Emmanuel C. Opara

Regenerative medicine as applied to solid organ transplantation: current status and future challenges

In the last two decades, regenerative medicine has shown the potential for “bench‐to‐bedside” translational research in specific clinical settings. Progress made in cell and stem cell biology, material sciences and tissue engineering enabled researchers to develop cutting‐edge technology which has lead to the creation of nonmodular tissue constructs such as skin, bladders, vessels and upper airways. In all cases, autologous cells were seeded on either artificial or natural supporting scaffolds. However, such constructs were implanted without the reconstruction of the vascular supply, and the nutrients and oxygen were supplied by diffusion from adjacent tissues. Engineering of modular organs (namely, organs organized in functioning units referred to as modules and requiring the reconstruction of the vascular supply) is more complex and challenging. Models of functioning hearts and livers have been engineered using “natural tissue” scaffolds and efforts are underway to produce kidneys, pancreata and small intestine. Creation of custom‐made bioengineered organs, where the cellular component is exquisitely autologous and have an internal vascular network, will theoretically overcome the two major hurdles in transplantation, namely the shortage of organs and the toxicity deriving from lifelong immunosuppression. This review describes recent advances in the engineering of several key tissues and organs.

Skeletal myogenic differentiation of urine-derived stem cells and angiogenesis using microbeads loaded with growth factors

To provide site-specific delivery and targeted release of growth factors to implanted urine-derived stem cells (USCs), we prepared microbeads of alginate containing growth factors. The growth factors included VEGF, IGF-1, FGF-1, PDGF, HGF and NGF. Radiolabeled growth factors were loaded separately and used to access the in vitro release from the microbeads with a gamma counter over 4 weeks. In vitro endothelial differentiation of USCs by the released VEGF from the microbeads in a separate experiment confirmed that the released growth factors from the microbeads were bioactive. USCs and microbeads were mixed with the collagen gel type 1 (2 mg/ml) and used for in vivo studies through subcutaneous injection into nude mice. Four weeks after subcutaneous injection, we found that grafted cell survival was improved and more cells expressed myogenic and endothelial cell transcripts and markers compared to controls. More vessel formation and innervations were observed in USCs combined with six growth factors cocktail incorporated in microbeads compared to controls. In conclusion, a combination of growth factors released locally from the alginate microbeads induced USCs to differentiate into a myogenic lineage, enhanced revascularization and innervation, and stimulated resident cell growth in vivo. This approach could potentially be used for cell therapy in the treatment of stress urinary incontinence.

Stability of alginate microbead properties in vitro.

Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3xa0weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy.

Engineered multilayer ovarian tissue that secretes sex steroids and peptide hormones in response to gonadotropins.

Although hormone replacement therapy is an option for the loss of ovarian function, hormone delivery through pharmacological means results in various clinical complications. The present study was designed to deliver sex steroids by a functional construct fabricated using encapsulation techniques. Theca and granulosa cells isolated from ovaries of 21-day old rats were encapsulated in multilayer alginate microcapsules to recapitulate the native follicular structure. Cells encapsulated in two other schemes were used as controls to assess the importance of the multilayer structure. The endocrine functions of the encapsulated cells were assessed in vitro for a period of 30 days. Encapsulated cells showed sustained viability during long-term in vitro culture with those encapsulated in multilayer capsules secreting significantly higher and sustained concentrations of 17 β-estradiol (E(2)) than the two other encapsulation schemes (p < 0.05, n = 6) in response to follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition, cells in the multilayer microcapsules also secreted activin and inhibin in vitro. In contrast, when granulosa and theca cells were cultured in 2D culture, progesterone (P(4)) secretion increased while E(2) secretion decreased over a 30-day period. In summary, we have designed a multilayer engineered ovarian tissue that secretes sex steroids and peptide hormones and responds to gonadotropins, thus demonstrating the ability to recapitulate native ovarian structure ex vivo.

