179 cm\({}^{-1}\), respectively [33]. Both CH\({}_{2}\) stretching absorbances shifted towards higher wavenumbers in the case of LPA derivatives, with an 18-carbon-atom acyl chain length compared with the 16-carbon-atom acyl chain length LPA. The increased CH\({}_{2}\) stretching frequencies indicate the higher disorder of the chains caused by the presence of double bonds in the acyl chains. The LPA derivatives are known to form complexes with HSA [19] in the 1:3 HSA:LPA ratio. Thus, the formed complexes were measured with the use of FTIR to investigate whether absorbance shifting occurred. HSA peaks dominate in the spectrum compared with the LPA peaks, which is presumably due to the significant difference in the molecular weights of the components. The two main characteristic bands for albumin are the amide I and II bands at 1648 cm\({}^{-1}\), and at 1537 cm\({}^{-1}\), respectively [34; 35]. According to our evaluation, there was a small spectral shift of the amide I band in presence of LPA. The spectral shift was 1 cm\({}^{-1}\) in the case of bound 18:1 LPA, and 3 cm\({}^{-1}\) in the case of 18:2 and 16:0 LPA variants. These variations indicate a subtle change in the secondary structure, while preserving the overall alpha-helical nature of the protein. A similar shift was observed in the case of the amide II band. These spectral changes in the amide bands might indicate the binding of LPA to HSA. Earlier, [PERSON] et al. showed that LPA protects hBM-dMSCs against hypoxia and serum deprivation-induced apoptosis in vitro, indicating that LPA is a potent survival factor for hBM-dMSCs in bone repair applications [36]. In addition, LPA was observed to promote H\({}_{2}\)O\({}_{2}\)-induced autophagy [37], the effect of which was shown to play important roles in cell survival [38]. These findings make LPA a promising candidate to improve the viability and proliferation of hBM-MSCs during bone healing. Our cell viability measurement data demonstrate that 18:1, 18:2 and 16:0 LPA species are non-toxic for hBM-dMSCs up to 10 \(\upmu\)M concentration. Although there is evidence for the advantages of using LPA in bone regenerative medicine [26; 27], there are still some challenges with the in vivo application of LPA. LPA is easily degraded in vivo by lipid phosphate phosphatases [39]; thus, LPA requires a carrier that allows the successful delivery of the lipid. In our experiments, all the three tested LPA species improved the proliferation of hBM-dMSCs, the effect of which was more pronounced when administered in the presence of HSA. This finding supports the assumption that HSA might serve as an ideal carrier for LPA in bone regenerative applications. Further, 18:1 and 16:0 LPA species did not significantly improve the cell proliferation without HSA.