WO2008098109A2 - Hydrogel compositions - Google Patents

Abstract

The invention provides compositions for making hydrogels with improved mechanical properties, particularly for biological cultures. It provides methods for making the hydrogels and for using them. Exemplary hydrogels are made from irradiated or oxidized alginate compositions. Such hydrogels are useful for, especially, culturing follicles in vitro.

Description

HYDROGEL COMPOSITIONS

Reference to Related Application

This application is a continuation-in-part of and claims full benefit of priority of US provisional application number 60/900,214, filed on 8 February 2007, which is herein incorporated by reference in its entirety.

Statement Regarding Government Funding

This invention was made with government support under Grant No. U54 41857 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

The present invention relates to hydrogels with improved properties that better mimic the properties of extracellular matrices and that are useful in, for instance, improved methods for growing follicles. Background

The ovarian follicle is the reproductive unit of the ovary, and consists of a centrally located oocyte surrounded by one or more layers of somatic granulosa and theca cells, which support follicle development. As follicles develop, the somatic cells surrounding the oocyte proliferate and differentiate, and the oocyte grows in preparation for ovulation and fertilization.

In vitro follicle culture systems provide a useful model in which to study folliculogenesis, and unravel some of the key questions that exist about selection of a dominant follicle, regulation of steroidogenesis, and maturation of the oocyte. These culture systems may be employed to identify the mechanisms underlying disorders such as polycystic ovarian syndrome, a condition that impacts nearly 10% of all reproductive age women and results in high levels of androgen production from follicles arrested at an intermediate stage of development. Moreover, the ability to grow follicles in vitro may provide a means to preserve reproductive potential for females facing premature infertility due to cancer therapies. To date, most systems designed to support follicle growth in vitro have been two- dimensional. (West ER, Shea LD, Woodruff TK, Semin Reprod Med, 2007; 25(4):287- 299; Cortvrindt R, Smitz J, Van Steirteghem AC, Hum Reprod, 1996; 11(12):2656-66; Eppig JJ, O'Brien MJ, Biol Reprod, 1996; 54(1): 197-207). In such systems, follicles attach to a two-dimensional culture surface, and somatic cells typically migrate away from the oocyte. This spreading alters the three-dimensional structure of the follicle, thereby disrupting the cell-cell interactions important to the exchange of necessary metabolic precursors from the somatic cells to the egg (Sugiura, K. Pendola FL, Eppig JJ. Developmental Biology 2005; 279: 20-30; Eppig JJ, Pendola FL, Wigglesworth K, Pendola JK. Biol. Reprod. 2005;73: 351-357).

Three dimensional culture systems maintain the three-dimensional structure of mouse follicles in vitro (Pangas SA, Saudye H, Shea LD, Woodruff TK.. Tissue Engineering 2003, 9(5): 1013-1021; Rreeger PK, Fernandes NN, Woodruff TK, Shea LD.. Biol Reprod 2005; 73(5):942-50), which more faithfully mimics the in vivo environment relative to two-dimensional systems (West ER, Shea LD, Woodruff TK. Semin Reprod Med 2007; 25(4):287-299). In fact, the three-dimensional culture systems in our laboratory support follicle survival and growth for 12 days in mouse and up to forty days in non- human primate follicles. Oocytes contained in encapsulated follicle cultures mature and are of sufficient quality to be fertilized. Live pups born from the in vitro-matured eggs are healthy and fertile (Xu M, Kreeger PK, Shea LD, Woodruff TK. Tissue Eng 2006; 12(10): 2739-2746).

We have employed alginate as a three-dimensional matrix for the encapsulation and maturation of ovarian follicles to produce mature fertilizable oocytes (Pangas SA, Saudye H, Shea LD, Woodruff TK; Tissue Engineering 2003, 9(5): 1013-1021; Kreeger PK, Fernandes NN, Woodruff TK, Shea LD.. Biol Reprod 2005;73(5):942-50). Alginate is a widely used biomaterial in tissue engineering applications (Lee KY, Mooney DJ. Chem Rev 2001; 101 (7): 1869-79), and is suitable for follicle culture due to its gentle gelling properties and biochemical characteristics (Gutowska A, Jeong B, Jasionowski M. Anat Rec 2001;263(4):342-9; Alsberg E, Kong HJ, Hirano Y, Smith MK, Albeiruti A, Mooney DJ. J Dent Res 2003;82(l l):903-8; Bouhadir KH, Hausman DS, Mooney DJ. Polymer 1999;40:3575-84; Rowley JA, Madlambayan G, Mooney DJ. Biomaterials 1999;20(l):45-53). Alginate is produced by brown algae, and is a linear polysaccharide copolymer of β-D-mannuronic acid and α-L-guluronic acid (Haug A, Larsen B, Smidsrod O. Acta Chem Scand 1967;21 :691-704; Haug A, Larsen B. Acta Chem Scand

1962; 16: 1908- 18). Alginate gels by ionic crosslinking in the presence of divalent cations, which are not harmful to the encapsulated follicles (Kreeger PK, Deck JW, Woodruff TK, Shea LD.. Biomaterials 2006;27(5):714-23). The alginate gel forms a mesh-like structure that permits diffusion of hormones and other proteins essential for follicle development. Follicle stimulating hormone (FSH), an essential hormone for follicle development, is able to diffuse through the alginate, and causes a dose-dependent increase in in vitro follicle growth (Kreeger PK, Fernandes NN, Woodruff TK, Shea LD. Biol Reprod 2005;73(5):942-50). While our first reports of follicle cultures demonstrated growth in vitro, encapsulated follicles did not achieve the sizes typically observed in vivo, and few follicles differentiate to form a stratified theca cell layer or fluid- filled antral cavity characteristic of mature follicles (Pangas SA, Saudye H, Shea LD, Woodruff TK.. Tissue Engineering 2003;9(5): 1013-1021; Kreeger PK, Fernandes NN, Woodruff TK, Shea LD.. Biol Reprod 2005;73(5):942-50). As a result there is a need for improved materials and methods for in vitro culture of biological materials such as follicles.

