Conformal Microcapsules Encapsulating Microcarrier/Cells Complexes For Treatments Of Liver Damage And Osteoporosis

The traditional cell microcapsule is faced with following burning questions:The total implant volume is so large that it is impossible to be transplanted to small body spaces;Transport efficiencies of nutrients and therapeutic secretions are rather poor.In this study,a novel conformal microcapsule was established using aqueous two-phase emulsification,with cells cultured on microcarriers designed as the core,alginate-chitosan-alginate(ACA)polyelectrolyte layers as the shell.Their feasibilities of treating liver damage and osteoporosis were investigated.Three sections were included:(1)Preparation and characteristics of ACA conformal microcapsules containing Cytodex-3 microcarriers.Conformal microcapsules were prepared using 0.3%,0.6%,or 1.2%(w/v)chitosan solution,respectively.They were co-incubated with BSA or IgG solution to explore protein ingresses into the ACA membranes;The bead agitation test and compression test were carried out during their storage in a month;Their surface image and roughness parameters were obtained using an atomic force microscope.It was showed that above mentioned three groups of conformal microcapsules permitted ingress of BSA but prevented IgG from entering.The conformal microcapsules were stable enough to withstand the shear force in bead agitation test and they presented a similar curve of resistance force versus time during the storage period of a month,with the highest resistance force being about 1.7-2.2 g per microcapsule.For the protein permeability and mechanical stability investigations,no siginificant differences were seen between the three groups.Small values of root mean square(8.23± 2.03 nm)and average deviation(6.73 ± 1.81 nm)were observed,which suggested surfaces of conformal microcapsules were as smooth as traditional microcapsules.(2)Conformal microcapsules encapsulating Cytodex-3 microcarrier/L02 cells complexes(microC/L02)for treatment of liver damage.Water soluble tetrazolium salt assay and 5,6-carboxyfluorescein diacetate succinimidyl ester/propidium iodide(CFDA-SE/PI)staining were used to evaluate cell viability in conformal microcapsules after encapsulation;Enzyme linked immunosorbent assay(ELISA)and urease-glutamate dehydrogenase kinetic method were employed to measure human albumin and urea concentrations in culture supernatants of eonformal microcapsules During 60 days post-transplantation,rat serum total bilirubin(TBIL),alanine transaminase(ALT)and albumin(ALB)levels were assayed for three groups,including transplant group,model group and normal group.Hematoxylin/eosin(HE)staining was performed on liver tissue samples for histopathologic analysisIt was demonstrated that almost all cells were alive after microencapsulation and their viability recovered to be normal in 16 h,suggesting the preparation process did little harm to cells.Human albumin and urea were secreted continually in vitro.After transplantation,Serum ALT,TBIL,and ALB levels in vivo could be maintained normal for 60 days.Most(>90%)of retrieved conformal microcapsules were intact and cells in them kept alive.Compared with rats in model group,rats in transplant group exhibited ameliorated histological structure of hepatic lobule(3)Conformal microcapsules encapsulating Cytodex-3 microcarrier/recombinant Chinese hamster ovary cells secreting insulin-like growth factor 1(microC/rCHO-IGF-1)for treatment of osteoporosis.Water soluble tetrazolium salt assay and CFDA-SE/PI staining were used to evaluate cell viability in conformal microcapsules after encapsulation;IGF-1 concentrations in culture supernatants of conformal microcapsules were assayed by ELISA;When their culture supertanants were co-incubated with primary rat calvarial osteoblasts,Alizarin red S staining was employed to evaluate their ability to promote osteoblast differentiation.On day 30 post-transplantation,rat serum calcium,phosphorus,alkaline phosphatase(ALP)levels were assayed;Bone mineral densities of rat femurs were determined by dual-energy X-ray absorptiometry;During 30 days post-transplantation,serum IGF-1 levels were measured.HE staining was performed on rat femurs for histopathologic analysis.It was showed that almost all cells were alive after microencapsulation and their viability recovered to be normal in 24 h.IGF-1 could be secreted from conformal microcapsules continuously and their culture supermatants could promote mineralization of primary calvarial osteoblasts.After transplantation,serum IGF-1 could be improved during 30 days.On day 30 post-transplantation,serum phosphorus,ALP levels in transplant and sham group were significantly lower than those of ovariectomy group,indicating transplanted microcapsules helped to alleviate osteoporosis.Most(>90%)of retrieved conformal microcapsules were intact and cells in them kept alive.Compared with ovariectomy group,bone mineral densities of rat femurs in transplant group were siginificantly higher and better trabecular bone microarchitecture was observed in corresponding HE staining image.In summary,both microC/LO2 and microC/rCHO-IGF-1 were encapsulated into ACA conformal microcapsules using aqueous two-phase emulsification.Their feasibilties of treating acetaminophen-induced liver damage or osteoporosis were investigated.Conformal microcapsules containing microC/L02 were able to ameliorate biochemical indexes and histological structure of hepatic lobule.Conformal microcapsules containing microC/rCHO-IGF-1 could secret IGF-1,improve bone mineral density,and ameliorate trabecular bone arrangement.This study might be promising for miniaturization and function reinforcement of cell microcapsules.