In this report, we investigate the fibrin-alginate inter penetrating network (FA-IPN) to provide dynamic cell-responsive mechanical properties, which we apply to the in vitro growth of ovarian follicles. The mechanical properties and polymerization rate of the gels were investigated by rheology, and the fiber structure was imaged by electron microscopy. Using a mouse model, twolayered secondary follicles were encapsulated in FA-IPNs, and growth, morphology, hormone production, fibrin degradation rate and the numbers of competent eggs were assessed. The initial mechanics of the FA-IPN are determined by the composite material, and subsequent degradation of fibrin by the encapsulated cells would produce a material with mechanical properties due to the alginate alone. The rate of meotically competent oocytes produced by culture in FA-IPN was 82%, which was significantly greater than in alginate alone. This increase in oocyte quality is an important step in identifying 3D culture systems that can provide a fundamental tool to investigate follicle maturation, and may be applied to promote the growth of human follicles, which can be used to provide reproductive options for women facing a cancer diagnosis

New degradable poly(ester anhydride)s were prepared by the melt polycondensation of diacid oligomers of poly(sebacic acid) (PSA) transesterified with ricinoleic acid. The transesterification of PSA with ricinoleic acid to form oligomers was conducted via a melt bulk reaction between a high molecular weight PSA and ricinoleic acid. A systematic study on the synthesis, characterization, degradation in vitro, drug release, and stability of these polymers was performed. Polymers with weight‐average molecular weights of 2000–60,000 and melting temperatures of 24–77 °C were obtained for PSA containing 20–90% (w/w) ricinoleic acid. NMR and IR analyses indicated the formation of ester bonds along the polyanhydride backbone. These new degradable copolymers have potential use as drug carriers. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1059–1069, 2003

Chemo- and radiation therapies used to treat cancer can have the unintended effect of making patients infertile. Clinically established fertility preservation methods, such as egg and embryo cryopreservation, are not applicable to all patients, which has motivated the development of strategies that involve ovarian tissue removal and cryopreservation before the first sterilizing treatment. To restore fertility at a later date, the early-stage follicles present in the tissue must be matured to produce functional oocytes, a process that is not possible using existing cell culture technologies. This review describes the application of tissue engineering principles to promote ovarian follicle maturation and produce mature oocytes through either in vitro culture or transplantation. The design principles for these engineered systems are presented, along with identification of emerging opportunities in reproductive biology.

Synthetic hydrogels with tunable properties are appealing for regenerative medicine. A critical limitation in hydrogel design at low solids concentration is the formation of defects, which increase gelation times and swelling, and reduce elasticity. Here, we report that tri-functional crosslinking peptides applied to 4-arm poly-(ethylene glycol) (PEG) hydrogels decreased swelling and gelation time relative to bi-functional crosslinkers. In contrast to bi-functional peptides, the third cross-linking site on the peptide created a branch point if an intramolecular crosslink formed, which prevented non-functional “dangling-ends” in the hydrogel network and enhanced the number of elastically active cross-links. The improved network formation enabled mouse ovarian follicle encapsulation and maturation in vitro. Hydrogels with bi-functional crosslinkers resulted in cellular dehydration, likely due to osmosis during the prolonged gelation. For tri-functional crosslinkers, the hydrogels supported a 17-fold volumetric expansion of the tissue during culture, with expansion dependent on the ability of the follicle to rearrange its microenvironment, which is controlled through the sensitivity of the cross-linking peptide to the proteolytic activity of plasmin. The improved network design enabled ovarian follicle culture in a completely synthetic system, and can advance fertility preservation technology for women facing premature infertility from anticancer therapies.

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Polyesteranhydrides synthesized by the transesterification of ricinoleic acid and sebacic acid followed by anhydride polymerization were examined as potential controlled delivery carrier for paclitaxel. Solid and liquid polymers were used. Polymers containing 30% ricinoleic acid are solid whereas polymers containing 70% ricinoleic acid are liquid at body temperature and semisolid at room temperature. It was found that upon addition of the liquid polymer to water it solidifies to form a stable semisolid. Paclitaxel, a potent antitumor agent, was incorporated in the polymers (5-20% w/w) and its release in buffer solution was monitored. Paclitaxel was released for over 100 days while the polymer carrier was being degraded. The release rate was affected by the paclitaxel content; the higher the content, the slower was the release. The toxicity of the polymers and formulations with paclitaxel was examined by subcutaneous injection of liquid polymer samples or implantation of solid polymer specimens to mice for different time periods. Histopathological examination of the tissue surrounding the implant showed minor inflammation 1 week after the injection and no inflammation 3 weeks after implantation. Injection of the polymer without paclitaxel showed no adverse effects.

Cellular phenotype is heavily influenced by the extracellular matrix (ECM), a complex and tissue-specific three-dimensional structure with distinct biophysical and biochemical properties. As naturally derived cell culture platforms are difficult to controllably modulate, engineered synthetic ECMs have facilitated our understanding of how specific matrix properties direct cell behavior. However, synthetic approaches typically lack fibrous topography, a hallmark of stromal and interstitial ECMs in vivo. To construct tunable biomimetic models with physiologic microstructure, we developed a versatile approach to generate modular fibrous architectures in 3D. Photo-cross-linkable polymers were electrospun, photopatterned into desired lengths, and coencapsulated alongside cells within natural biopolymer, semisynthetic, and synthetic hydrogels. Cells encapsulated within fiber-reinforced hydrogel composites (FHCs) demonstrated accelerated spreading rates compared to in gels lacking such fibrous topography. Furthermore, increases in fiber density at constant bulk hydrogel elastic modulus produced morphologically distinct cell populations and modulated cellular mechanosensing in 3D, as evidenced by increased nuclear localization of the mechanosensitive transcription factor, Yes-associated protein (YAP). This work documents the impact of physical guidance cues in 3D and establishes a novel approach to generating more physiologic tissue-and disease-specific biomimetic models.

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of folliclestimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers.This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.