Recent results in our lab suggested to us that the physical properties of the matrix may influence follicle development. (Xu M, West E, Shea LD, Woodruff TK. Biol Reprod 2006;75(6):916-23). The mechanical properties and density of natural and synthetic extracellular matrices are known to affect cellular processes and regulate tissue formation. As described herein, these factors were independently investigated for their role in ovarian follicle development. The matrix composition was controlled through decreasing the solids concentration or the molar mass of the encapsulating biomaterial, alginate. We found, in accordance with the invention herein described that appropriate modification of the gel material and the resulting hydrogel provided substantial benefits for in vitro culture of follicles. As described further herein, decreasing matrix stiffness and solids concentration enhanced follicle growth, and coordinated differentiation of the follicle cell types, as evidenced by antral cavity formation, theca cell differentiation, oocyte maturation, and relative hormone production levels. While a stiff environment favored high progesterone and androgen secretion, decreasing alginate stiffness resulted in estrogen production which exceeded progesterone and androgen accumulation. These studies reveal, for the first time, a direct link between the biomechanical environment and follicle function, indicate a novel non-hormonal mechanism regulating follicle development, and provide improved materials and methods for follicle culture based on these insights, exemplified by the modified alginate hydrogels and follicular maturation methods described herein below. Summary of the Invention

In embodiments the invention provides alginate hydrogel compositions comprising alginate treated with gamma irradiation. In embodiments the compositions further comprise alginate that has not been exposed to gamma radiation. In embodiments the gamma irradiated alginate mixed with a 50% solution of untreated alginate. In embodiments the compositions are useful for making hydrogels for culturing follicles, particularly preantral follicles. In embodiments the alginate is exposed to a dose of gamma radiation of between 0.25 and 5 Mrad. In embodiments the alginate treated with gamma radiation is in a solution comprising between about 55% and 65% guluronic acid. In embodiments, the hydrogel has a shear modulus of between about 100 Pa and 20 kPa.

In embodiments the invention provides methods for making alginate compositions useful for making alginate hydrogels comprising treating alginate with gamma irradiation. In embodiments the methods are useful for making hydrogels for culturing follicles, particularly preantral follicles. In embodiments the alginate is exposed to a dose of gamma radiation of between 0.25 and 5 Mrad. In embodiments the alginate treated with gamma radiation is in solution comprising between about 55% and 65% guluronic acid. In embodiments, the hydrogel has a shear modulus of between about 100 Pa and 20 kPa. In embodiments the invention provides a natural or synthetic alginate hydrogel composition for maturing and/or developing an encapsulated preantral follicle, wherein the hydrogel is produced by (a) treating an alginate solution with gamma radiation; and (b) crosslinking the treated alginate solution. In embodiments the alginate solution is exposed to a dose of gamma radiation of between about 0.25 and 5 Mrad. In embodiments the alginate solution treated with gamma radiation comprises between about 55% and 65% guluronic acid. In embodiments, the hydrogel has a shear modulus of between about 100 Pa and 20 kPa.

In embodiments the invention provides an alginate hydrogel composition for maturing and/or developing an encapsulated preantral follicle, wherein the hydrogel is produced by oxidizing an alginate solution to a theoretical extent of between about 0.25% and 5%, prior to encapsulating the preantral follicle and subsequently crosslinking the solution.

In embodiments the alginate solution is oxidized by treatment with an oxidizing agent. In embodiments the oxidizing agent is any one or more of the following (any one or more selected from the group consisting of): sodium periodate, Ammonium Cerium (IV) Nitrate, Bleach, N-Bromosuccinimide, Η-tert -Butylbenzenesulfϊnimidoyl chloride, tert-Butyl hydroperoxide, CAN, Cerium Ammonium Nitrate, 3-Chloroperoxybenzoic acid, Chromium Compounds, CMHP, Copper Compounds, Cumene hydroperoxide, Dess- Martin Periodinane, Dimethyl sulfoxide, Ferric Nitrate, Formic Acid, Hydrogen peroxide, Hydrogen peroxide urea adduct, Hypervalent iodine compounds, IBX, Iodine, lodosobenzene diacetate, lodosylbenzene, 2-Iodoxybenzoicacid, Iron(III), (V) and (IV), Manganese Compounds, Manganese (IV) oxide, MCPBA, meto-Chloroperbenzoic acid, JV-Methylmorpholine-iV-Oxide, Molybdenum Compounds, N-Bromosuccinimide, N- Methylmorpholine-iV-Oxide, NMO, N-tert -Butylbenzenesulfϊnimidoyl chloride, Osmium tetroxide, Oxalyl chloride, Oxone, Oxygen, Ozone, Peracetic acid, Periodic acid, Peroxy acids, Potassium Permanganate, Pivaldehyde, Potassium peroxomonosulfate, Ruthenium(III) and (IV), Sodium hypochlorite, Sodium periodate, TEMPO, N-tert - Butylbenzenesulfϊnimidoyl chloride, tert-bvXy\ hydroperoxide, Tetrabutylammonium peroxydisulfate, 2,2,6, 6-Tetramethylpiperidinyloxy, Triacetoxyperiodinane, Trifluoroacetic peracid, Trimethylacetaldehyde, UHP, Urea hydrogen peroxide adduct, and Vanadium compounds. In embodiments the invention provides an alginate hydrogel composition for maturing and/or developing an encapsulated cell, comprising a solution crosslinked/prepared from an alginate solution comprising between about 55% and 65% guluronic acid, wherein the hydrogel has a shear modulus of between about 100 Pa and 20 kPa. In embodiments the alginate solution is gamma irradiated. In embodiments the alginate solution is treated with an oxidizing agent. In embodiments the oxidizing agent is any one or more selected from the immediately foregoing group of oxidizing agents.

