Production of osteogenic and angiogenic factors by microencapsulated adipose stem cells varies with culture conditions.

Growth factors produced by stem cells aid in the bone repair process. We investigated the ability of encapsulated rat adipose-derived stem cells (rASCs) treated with osteogenic media (OM) to produce growth factors, and determined the optimal combination of OM components that will lead to the production of both osteogenic and angiogenic factors. Our results demonstrate that microencapsulated stem cells were able to produce vascular endothelial growth factor (VEGF), fibroblast growth factor-2, and bone morphogenetic protein-2 (BMP2) necessary for bone regeneration. OM led to the reduction of angiogenic factors; however, the removal of dexamethasone restored angiogenic factor production. Additionally, we determined whether the effect of dexamethasone on VEGF and BMP2 varied among rat, rabbit, mouse, and humans. Dexamethasone led to a reduction in VEGF levels in ASCs derived from rats, mice, and humans, while this reduction was absent in rabbit ASCs (rbASCs). Human ASCs (hASCs) from donors of different race and sex showed a similar response to dexamethasone with secreted VEGF levels. BMP2 levels secreted by rbASCs, mouse ASCs (mASCs), and hASCs were independent of the media treatments, while rASCs responded differently in the surrounding media and within the microbeads. In conclusion, microencapsulated ASCs can be treated to produce osteogenic and angiogenic factors for tissue regeneration applications, but outcomes may vary with culture conditions.

Microencapsulated rabbit adipose stem cells initiate tissue regeneration in a rabbit ear defect model.

Cell-based tissue engineering can promote cartilage tissue regeneration, but cell retention in the implant site post-delivery is problematic. Alginate microbeads containing adipose stem cells (ASCs) pretreated with chondrogenic media have been used successfully to regenerate hyaline cartilage in critical size defects in rat xiphoid suggesting that they may be used to treat defects in elastic cartilages such as the ear. To test this, we used microbeads made with low viscosity, high mannuronate medical grade alginate using a high electrostatic potential, and a calcium cross linking solution containing glucose. Microbeads containing rabbit ASCs (rbASCs) were implanted bilaterally in 3 mm critical size midcartilage ear defects of six skeletally mature male New Zealand White rabbits (empty defect; microbeads without cells; microbeads with cells; degradable microbeads with cells; and autograft). Twelve weeks post-implantation, regeneration was assessed by microCT and histology. Microencapsulated rbASCs cultured in chondrogenic media expressed mRNAs for aggrecan, Type II collagen, and Type X collagen. Histologically, empty defects contained fibrous tissue; microbeads without cells were still present in defects and were surrounded by fibrous tissue; nondegradable beads with rbASCs initiated cartilage regeneration; degradable microbeads with cells produced immature bone-like tissue, also demonstrated by microCT; and autografts appeared as normal auricular cartilage but were not fully integrated with the tissue surrounding the defect. Elastin, the hallmark of auricular cartilage, was not evident in the neocartilage. This delivery system offers the potential for regeneration of auricular cartilage, but vascularity of the treatment site and use of factors that induce elastin must be considered.

Microencapsulation of Stem Cells for Therapy.

An increasing demand to regenerate tissues from patient-derived sources has led to the development of cell-based therapies using autologous stem cells, thereby decreasing immune rejection of scaffolds coupled with allogeneic stem cells or allografts. Adult stem cells are multipotent and are readily available in tissues such as fat and bone marrow. They possess the ability to repair and regenerate tissue through the production of therapeutic factors, particularly vasculogenic proteins. A major challenge in cell-based therapies is localizing the delivered stem cells to the target site. Microencapsulation of cells provides a porous polymeric matrix that can provide a protected environment, localize the cells to one area, and maintain their viability by enabling the exchange of nutrients and waste products between the encapsulated cells and the surrounding tissue. In this chapter, we describe a method to produce injectable microbeads containing a tunable number of stem cells using the biopolymer alginate. The microencapsulation process involves extrusion of the alginate suspension containing cells from a microencapsulator, a syringe pump to control its flow rate, an electrostatic potential to overcome capillary forces and a reduced Ca++ cross-linking solution containing a nutrient osmolyte, to form microbeads. This method allows the encapsulated cells to remain viable up to three weeks in culture and up to three months in vivo and secrete growth factors capable of supporting tissue regeneration.

Development of a cell delivery system using alginate microbeads for tissue regeneration.

Alginate microbeads incorporating adipose-derived stem cells (ASCs) have potential for delivering viable cells capable of facilitating tissue regeneration. These microbeads are formed in calcium crosslinking solutions containing organic osmolytes to ensure physiological osmolality, but the comparative effects of these osmolytes on the microencapsulated cells are not known. In addition, delivery parameters needed to use microencapsulated cells for tissue regeneration remain unknown. We investigated the following parameters: (1) osmolyte effects on microbead diameter, cell viability and growth factor production; (2) the effect of the number of cells per microbead and the number of microbeads per unit volume on cell viability, growth factor production, and microbead degradation; (3) the ability of both degradable and non-degradable alginate microbeads to localize cells at the delivery site in vivo; and (4) whether alginate microbeads containing alginate-lyase elicit an inflammatory response after repeated exposure. Smallest microbead diameters were achieved using glucose as the osmolyte but cell viability and growth factor production did not depend on osmolyte type. As cell number per microbead or microbead number per well increased, growth factor production per cell decreased although percent cell viability was unchanged. The rate of cell release varied with the number of beads per well and with the number of cells per microbead. At the highest microbead density and at the lowest density of cells per microbead, cell release was delayed. Therefore fewer microbeads may be sufficient for clinical applications. Both degradable (0.22 U g-1) and non-degradable (0 U g-1) alginate microbeads localized cells at the delivery site. Degradable alginate microbeads delivered subcutaneously elicited a mild chronic inflammatory response on second exposure, but how this might impact repeated use of the technology remains to be determined.

Controlled release of rat adipose-derived stem cells from alginate microbeads.

Cell-based therapies have potential for tissue regeneration but poor delivery methods lead to low viability or dispersal of cells from target sites, limiting clinical utility. Here, we developed a degradable and injectable hydrogel to deliver stem cells for bone regeneration. Alginate microbeads <200 µm are injectable, persist at implantation sites and contain viable cells, but do not readily degrade in-vivo. We hypothesized that controlled release of rat adipose-derived stem cells (ASCs) from alginate microbeads can be achieved by incorporating alginate-lyase in the hydrogel. Microbeads were formed using high electrostatic potential. Controlled degradation was achieved through direct combination of alginate-lyase and alginate at 4 °C. Results showed that microbead degradation and cell release depended on the alginate-lyase to alginate ratio. Viability of released cells ranged from 87% on day 2 to 71% on day 12. Monolayer cultures of released ASCs grown in osteogenic medium produced higher levels of osteocalcin and similar levels of other soluble factors as ASCs that were neither previously encapsulated nor exposed to alginate-lyase. Bmp2, Fgf2, and Vegfa mRNA in released cells were also increased. Thus, this delivery system allows for controlled release of viable cells and can modulate their downstream osteogenic factor production.