FGF-1 delivery from multilayer alginate microbeads stimulates a rapid and persistent increase in vascular density

In recent years, great advances have been made in the use of islet transplantation as a treatment for type I diabetes. Indeed, it is possible that stimulation of local neovascularization upon transplantation could improve functional graft outcomes. In the present study, we investigate the use of multilayered alginate microbeads to provide a sustained delivery of FGF-1, and whether this results in increased neovascularization in vivo. Multilayered alginate microbeads, loaded with either 150ng or 600ng of FGF-1 in the outer layer, were surgically implanted into rats using an omentum pouch model and compared to empty microbead implants. Rats were sacrificed at 4days, 1week, and 6weeks. Staining for CD31 showed that both conditions of FGF-1 loaded microbeads resulted in a significantly higher vessel density at all time points studied. Moreover, at 6weeks, alginate microbeads containing 600ng FGF-1 provided a greater vascular density compared to both the control group and the microbeads loaded with 150ng FGF-1. Omenta analyzed via staining for smooth muscle alpha actin showed no variation in mural cell density at either 4days or 1week. At 6weeks, however, omenta exposed to microbeads loaded with 600ng FGF-1 showed an increase in mural cell staining compared to controls. These results suggest that the sustained delivery of FGF-1 from multilayered alginate microbeads results in a rapid and persistent vascular response. An increase in the local blood supply could reduce the number of islets required for transplantation in order to achieve clinical efficacy.

Immunoisolation: where regenerative medicine meets solid organ transplantation

Immunoisolation refers to an immunological strategy in which nonself antigens present on an allograft or xenograft are not allowed to come in contact with the host immune system, and it is implemented to prevent allorecognition and avoid immunosuppression. In this setting, the two most promising technologies, encapsulation of pancreatic islets (EPI) and immunocloaking (IC), are used. In the case of EPI, islets are inserted in capsules that, allow exchange of oxygen, nutrients and other molecules. In the case of IC, a natural nanofilm is injected prior to renal transplantation within the vasculature of the graft with the intent to pave the inner surface of the vascular lumen and camouflage the antigens located on the membrane of endothelia cells. Significant progress achieved in experimental models is leading EPI and IC to clinical translation.

Effects of Allogeneic Bone Marrow Derived Mesenchymal Stromal Cell Therapy on Voiding Function in a Rat Model of Parkinson Disease

PURPOSEnCellular therapy induced transient urodynamic improvement in a rat model of Parkinson disease in which bladder dysfunction was noted after unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. We sought to prolong the effect by injecting allogeneic rat bone marrow mesenchymal stromal cells before and after microencapsulation into the substantia nigra pars compacta.nnnMATERIALS AND METHODSnFemale rats underwent unilateral stereotactic injection of 6-hydroxydopamine in the medial forebrain bundle. Injection was performed in the ipsilateral substantia nigra pars compacta using vehicle alone or vehicle with nonmicroencapsulated or microencapsulated rat bone marrow derived mesenchymal stromal cells. Rats were evaluated by cystometry 7, 14, 28 and 42 days after treatment. Brains were extracted for immunostaining.nnnRESULTSnAt 42 days the nonmicroencapsulated group had lower threshold and intermicturition pressure, spontaneous activity and AUC than vehicle treated animals. Rats that received microencapsulated cells had lower threshold pressure at 28 days and lower spontaneous activity at 42 days than vehicle treated rats. Microencapsulated and nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells were noted in the substantia nigra pars compacta up to 42 days after transplantation. At 42 days tyrosine hydroxylase positive neurons were more numerous in the substantia nigra pars compacta of the nonmicroencapsulated group, followed by the microencapsulated and vehicle treated groups.nnnCONCLUSIONSnUrodynamic effects of the 6-hydroxydopamine lesion persisted up to 42 days after vehicle injection. Transplantation of nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells improved urodynamic pressure by 42 days after treatment more markedly than microencapsulated cells. This was associated with more tyrosine hydroxylase positive neurons in the treated substantia nigra pars compacta of the nonmicroencapsulated group, suggesting that functional improvement requires a juxtacrine effect.