In embodiments the invention provides an in vitro method for maturing a preantral follicle comprising: (A) irradiating a non-crosslinked alginate solution with gamma radiation; (B) suspending a preantral follicle into irradiated non-crosslinked solution; (C) crosslinking the suspension, thereby forming a preantral follicle-single three dimensional gel matrix, wherein the follicle is encapsulated within the matrix, and wherein the gel matrix has a shear modulus of between about 100 Pa and 20 kPa; (D) culturing the preantral follicle in the three dimensional matrix, wherein the preantral follicle forms an antral cavity, and (E) releasing the antral follicle from the three dimensional gel matrix.

Brief Description of the Figures

Figure 1. Shear elastic modulus (G) of each alginate condition. Data represented as average±SEM from three or more independent measurements. In this and other figures, 3% = 3% untreated, 1.5% = 1.5% untreated, 0.7% = 0.7% untreated, ox = oxidized, and irr = irradiated, and significant differences between conditions are indicated by different letters (p<0.05).

Figure 2. Two-layered secondary follicle growth: (A) Growth over a 12 day culture period. (B) Percent increase in follicle diameter at day 12 (relative to day 0) of culture (p<0.05). (C-F) A representative follicle at day 0 of culture (C), and at day 12 of culture in 3% (D), 1.5% (E), and irradiated (F) alginate. Oo = oocyte, scale bar = 50 μm. Data represented as average ± SEM from two or more independent experiments for each alginate condition. Figure 3. Multilayered secondary follicle growth: (A) Growth in alginate over an

8-day culture period. (B) Percent increase in follicle diameter at day 8 (relative to day 0) of culture. (CF) A representative follicle at day 0 of culture (C), and at day 8 of culture in 3% (D), 1.5% (E), and 0.7% (F) alginate. Oo = oocyte, a = antral cavity, scale bar =100 μm. Significant differences between alginate conditions are indicated by different letters (p<0.05). Data represented as average ± SEM from two or more independent experiments for each alginate condition.

Figure 4. Antral cavity formation: Percent of (A) two-layered and (B) multilayered secondary follicles that form an antral cavity by the end of culture. (p<0.05)

Figure 5. Theca layer formation. After 12 days of culture, a stratified theca layer surrounds a follicle encapsulated as a two-layered secondary follicle in irradiated alginate. Oo = oocyte, gc = granulosa cells, tc = theca cells, scale bar = 50 μm.

Figure 6. Steroid production by two-layered and multilayered secondary follicles. Estradiol, androstenedione, and progesterone concentrations in culture media during culture and at the final day of culture for two-layered (A-C) and multilayered (D-F) secondary follicles. Significant differences between alginate conditions are indicated by different letters (p<0.05). Data represented as average ± SEM from two or more independent experiments for each alginate condition.

Figure 7. Oocyte quality: Oocytes collected from (A) two-layered and (B) multilayered secondary follicle cultures classified as MR following hCG treatment. (p<0.05).

Figure 8. Proposed model for mechanical regulation of ovarian function and disease: (A) Immature follicles reside in the cortex in the human ovary. Unknown signals stimulate follicle development from the primordial to primary to secondary stage of development. We suggest that the biomechanics of the environment contribute to follicle development and the relative dense cortex maintains quiescence while the perimedullar interior of the ovary represents a more permissive biomechanical environment. (B) The follicles found in a PCOS ovary are usually small, accumulate in the cortex, and secrete high levels of androgen and relatively low levels of estrogen. The underlying etiology of this disease is unknown. Based on our work, we hypothesize that the relatively dense cortex creates a biomechanically non-permissive environment and is one of the contributing factors in the disease.

Glossary Words, terms and phrases generally are used herein in accordance with their meanings to those skilled in the pertinent arts and, unless defined otherwise herein, all technical and scientific terms used herein have the same meanings as would be commonly understood by one of skill in the art to which the disclosed invention and the term as used herein pertains. Illustrative descriptions of the meanings of certain terms used herein are set forth for clarity below.

