| BackgroundBreast cancer, one of the malignant tumors, has seriously threatened the life and health of women. The cancer epidemiology survey results showed that the incidence of breast cancer has been the first place in the female cancer incidence. In China, the incidence of breast cancer has increased 18/10,000,000 to 30-40/10,000,000 in 30 years. In the major cities such as Shanghai, Beijing, Guangzhou and Tianjin, breast cancer has become the "top murder" of women ranging from 30 to 54 yea^old.Most of the patients with breast cancer will receive the surgery to remove tumors. However, the radical hysterectomy or the partial mastectomy will lead to breast deformation, over 90% of patients will look for the breast reconstruction to maintain perfect second female characteristics. Due to the development of surgery, chemotherapy, endocrine therapy, biological therapy and radiotherapy, nearly all patients after surgery can receive breast reconstruction at the time to improve their life quality.At present, there are three major means for breast reconstruction:prosthesis implantation, autologous skin flap transplantation and autologous fat tissue transplantation. Following the developing of liposuction, the autologous fat tissue transplantation has become the most popular one because of its characteristic: easy-to-get, minimal invasiveness and shaping naturally. However, the transplantation will be absorbed and the reduction of the volume will be as high as 40 ～60% with the extension of time because the nutrition and oxygen of the transplantation cannot be guaranteed.Because of the limitation of autologous fat tissue transplantation, regenerative medicine and tissue engineering researchers began to focus on the new materials for breast reconstruction. These new materials should form a three-dimensional scaffold that supports the cell adherence and proliferation and also provides an environment which is similar to the real one in vivo. Cells in the scaffold should not only survive and proliferate, but also differentiate as usual and express original cell phenotype. In addition, these scaffold carried with cells can become the transplantation for breast reconstruction.As a new tissue engineering scaffold, the self-assembling peptides had received extensive attention in recent years. Such scaffolds could be established a growth environment to simulate that in vivo. The hydrogel can support not only the mechanical properties of the adhesion and growth of cells, but also conducive to the exchange of information and material. It has been found that self-assembled peptide hydrogel can provide excellent microenvironment for the growth and differentiation of precursor cells and stem cells. In addition, this kind of peptide hydrogel has a superior biocompatibility, and can be designed according to the actual application needs. These characteristics are benefit to build individual materials to meet the needs of the use. The hydrogel contains higher than 99% of water, which is suitable for carrying drugs or therapeutic proteins. If it is needed to prepare a special drug loading system, it can also be achieved by changing the composition of the peptide.RADA16-Ⅰ (AcN-RADARADARADARADA-CNH) is composed of natural amino acids, and it is a kind of injectable self-assembled polypeptide which can form a hydrogel scaffold with a large number of nano-fibers under certain conditions. The structure of RAD A16-1 hydrogel scaffold was similar to the extracellular matrix, which could provide a good culture condition for the cells. At present, the RADA16-Ⅰ hydrogel have been applied to the regeneration of bone, muscle, nerve, blood vessel and so on, and has been widely concerned.With the development of the research of stem cell in the medical field, it has been favored by the majority of researchers because of its huge potential. The Stem cells not only have the ability of self replication, but also can be differentiated into a variety of functional cells under certain conditions. Compared to other sources of stem cells, adipose derived stem cell is one of the easiest one to get material source. So it is the ideal stem cells extracted from the source. A large number of studies have reported that adipose derived stem cell transplantation can repair damaged tissue structure and function.In this work, we explore the feasibility of tamoxifen (TAM)-loaded RADA16-Ⅰ peptide hydrogel for the therapeutic breast tissue.ObjectiveTo study the feasibility of constructing the therapeutic breast tissue by RADA16-Ⅰloaded with adipose derived stem cells which is isolated from liposuction surgery, and the antitumor drug tamoxifen.MethodsThe fat got from liposuction was washed by phosphate buffered saline (PBS) containing penicillin streptomycin twice in a centrifuge tube and centrifuged to get the fat.0.15%collagenase I was add in the tube as same volume as the fat then put the tube into the shaker at 37℃ to digest for at least 60 min. The pellets were centrifuged to obtain SVF cell pellets. After the SVF was cultured in the flask for 3 days, we can observe the ADSCs. The cells were trypsinized when the fusion rate of the cells arrived above 90%.The surface markers of the ADSCs were detected by flow cytometry. The ADSCs was induced to adipogenic differentiation and assessed by Oil Red O staining. The control blank hydrogel scaffold was made by the RADA16-Ⅰpowder dissolved with ultrapure water (0.1%(v/w)) and put in the thermostats at the temperature of 37℃ to self-assemble. The TAM-loaded RADA16-Ⅰ hydrogel was prepared by dissolving the peptide powder and TAM in ultrapure water.Scanning electron microscopy (SEM) was used to study the surface morphology of the RADA16-Ⅰ peptide hydrogel and the TAM-loaded peptide hydrogel. The effect of TAM on the physical-chemical properties of hydrogel was studied by FTIR (Fourier transform infrared spectroscopy) and the DSC (Differential scanning calorimetry).The inhibition rate of cells in each groups were assessed by MTT method. The cell apoptosis was evaluated by flow cytometry. The expression of apoptosis related proteins were assessed by western blot assay. The complex that loaded ADSCs and RADA16-Ⅰ hydrogel or loaded TAM-ADSCs RADA16-Ⅰ hydrogel was subcutaneously implanted into the nude mice respectively. The complex maintenance time in vivo and the influence on the overall survival and weight of the nude mouse was evaluated..ResultsSurface antigen assay confirmed that ADSCs were successfully isolated from the fat tissue and could be induced into adipocyte. The TAM-loaded RADA-Ⅰ peptide hydrogel formed a transparent viscous hemisphere-shaped hydrogel (0.1%v/w). FTIR results displayed that there was no significant difference between TAM-loaded hydrogel and control hydrogel. In 2D culture, ADSCs and MCF-7 cells were inhibited by 44.76% and 80.25% respectively as treated by 25μmol/L TAM for 72 h. Meanwhile, in 3D culture, ADSCs and MCF-7 cells were inhibited by 25.36% and 81.86% respectively as treated by TAM-loaded hydrogel. The growth inhibition rate of the ADSCs caused by TAM in 2D culture were higher than those in 3D culture (P<0.05), suggesting that TAM released from RADA16-Ⅰ hydrogel might weaken the inhibition and apoptosis effect on ADSCs. However, the growth inhibition rate of MCF-7 cells caused by TAM in 2D culture was similar with that in 3D culture (P>0.05), suggesting that both TAM in 2D culture medium and TAM released from 3D hydrogel retain the same cytotoxic effect on MCF-7. Theexpression of Bcl-2 also confirmed the results. The apoptosis study even showed that TAM released from 3D hydrogel may have more effect on MCF-7 especially for the late apoptosis of cell. On the other hand, these phenomena are not observed in ADSCs, the apoptosis caused by TAM may be reduced by hydrogel. After subcutaneously implantation of ADSCs and RADA16-Ⅰ hydrogel complex or TAM-loaded ADSCs-RADA16-Ⅰ hydrogel complex respectively into the nude mice, round mass was formed with regular shape and clear edge. The mass could maintain up to 7 days and retain its shape after being pressed slightly. There is no difference between 2 groups on body weight of mice.ConclusionOur results suggested that TAM-loaded RADA16-Ⅰ hydrogel both provide a scaffold for ADSCs cells attachment and retain cytotoxic effect on MCF-7 cells. It is feasible that the TAM-loaded RADA16-Ⅰ hydrogel scaffold is promising for therapeutic breast tissue following lumpectomy. |
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