Microencapsulation of porcine thyroid cell organoids within a polymer microcapsule construct

Hypothyroidism is a common condition of hormone deficiency, and oral administration of thyroid hormones is currently the only available treatment option. However, there are some disadvantages with this treatment modality including compliance challenges to patients. Therefore, a physiologically based alternative therapy for hypothyroidism with little or no side-effects is needed. In this study, we have developed a method for microencapsulating porcine thyroid cells as a thyroid hormone replacement approach. The hybrid wall of the polymer microcapsules permits thyroid hormone release while preventing immunoglobulin antibodies from entry. This strategy could potentially enable implantation of the microcapsule organoids containing allogeneic or xenogeneic thyroid cells to secret hormones over time without the need for immunosuppression of recipients. Porcine thyroid cells were isolated and encapsulated in alginate-poly-L-ornithine-alginate microcapsules using a microfluidic device. The porcine thyroid cells formed three-dimensional follicular spheres in the microcapsules with decent cell viability and proliferation. Thyroxine release from the encapsulated cells was higher than from unencapsulated cells (Pu2009<u20090.05) and was maintained during the entire duration of experiment (>28 days). These results suggest that the microencapsulated thyroid cell organoids may have the potential to be used for therapy and/or drug screening.

Evaluation of the Tissue Response to Alginate Encapsulated Islets in an Omentum Pouch Model

Islet transplantation is currently in clinical use as a treatment for type I diabetes, but donor shortages and long-term immunosuppression limit broad application. Alginate microcapsules coated with poly-l-ornithine can be used to encapsulate islets in an environment that allows diffusion of glucose, insulin, nutrients, and waste products while inhibiting cells and antibodies. While clinical trials are ongoing using islets encapsulated in alginate microbeads, there are concerns in regards to long-term stability. Evaluation of the local tissue response following implantation provides insight into the underlying mechanisms contributing to biomaterial failure, which can be used to the design of new material strategies. Macrophages play an important role in driving the response. In this study, the stability of alginate microbeads coated with PLO containing islets transplanted in the omentum pouch model was investigated. Biomaterial structure and the inflammatory response were characterized by X-ray phase contrast (XPC) μCT imaging, histology, and immunostaining. XPC allowed evaluation of microbead 3D structure and identification of failed and stable microbeads. A robust inflammatory response characterized by high cell density and the presence of pro-inflammatory macrophages was found around the failed grafts. The results obtained provide insight into the local tissue response and possible failure mechanisms for alginate microbeads.

This paper is a winner in the Undergraduate category for the SFB awards: Evaluation of the tissue response to alginate encapsulated islets in an omentum pouch model.

Islet transplantation is currently in clinical use as a treatment for type I diabetes, but donor shortages and long-term immunosuppression limit broad application. Alginate microcapsules coated with poly-l-ornithine can be used to encapsulate islets in an environment that allows diffusion of glucose, insulin, nutrients, and waste products while inhibiting cells and antibodies. While clinical trials are ongoing using islets encapsulated in alginate microbeads, there are concerns in regards to long-term stability. Evaluation of the local tissue response following implantation provides insight into the underlying mechanisms contributing to biomaterial failure, which can be used to the design of new material strategies. Macrophages play an important role in driving the response. In this study, the stability of alginate microbeads coated with PLO containing islets transplanted in the omentum pouch model was investigated. Biomaterial structure and the inflammatory response were characterized by X-ray phase contrast (XPC) μCT imaging, histology, and immunostaining. XPC allowed evaluation of microbead 3D structure and identification of failed and stable microbeads. A robust inflammatory response characterized by high cell density and the presence of pro-inflammatory macrophages was found around the failed grafts. The results obtained provide insight into the local tissue response and possible failure mechanisms for alginate microbeads.