"a" and "an" as used herein, including in the claims, include both the singular and the plural; that is, as used herein they have the same meaning as "one or more than one" and the same meaning as "at least one." Description of the Invention

Mechanical stresses are known to affect cell behavior in a variety of tissues (Discher DE, Janmey P, Wang YL.Science 2005;310(5751): 1139-43; Brandl F, Sommer F, Goepferich A. Biomaterials 2007;28(2): 134-46), including those which are not thought to be mechanically challenged, and this is the case for the ovary (Paszek MJ, Weaver VM. J Mammary Gland Biol Neoplasia 2004;9(4):325-42; Marti A, Feng Z, Altermatt HJ, Jaggi R. Eur J Cell Biol 1997;73(2): 158-65; Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, et al. Cancer Cell 2005;8(3):241-54; Oster GF, Murray JD, Harris AK. J Embryol Exp Morphol 1983;78:83-125). In accordance with this in vivo behavior, it has been observed that the mechanical properties of hydrogels can regulate cellular processes in cells in vitro, such as cell proliferation and extracellular matrix production, leading to tissue formation in engineered tissues (Bryant SJ, Chowdhury TT, Lee DA, Bader DL, Anseth KS. Ann Biomed Eng 2004;32(3):407-17;Bryant SJ, Durand KL, Anseth KS. J Biomed Mater Res A 2003;67(4): 1430-6; Bryant SJ, Anseth KS. J Biomed Mater Res 2002;59(l):63-72).

Embodiments of the present invention provides hydrogels with mechanical characteristics that enhance in vitro bio-culture, particularly follicular growth. In particular, embodiments of the present invention provides alginate hydrogels ranging in stiffness, formed by, for instance, varying the alginate solids concentration, or by treating the polymer with radiation or chemical oxidation, that provide improved hydrogels for, for instance, biological cultures, such as, in particular, follicle culture (Kong HJ, Smith MK, Mooney DJ. Biomaterials 2003;24(22):4023-9; Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ. Biotechnol Prog 2001;17(5):945-50; Kong HJ, Lee KY, Mooney DJ. Polymer 2002;43:6239-46).

In embodiments of the invention, matrices were developed to represent conditions with constant density with varied stiffness, as well as constant stiffness with varied density. The mechanical properties of each condition were characterized by measurement of the shear elastic modulus. Using a mouse model, two-layered and multilayered secondary follicles were isolated and encapsulated in each hydrogel condition, and growth, morphology, and hormone production were assessed. At the end of culture, the quality of the oocytes was assessed based upon their ability to resume meiosis. Identifying the mechanical properties and solids concentration of the hydrogel that maximize follicle development provides a novel method for optimizing hydrogels for biological culture, such as for follicle development, provides improved hydrogels for this purpose and also provides improved in vitro culture methods. The optimization methods, moreover, represent a critical step toward developing a system that will enable follicle growth following ovarian tissue banking. The stiffness of the alginate matrix influences the growth and differentiation of multiple cellular compartments with the follicle. Three methods were employed to control the stiffness of alginate: irradiation, oxidation, and variation of solids concentration. The resulting shear elastic moduli (G)' represent a range of three significantly different conditions: high G' (3% alginate), intermediate G' (1.5% alginate), and low G' (0.7%, oxidized, and irradiated alginate). Follicles matured in these matrices demonstrated increased growth with decreasing alginate stiffness. Follicles cultured in low G' alginate also had higher rates of antrum and theca layer formation than follicles cultured in more stiff hydrogels. Additionally, androgen and progesterone were converted to estrogen in less stiff conditions, whereas androgen or progesterone exceeded estradiol levels in the more stiff conditions. Most importantly, oocytes cultured in low G' alginate were of higher quality, as measured by their ability to resume meiosis, than oocytes cultured in relatively stiff alginate conditions. Taken together, these results suggest that the physical properties of the follicle environment influence cellular processes and the coordination between the cellular compartments necessary for successful maturation. This mechanical regulation of follicle development is new to follicle biology, as endocrine hormones have been widely characterized as the primary factors regulating development. In addition, these findings emphasize the broad impact that the physical properties of a biomaterial may have on engineered tissues, particularly for tissues that are not traditionally thought to be mechanically stimulated.

Multiple alginate conditions can be selected to isolate the role of matrix stiffness and solids concentration on follicle development. The roles of matrix stiffness and solids concentration were isolated by manipulating the alginate molecular weight and composition of the hydrogels. A previous report has shown that decreasing the solids concentration leads to enhancement in follicle growth (Xu M, West E, Shea LD, Woodruff TK., Biol Reprod 2006;75(6):916-23); however, decreasing the percentage of alginate in solution both decreases shear elastic modulus, and may also facilitate diffusion of molecules through the alginate. To examine this question more thoroughly, the stiffness of the matrix was decreased by varying the molecular weight of the polymer while maintaining a constant solids concentration. Preparations of untreated, oxidized, and irradiated alginate had similar solids concentration (1.5% w/v); however, the shear modulus ranged from 200 Pa to 1300 Pa for these formulations. The shear modulus of the present invention may range from about 100 Pa to about 20 kPa. High doses of radiation cause breakage in the guluronic acid blocks, thereby decreasing the ability of the alginate to form crosslinks (Kong HJ, Smith MK, Mooney DJ. Biomaterials 2003;24(22):4023-9).

