br The sti ness Fig d
The stiﬀness (Fig. 1d and S2) and compressive strength (Supplemental Table 1) of the three CA scaﬀold compositions were measured in dry and wet states. Two stiﬀness values, bulk stiﬀness and wall stiﬀness, were obtained due to the porous structure of the CA Biomaterials 217 (2019) 119311
a significant increase in scaﬀold stiﬀness and compressive strength in both dry and wet conditions (Fig. 1d, S2; Table S1). The stiﬀness and compressive strength of PCa PDX tumors were measured with the same method and were 2.447 ± 0.101 MPa and 276.11 ± 34.56 kPa (Fig. 1d, S2), respectively, indicating that the wet 4 wt% CA scaﬀolds most closely approximated the PCa tumor stiﬀness out of the three CA scaﬀold compositions. While the normal prostate tissue and bone were not measured with the same technique for comparison to the scaﬀolds, the 2 wt% CA scaﬀolds resembled the stiﬀness of the native prostate tissue and the 6 wt% CA scaﬀolds approximated the stiﬀness of the bone microenvironment. Various methods, including shear wave elasto-graphy and ultrasound, have been reported to measure the stiﬀness and change in stiﬀness of prostate tissue, as increases in prostate stiﬀness are correlated with carcinogenesis . While these diﬀerent methods have been used to assess the prostate stiﬀness, it Prostaglandin J2 is challenging to correlate data from diﬀerent techniques without introducing artifacts, due to diﬀerent sample sizes, diﬀerent methods, and other test re-strictions.
The PEC formation in the CA scaﬀolds was assessed with FTIR analysis. The FTIR spectra for chitosan, alginate, and the three CA scaﬀold compositions are presented in Fig. S3. Characteristic peaks in the alginate spectra include the peaks at 1600 cm−1 (COO− antisym-metric stretch), 2920 cm−1 (C–H stretch), and 3430 cm−1 (O–H stretch). Characteristic peaks in the chitosan spectra includes the peaks at 2900 cm−1 (C–H stretch), 1650 cm−1 (amide I, C = O), and 1590 cm−1 (amide II, N–H). The chitosan used for scaﬀold synthesis is partially deacetylated (≥75% deacetylated), resulting in the two amide peaks at 1650 cm−1 and 1590 cm−1 . CA PEC formation is in-dicated by the combination of amide I (1650 cm−1) and N–H bending of amide II (1590 cm−1) chitosan peaks and the COO− antisymmetric (1600 cm−1) alginate peak into one peak at 1630 cm−1, which is shown on the 2, 4, and 6 wt% CA spectra . Similar findings have been reported that the formation of this new peak indicates CA PEC forma-tion, although the peak wavelength varies between reports [26,39,40]. The verification of PEC formation for each scaﬀold composition con-firms that the CA molecules interacted. The formation of CA PECs provides greater scaﬀold stability than a mixture of the two molecules without complex formation.
3.2. PCa cell lines respond diﬀerentially to scaﬀold stiﬀness
Three PCa cell lines, PC-3, C4-2B and 22Rv1, were cultured on the 2, 4, and 6 wt% CA scaﬀold compositions. The three PCa cell lines have diﬀerent profiles: PC-3 cells were derived from a lumbar metastasis and are androgen independent; C4-2B cells were derived from a bone
Fig. 1. Chitosan-alginate (CA) scaﬀold properties. a) Appearance of 2, 4, and 6 wt% CA scaﬀolds discs in dry (top) and wet (bottom) states. b) Pore morphology of 2, 4, and 6 wt% CA scaﬀolds, low (top) and high (bottom) magnification images. c) Percent porosity, average pore size and compressive strength for 2, 4, and 6 wt% CA scaﬀolds. d) Wall stiﬀness of 2, 4, and 6 wt% CA scaﬀolds, along with PDX tumor stiﬀness, in dry and wet conditions. The * denotes statistically significant diﬀerences (p < 0.05).
metastasis of the LNCaP parental line and has a full length AR; and 22Rv1 cells were derived from a human prostatic carcinoma xenograft, CWR22R, and has a truncated AR . Additionally, the PC-3 cells display osteolytic behavior, while the C4-2B and 22Rv1 cells display osteoblastic behavior [42,43]. These three cell lines provided a com-parison of PCa osteolytic and osteoblastic phenotypes and androgen responses that are observed in human patients to examine on the three CA scaﬀold compositions. The three cell lines had a cell seeding eﬃ-ciency of > 80% on all scaﬀold compositions (Table S2). The cell number in the CA scaﬀold cultures was assessed at 5, 10, and 15 d time points (Fig. 2). At 5 d, either the 2 wt% or 4 wt% CA scaﬀolds had the greatest cell number, while the 6 wt% CA had the lowest cell number for all three cell lines. At 10 d, 4 wt% CA had the greatest cell number for C4-2B, while 6 wt% CA had the greatest cell number for PC-3 and 22Rv1. At 15 d, 6 wt% CA has the greatest cell number for all cell lines. A maximum cell number appears to have been reached at 10 d for all cell lines, followed by consistent or decreased cell numbers in all cul-tures at 15 d. This decreased cell number at 15 d could be due to several