br cell line samples is background staining
cell line samples is background staining of the scaﬀold, not positive staining for mineralization. The background staining of the scaﬀold was determined by comparing the samples to uncultured scaﬀold controls that were stained with Alizarin Red (Fig. S10).
To confirm that the observed mineralization was bone mineral and not pathological calcification, osteocalcin expression was assessed with IF. Osteocalcin is a non-collagenous protein found in bone matrix that plays a role in bone mineralization . Analysis of the osteocalcin expression for PC-3, C4-2B and 22Rv1 cell lines on 2, 4, and 6 wt% CA scaﬀolds (Fig. 9) demonstrated that C4-2B and 22Rv1 scaﬀold cultures expressed osteocalcin, while PC-3 cultures did not. None of the 2D cultures for any cell line expressed osteocalcin, demonstrating that the osteocalcin expression was promoted by scaﬀold culture. This confirms the presence of mineralized bone matrix for the osteoblastic C4-2B and 22Rv1 cultures, and indicates that the osteolytic PC-3 cultures did not exhibit mineralized bone matrix. Osteocalcin expression demonstrates the presence of bone matrix and confirms that the mineralization ob-served with SEM and Alizarin Red analysis is bone mineral and not pathological calcification.
The mineralization results agree with the literature. PC-3 Malonyl Coenzyme A in-duced osteoblast-like cells to express osteoclastogenesis factors after co-culture  or when cultured with PC-3 conditioned media . The osteolytic behavior of PC-3 cells was demonstrated by injecting PC-3 cells into humanized tissue engineered bone constructs implanted in mice, where the bone was resorbed . Conversely, C4-2B cells en-hanced the mineralization of hematopoietic progenitor cells . Ad-ditionally, the mineralization in the C4-2B cultures and the osteoblastic phenotype may contribute to the downregulation of PSA expression observed in the C4-2B scaﬀold cultures. PSA expression for C4-2 cells was downregulated when cultured with conditioned media (CM) from osteoblast cell lines due to soluble factors in CM .
This study demonstrated that osteoblastic and osteolytic PCa cell lines responded in diﬀerent manners to CA scaﬀolds with diﬀerent stiﬀnesses. Further research is needed to evaluate the eﬀect of scaﬀold stiﬀness on PCa cells when co-cultured with fibroblasts or other cells found in the PCa tumor microenvironment. Additionally, incorporation of proteins into or conjugation on to the CA scaﬀolds may provide a greater response to scaﬀold stiﬀness by enabling integrin binding. Since the CA scaﬀold cultures revealed diﬀerences in the PCa cell line re-sponses, the CA scaﬀolds have potential for use to evaluate patient-derived primary PCa cells. These CA scaﬀold cultures may be used to determine the expression of phenotypic markers and screen prospective chemotherapies, allowing these cultures to supplement pathology data from tumor biopsies. Further research is needed to determine if CA scaﬀold cultures recapitulate the responses of the patient tumor to
Fig. 8. Alizarin Red staining to assess mineralization. a) Comparison of PC-3, C4-2B and 22Rv1 cell lines on 4 wt% CA scaﬀolds at 10 d. Dark red staining denotes mineralization, indicated with arrows. b) Quantification of Alizarin Red staining in scaﬀolds compared to uncultured scaﬀold controls, * denotes statistically significant diﬀerences (p < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
therapies, which would potentially enable their use in personalized cancer treatment.