Oxidation treatment renders alginate susceptible to hydrolysis and cleavage of alginate polymer chains, thereby decreasing the molar mass. Alginate may be oxidized by any number of oxidizing agents known in the art. Oxidizing agents include, but are not limited to, sodium periodate, Ammonium Cerium (IV) Nitrate, Bleach, N-

Bromosuccinimide, ^~N-tert -Butylbenzenesulfinimidoyl chloride, tert-QvXy\ hydroperoxide, CAN, Cerium Ammonium Nitrate, 3-Chloroperoxybenzoic acid, Chromium Compounds, CMHP, Copper Compounds, Cumene hydroperoxide, Dess-Martin Periodinane, Dimethyl sulfoxide, Ferric Nitrate, Formic Acid, Hydrogen peroxide, Hydrogen peroxide urea adduct, Hypervalent iodine compounds, IBX, Iodine, lodosobenzene diacetate, lodosylbenzene, 2-Iodoxybenzoicacid, Iron(III), (V) and (IV), Manganese Compounds, Manganese (IV) oxide, MCPBA, metø-Chloroperbenzoic acid, JV-Methylmorpholine-iV- Oxide, Molybdenum Compounds, iV-Bromosuccinimide, JV-Methylmorpholine-iV-Oxide, NMO, N-tert -Butylbenzenesulfmimidoyl chloride, Osmium tetroxide, Oxalyl chloride, Oxone, Oxygen, Ozone, Peracetic acid, Periodic acid, Peroxy acids, Potassium Permanganate, Pivaldehyde, Potassium peroxomonosulfate, Ruthenium(III) and (IV), Sodium hypochlorite, Sodium periodate, TEMPO, N-tert -Butylbenzenesulfϊnimidoyl chloride, tert-bvΛy\ hydroperoxide, Tetrabutylammonium peroxydisulfate, 2,2,6,6- Tetramethylpiperidinyloxy, Triacetoxyperiodinane , Trifluoroacetic peracid, Trimethylacetaldehyde, UHP, Urea hydrogen peroxide adduct, and Vanadium compounds. Follicle growth, antrum formation, theca development, steroid production, and the ability of the oocytes to resume meiosis were all enhanced by culture within the oxidized and irradiated alginate relative to the unmodified alginate of the same solids concentration. Decreasing the stiffness of the matrix is hypothesized to allow for the maintenance of tensional homeostasis within the cellular compartments, thereby promoting the cellular processes that create a local paracrine milieu that increases oocyte quality.

The role of solids concentration on follicle development was investigated by comparing 0.7% untreated alginate with the oxidized and irradiated alginate. The oxidized, irradiated, and 0.7% alginate had similar shear moduli but differed in the solids concentration (1.5% vs. 0.7%). Decreasing the solids concentration of the matrices did not significantly affect growth or oocyte quality relative to oxidation or radiation treatment, but antrum formation was significantly enhanced in low concentration alginate for both stages of follicles. Decreasing the solids concentration may influence diffusion, either increasing availability of factors that promote cellular processes or allowing inhibitory factors to escape. In addition to antrum formation, decreasing the solids concentration led to an increase in estradiol production and decreased relative levels of androstenedione and progesterone. These relative hormone levels determined at 0.7% alginate represent the steroid profile of a healthy, growing follicle in vitro. Taken together, these experiments demonstrate matrix stiffness substantially enhances follicle development and steroid production, which is further enhanced by the solids concentration of the matrix. Our results demonstrate that ovarian mechanics function to coordinate the differentiation of the compartments of the follicle (oocyte, granulosa, and theca cells). Follicular cells communicate via secreted factors and gap junctions; thus, a mechanical stimulus or signal is quickly transmitted throughout the follicle population, a phenomenon observed in other engineered systems (Swartz MA, Tschumperlin DJ, Kamm RD, Drazen JM. Proc Natl Acad Sci USA 2001;98(l l):6180-5). While mechanotransduction is a regulator of tissue development, the molecular basis of this process has only been investigated relatively recently, and much remains unknown (Discher DE, Janmey P, Wang YL. Science 2005;310(5751): 1139-43; Ingber DE. Ann Med 2003;35(8):564-77). Mechanotransduction maybe attributed to autocrine mechanoregulatory circuits in mechanoresponsive tissues (Paszek MJ, Weaver VM. J Mammary Gland Biol Neoplasia 2004;9(4):325-42; Tschumperlin DJ, Dai G, MaIy IV, Kikuchi T, Laiho LH, McVittie AK, et al. Nature 2004;429(6987):83-6), and similar mechanisms may be in place in the ovary. Expression of other factors including fibroblast growth factor, PI 3 -kinase, Akt, transforming growth factor beta (TGFβ) and matrix metalloproteinases (MMPs), among others, is also mediated by mechanical loading (Paszek MJ, Weaver VM. J Mammary Gland Biol Neoplasia 2004;9(4):325-42). Additionally, tensional forces exerted by gel matrices may regulate cell aggregate morphogenesis via a GTPase-dependent mechanism (Paszek MJ, Weaver VM. J Mammary Gland Biol Neoplasia 2004;9(4):325-42; Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, et al. Cancer Cell 2005;8(3):241-54). Any combination of mechanically-activated signaling pathways may contribute to the role of the matrix stiffness on follicle development and disease, suggesting a series of new lines of investigation. This study represents the seminal investigation into the role of mechanotransduction in ovarian follicle development.

