Novel multiarm PEG-based hydrogels for tissue engineering.

Injectable scaffolds are promising substrates for regenerative medicine applications. In this study, multiarm amino-terminated poly(ethylene glycol) (PEG) hydrogels were crosslinked with genipin, a compound naturally derived from the gardenia fruit. Four- and eight-arm amino-terminated PEG hydrogels crosslinked with varying concentrations of genipin were characterized. Both surface and cross-sectional structures of PEG-based hydrogels were observed by scanning electron microscopy. In vitro gelation time, water uptake, swelling, and weight loss of PEG hydrogels in phosphate buffered saline at 37 degrees C were studied. The results showed that the eight-arm PEG demonstrated a much slower gelation time compared with the four-arm PEG, which may be due to the differing structures of the multiarm PEG hydrogels, which in turn affects the ability of genipin to react with the amine groups. Human adipose-derived stem cells were seeded onto the four- and eight-arm PEG hydrogels in vitro to assess the biological performance and applicability of the gels as cell carriers. The four-arm PEG hydrogel resulted in enhanced cell adhesion when compared with the eight-arm PEG hydrogel. Overall, these characteristics provide a potential opportunity for multiarm PEG hydrogels as injectable scaffolds in a variety of tissue engineering applications.

FGF-2 enhances vascularization for adipose tissue engineering.

BACKGROUND: Current therapies for soft-tissue reconstruction include autologous tissue flaps and alloplastic implants. Although autologous fat transplantation using a minimally invasive cannula harvest has less donor-site morbidity than tissue flaps, there is a variable degree of fat resorption over time. Preadipocytes isolated from harvested fat are better able to withstand the mechanical trauma from the suction cannula and subsequently may result in improved cell survival and generation of new fat tissue after transfer to another anatomic site. The authors hypothesized that particulate small intestinal submucosa could be useful as injectable cell delivery vehicles for preadipocytes, and that the release of fibroblast growth factor (FGF)-2 would enhance vascularization. METHODS: Preadipocytes were isolated from discarded human adipose tissue and cultured on small intestinal submucosa particles in a stirred bioreactor (spinner flask). Preadipocytes attached and proliferated on small intestinal submucosa microparticles and maintained high viability over several weeks of culture. FGF-2 was encapsulated in poly(lactic-co-glycolic acid) microspheres and injected in conjunction with the preadipocyte/small intestinal submucosa particles into a mouse subcutaneous model. RESULTS: Preadipocytes attached and proliferated on small intestinal submucosa particles in vitro. In vivo, vascularization was significantly enhanced with the incorporation of FGF-2-loaded poly(lactic-co-glycolic acid) microspheres. In addition, cell survival during the 14-day in vivo observation period was confirmed by fluorescent dye labeling. CONCLUSIONS: Small intestinal submucosa particles are a favorable scaffold for preadipocytes, allowing ex vivo proliferation on particles small enough to be injected. Delivery of FGF-2 from poly(lactic-co-glycolic acid) microspheres resulted in cell survival and enhanced vascularization.

Controlled in vivo degradation of genipin crosslinked polyethylene glycol hydrogels within osteochondral defects.

Polyethylene glycol (PEG) hydrogels show promise as scaffolds for growth factor delivery to enhance cartilage repair. However, methods to control growth factor release in vivo are needed. We have recently shown that in vitro polymer degradation and in vitro growth factor release kinetics can be altered using PEG crosslinked with different concentrations of genipin. However, the degradation and behavior of PEG-genipin in vivo within the cartilage repair site are unknown. This study was conducted to test the hypotheses that the degradation of PEG-genipin can be altered in vivo within osteochondral defects by changing the concentration of genipin, and that PEG-genipin is biocompatible within the mammalian diarthrodial environment. PEG-genipin cylindrical polymers crosslinked using 8mM, 17.6 mM, or 35.2 mM of genipin were implanted into osteochondral defects made in the trochlea of 24 male Sprague- Dawley rats (48 knees). Rats were sacrificed at 5 weeks and gross, cross-sectional, and histologic assessments were performed. Altering the genipin concentration changed the in vivo degradation properties of the hydrogel ( p < 0.01). Consistent with in vitro findings, polymer degradation was inversely related to the concentration of genipin. Near-complete degradation was seen at 8 mM, intermediate degradation at 17.6 mM, and minimal degradation at 35.2 mM. The results of this study show the degradation of PEGgenipin can be altered in vivo within osteochondral defects by changing the concentration of genipin and that PEG-genipin is biocompatible within osteochondral defects. This new in vivo data support potential use of PEG-genipin polymer as an innovative delivery system to control in vivo release of growth factors for improving articular cartilage repair.

Controlled release of bioactive doxorubicin from microspheres embedded within gelatin scaffolds.

We have encapsulated the chemotherapeutic agent doxorubicin into biodegradable polymer microspheres, and incorporated these microspheres into gelatin scaffolds, resulting in a controlled delivery system. Doxorubicin was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) using a double emulsion/solvent extraction method. Characterization of the microspheres including diameter, surface morphology, and in vitro drug release was determined. The release of doxorubicin up to 30 days in phosphate buffered solution was assessed by measuring the absorbance of the releasate solution. Gelatin scaffolds were crosslinked using glutaraldehyde and microspheres were added to gelatin during gelation. The murine mammary mouse tumor cell line, 4T1, was treated with various doses of doxorubicin. A propidium iodide assay was utilized to visualize dead cells. Using a Transwell basket assay, PLGA microspheres and gelatin constructs were suspended above 4T1 cells for 48 h. Viable cells were determined using the CyQUANT cell proliferation assay. Results indicate that the release was controlled by the incorporation of PLGA microspheres into gelatin constructs. A significant difference was seen in the cumulative release over days 5-16 (p < 0.05). The bioactivity of doxorubicin released from the microspheres and scaffolds was maintained as proven by significant reduction in viable cells after treatment with PLGA microspheres as well as with the gelatin constructs (p < 0.001). The drug-polymer conjugate can be used as a controlled drug delivery system in a biocompatible scaffold that could potentially promote preservation of soft tissue contour.

Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels.

Transforming growth factor-beta1 (TGF-beta1) is of great relevance to cartilage development and regeneration. A delivery system for controlled release of growth factors such as TGF-beta1 may be therapeutic for cartilage repair. We have encapsulated TGF-beta1 into poly(DL-lactide-co-glycolide) (PLGA) microspheres, and subsequently incorporated the microspheres into biodegradable hydrogels. The hydrogels are poly(ethylene glycol) based, and the degradation rate of the hydrogels is controlled by the non-toxic cross-linking reagent, genipin. Release kinetics of TGF-beta1 were assessed using ELISA and the bioactivity of the released TGF-beta1 was evaluated using a mink lung cell growth inhibition assay. The controlled release of TGF-beta1 encapsulated within microspheres embedded in scaffolds is better controlled when compared to delivery from microspheres alone. ELISA results indicated that TGF-beta1 was released over 21 days from the delivery system, and the burst release was decreased when the microspheres were embedded in the hydrogels. The concentration of TGF-beta1 released from the gels can be controlled by both the mass of microspheres embedded in the gel, and by the concentration of genipin. Additionally, the scaffold permits containment and conformation of the spheres to the defect shape. Based on these in vitro observations, we predict that we can develop a microsphere-loaded hydrogel for controlled release of TGF-beta1 to a cartilage wound site.