The physical properties of the encapsulating alginate matrix represent a novel mechanism for the regulation of follicle development, namely the extracellular matrix stiffness and density. To date, the factors known to regulate follicle development in vivo have been diffusible gonadotropins. However, the activity of these factors is likely context dependent, and the presentation of these factors to follicles entrapped within a matrix that has a non-permissive stiffness or density may be the fundamental mechanism that underlies some ovarian disorders. Specifically, the steroid profiles reported herein for stiff and dense matrices resemble trends observed in ovarian disease. The high level of androstenedione and progesterone in the immature follicles cultured under the 'non- permissive' condition (3%) suggests inadequate steroid conversion and is consistent with poor follicle health (Erickson GF, Magoffm DA, Garzo VG, Cheung AP, Chang RJ. Hum Reprod 1992;7(3):293-9) and follicles from women with PCOS (Willis DS, Watson H, Mason HD, Galea R, Brincat M, Franks S. J Clin Endocrinol Metab 1998; 83(11):3984- 3991; Joseph-Home R, Mason H, Batty S, White D, Hillier S, Urquhart M, et al. Hum Reprod 2002;17(6):1459-1463). Additionally, PCOS follicles have a significant amount of stroma and theca cell hypertrophy, and PCOS ovarian cortexes are thicker and more collagenized than normal ovaries (Hughesdon PE. Obstet Gynecol Surv 1982;37(2):59- 77). A provocative interpretation is that the biomechanical environment of the PCOS ovary creates the in vivo equivalent of a 'non-permissive' mechanical environment and phenocopies the 'stiff 3% alginate conditions (Figure 7). Equally intriguing is the notion that follicle selection may rely on a biomechanical signal from the surrounding cortex. Thus, the in vitro follicle culture system permits a more detailed evaluation of this concept and may provide new clues regarding follicle development in both the normal ovary and in PCOS. In conclusion, the results of this study demonstrate that low- stiffness alginate is permissive to follicle maturation, as demonstrated by follicle growth, differentiation, appropriate steroid production, and good oocyte quality. While effects of matrix stiffness on cells in three-dimensional culture have been well-documented, little is known about the forces and signaling that regulate ovarian morphology and coordinated follicle function. This study documents a previously unappreciated role of these forces on follicle function and opens a new line of investigation on normal follicle selection and possible explanations for ovarian diseases.

The following Example are provided by way of illustration and not limitation of the invention herein described. It is to be understood that a full understanding of the invention herein described is to be had by reading the entirety of the disclosure in the light of the knowledge and understanding of a person skilled in the arts to which it pertains.

Examples

EXAMPLE 1. Materials and Methods used in Examples 2-6 Animals and materials Male CBA and female C57BL/6 mice (Harlan, Indianapolis, IN) were housed as breeding pairs in a temperature- and light-controlled environment and provided with food and water ad libidum. Animals were fed phytoestrogen- free Teklad Global irradiated 2919 chow. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and protocols were approved by the IACUC at Northwestern University. Unless otherwise specified, all chemicals were purchased from Sigma- Aldrich (St. Louis, MO) and media formulations from Invitrogen (Carlsbad, CA).

Alginate preparation Sodium alginate (55-65% guluronic acid) was generously provided by FMC

BioPolymers (Philadelphia, PA). Radiation-treated alginate was placed in a Cesium- 137 irradiator (M38-1 Gammator, Radiation Machinery Corp., Parsippany, NJ) and exposed to doses of gamma radiation ranging from 0.25 to 5 Mrad. Prior to encapsulation, the selected 5 Mrad-treated alginate was blended with 50% untreated alginate so that the resulting gel maintains sufficient integrity for follicle encapsulation. Oxidized alginate was treated to a theoretical extent ranging from 0.25-5% (i.e. 0.25-5% of alginate monomers oxidized) with sodium periodate as previously described (26) and dialyzed four days to remove the remaining reactants. The 1% oxidized alginate was used for all follicle encapsulations. For purification, alginate was dissolved at a concentration of 1% (w/v) in deionized water. Activated charcoal (Fisher Scientific, Hampton, NH) was then added (0.5 g charcoal/g alginate) and stirred in the alginate solution for 30 min to remove organic impurities. The solutions were then filtered through 0.22 μm filters and lyophilized within Steriflip conical tubes (Millipore, Billerica, MA). Prior to use, aliquots of alginate were reconstituted overnight with sterile 1 x phosphate buffered saline to 0.7%, 1.5%, or 3% (w/v) concentrations.

Alginate characterization

Alginate molar mass was measured by gel permeation chromatography (GPC) to characterize the extent of polymer degradation following radiation and oxidation. Briefly, each alginate solid was dissolved in water at a concentration of 2 mg/ml, and injected into a series of three size-exclusion columns (Shodex SB-806 HQ, SB-804 HQ, and SB-802.5 HQ, Showa Denko, Kawasaki, Japan). In this tandem GPC-MALLS mode, the effluent from the GPC system flows directly into a DAWN DSP Laser Photometer and Optilab DSP Interferometric Refractometer connected in series (both Wyatt Technology, Santa Barbara, CA). The flow rate was 0.3 ml/min and the mobile phase consisted of 100 mM NaCl, 50 mM NaH₂PO₄, and 200 ppm NaN₃. The tandem GPC-MALLS data were processed using ASTRA software (Wyatt Technology, Santa Barbara, CA) to determine weight average molar mass (M_w), radius of gyration (R_g), and polydispersity index (PDI). Three separate measurements were collected for each alginate condition and the results were averaged.

Alginate shear modulus was measured at 25°C using a Paar Physica MCR Rheometer (Anton Paar, Graz, Austria) using a parallel plate geometry (diameter of 50 mm, gap of 1 mm) and Paar Physica US200 software (Anton Paar, Graz, Austria). Alginate and a 40% (w/v) CaSO₄ slurry were quickly blended (40 μl CaSO₄ slurry/ml alginate) and extruded onto the lower plate of the rheometer. The upper plate was quickly lowered to create a 1 mm gap, and the alginate crosslinked between the plates for two hours. Storage moduli (G) were determined by oscillatory shear experiments performed with a strain amplitude of 1% and oscillation frequency of 10 rad/s, which is within the linear viscoelasticity region, as verified by frequency and strain sweeps following each measurement.

Follicle isolation, encapsulation, and culture

Two-layered (100-130 μm) and multilayered (150-180 μm) secondary follicles (4- 5a and 5b size follicles, respectively, according to the Pedersen and Peters classification (28)) were mechanically isolated from the ovaries of 12- and 16-day old mice, respectively, as previously described (Kreeger PK, Deck JW, Woodruff TK, Shea LD.Biomaterials 2006;27(5):714-23) . Follicles were then transferred into beads of alginate solution on a polypropylene mesh (0.1 mm opening) and immersed in a calcium- containing solution (50 mM CaCl₂, 140 mM NaCl) for two minutes to crosslink the alginate solution as previously described (Kreeger PK, Deck JW, Woodruff TK, Shea LD.Biomaterials 2006;27(5):714-23; Kreeger PK, Fernandes NN, Woodruff TK, Shea LD. Biol Reprod 2005;73(5):942-50). The beads were then rinsed and transferred to growth medium composed of alpha minimum essential medium (αMEM) supplemented with 10 mIU/ml recombinant human follicle stimulating hormone (Organon, Roseland, NJ), 3 mg/ml bovine serum albumin (BSA), 1 mg/ml bovine fetuin, 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml selenium. After encapsulating all follicles, beads were placed in individual wells of a 96-well plate containing 100 μl growth medium, and cultured for eight to twelve days. Half the medium (50 μl) was exchanged every two days. At the end of follicle culture, the conditioned media was stored at -80⁰C for hormonal analysis. Follicle images were collected using a Leica DM IL light microscope (Leica, Wetzlar,Germany) equipped with phase objectives and a heated stage, a Spot Insight 2 Megapixel Color Mosaic camera, and Spot software (Spot Diagnostic Instruments, Sterling Heights, MI). Follicle diameters were measured using ImageJ software (National Institutes of Health, USA).

Oocyte meiotic competence

Oocyte meiotic competence was assessed by maturation after twelve days of culture for two-layered secondary follicles and after eight days of culture for multilayered secondary follicles. Follicles were removed from beads by incubating the beads in a 10 IU/ml solution of alginate lyase in pre-equilibrated αMEM at 37°C for one hour, which enzymatically degrades the alginate bead. Follicles were then transferred to αMEM containing 0.25 pg/ml epidermal growth factor and 45 mIU/ml human chorionic gonadotropin and incubated at 37°C for 14-16 hours. Oocytes were removed from follicles, and images collected by Hoffman Modulation Contrast microscopy using a Leica DM IL microscope. Oocyte state was assessed from the images, and characterized as DG (degenerated), GV (intact germinal vesicle), or GVBD (germinal vesicle breakdown) based on the presence or absence of a germinal vesicle and polar body. Hormone assays

Androstenedione, 17β-estradiol, and progesterone were measured using commercially available radioimmunoassay kits (androstenedione and 17β-estradiol, Diagnostic Systems Laboratories, Inc., Webster, TX; progesterone, Diagnostic Products Corporation, Los Angeles, CA). The sensitivities for the androstenedione, estradiol, and progesterone assays are 0.1 ng/ml, 10 pg/ml, and 0.1 ng/ml, respectively. Assays were performed at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core. Media collected from follicles every other day of culture was stored at -80⁰C prior to analysis. To obtain sufficient media for each assay, media collected from follicles in identical alginate conditions was pooled for each time point (12- 20 samples pooled per measurement). Statistics

Statistical calculations were performed using JMP 4.0.4 software (SAS Institute, Gary, NC). Statistical significance for follicle size measurements and steroid levels was analyzed using a two-way ANOVA with repeated measures, or one-way ANOVA followed by Tukey-HSD for single time points, χ² analysis was used to analyze categorical data. EXAMPLE 2. Alginate characterization

The molar mass of alginate decreased with increasing radiation and oxidation treatment as shown in Table 1. The oxidation and radiation conditions selected (1% and 5 Mrad, respectively) decreased the molecular weight by a factor of 1.4 and 5.0, respectively. The polydispersity of the polymer increased with both oxidation and radiation indicating a broader range of polymer molar masses present. Polydispersity ranged from 1.90 to 3.12. The objective of this report was to investigate ovarian follicle maturation as a function of the physical properties of the hydrogel, thus, the shear modulus of each alginate formulation was measured. Formulations consisted of the following: 1) 3% alginate 2) 1.5% alginate, 3) 0.7% alginate, 4) 1.5% oxidized alginate, and 5) 1.5% irradiated alginate. The shear elastic modulus (G) was decreased by decreasing alginate solids concentration as well as by oxidation and radiation treatment (Figure 1). The oxidized, irradiated, and 0.7% alginate conditions had significantly lower moduli than the 1.5% and 3% alginate conditions, and do not differ significantly from one another. 3% alginate has a shear elastic modulus significantly higher than all other alginate conditions, while 1.5% alginate has an intermediate shear elastic modulus.

Oxidized alginate can degrade over time via hydrolysis (Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney DJ. Biotechnol Prog 2001;17(5):945-50), however in our system, alginate elastic modulus measurements varied minimally over 12 days. This observation can likely be attributed to the extensive dialysis following oxidation that hydrolyzes the polymer prior to follicle encapsulation.

EXAMPLE 3. Two-layered and multilayered secondary follicle encapsulation and growth

Follicle growth was subsequently investigated in hydrogels with varied stiffness. Media conditions were held constant. A minimum of 130 two-layered secondary follicles were encapsulated for each alginate conditions and cultured for 12 days. At the end of culture, 15-42% of follicles remained viable. Follicles cultured in hydrogels with decreased stiffness yielded greater increases in follicle diameter (Figure 2a-b). Two- layered follicles cultured in high-modulus 3% alginate increased 16.6% in diameter during 12 days of culture, which is significantly less than the percent increases in diameter of 97.8, 106.4, and 107.4 in the case of 0.7% alginate, oxidized alginate, and irradiated alginate, respectively (Figure 2b). Follicles cultured in the mid-range shear modulus alginate (1.5% alginate) increased 79.0% in diameter, which is a significant decrease relative to the growth in irradiated and oxidized alginate. Varying the method of alginate treatment to decrease shear modulus (decreasing alginate %, oxidation, and irradiation) did not significantly affect growth of follicles.

Multilayered secondary follicles demonstrated a similar trend for follicle growth in hydrogels. A minimum of 60 multilayered secondary follicles were encapsulated in alginate of each condition and 83-91% of the follicles remained viable following eight days of culture. Similarly to the two-layered follicles, all surviving multilayered secondary follicles in alginate grew during the culture period, and hydrogels with a decreased stiffness yielded greater increases in follicle diameter (Figure 3 a, b). Follicles cultured in 3% alginate averaged a 24.7% increase in follicle diameter, while those cultured in 1.5% alginate increased 92.1% in diameter (Figure 3b). Follicles in the lowest shear elastic moduli alginate conditions demonstrated significantly greater increases in follicle diameter relative to 3% and 1.5% alginate (Figure 3a,b). Follicles in 0.7% alginate, oxidized alginate, and irradiated alginate increased in diameter 131.5%, 147.2%, and 139.8%, respectively. Varying the method of alginate treatment to decrease shear modulus did not significantly affect the growth of multilayered secondary follicles. EXAMPLE 4. Follicle morphology and cell differentiation The formation of an antrum, a fluid- filled central cavity, is a characteristic of follicle development in vivo. Follicles encapsulated in 1.5% alginate were not able to form an antrum (Kreeger PK, Fernandes NN, Woodruff TK, Shea LD. Biol Reprod

2005;73(5):942-50), however recent reports indicate that mechanically permissive alginate enhances formation of the antral cavity (Xu M, West E, Shea LD, Woodruff TK., Biol Reprod 2006;75(6):916-23). Antrum formation was observed in the alginate culture system (Figure 3f), and the percentage of both two-layered and multilayered secondary follicles forming antra increased with decreasing shear elastic modulus of the encapsulating alginate (Figure 4). A larger percentage of multilayered follicles than two- layered secondary follicles were able to form antra in all alginate conditions. In both follicle classes, the rate of antrum formation was significantly higher in the 0.7% alginate condition than in other conditions (36.8% of two-layered secondary follicles and 80.6% of multilayered secondary follicles). Irradiated and oxidized alginate also supported increased rates of antrum formation relative to 3% and 1.5% alginate, although this increase is only significant in the case of multilayered secondary follicles. Theca formation was also enhanced in low- stiffness alginate. Both two-layered and multilayered secondary follicles formed stratified theca layers in low-stiffness conditions (Figure 5). A theca layer was observed in 16.4% of two-layered secondary follicles cultured in irradiated alginate and 10.7% of multilayered secondary follicles cultured in 0.7% alginate. The theca layer was most often observed surrounding relatively small follicles, perhaps indicating that the theca is present and proliferating in more follicles than observed by visual inspection, but is not discernable because the theca cells are distributed over an increased surface area, thereby decreasing the thickness of the layer. EXAMPLE 5. Steroid secretion Follicular cells secrete ovarian hormones during normal development, and therefore measurements of estradiol, androstenedione, and progesterone were performed on culture media collected from cultured follicles. While significant differences in steroid production levels were not detected in two-layered secondary follicles, measurements over the culture period demonstrate that follicles in 0.7% alginate have enhanced steroid production as early as day 6 of culture. Multilayered secondary follicles secreted significantly more estradiol and androstenedione with decreased alginate stiffness (Figure 6). Measurements at earlier time points were below the detectable limits of the assay. In both follicle size classes, estradiol production was highest in 0.7% alginate, which correlates with the enhanced antrum formation in this low solids concentration alginate condition. Estradiol and androstenedione are produced primarily by granulosa cells and theca cells, respectively, in vivo during development, and production of these steroids in vitro demonstrates that follicular cells are able to carry out their steroidogenic roles in vitro. Progesterone levels did not differ significantly between alginate conditions in two- layered or multi-layered secondary follicles. Appropriate steroid biosynthesis is reflected in the ratio of secreted estradiol, androstenedione, and progesteron (Table 2). The ratio of progesterone and/or androstenedione to estradiol decreases with decreasing alginate stiffness in the immature follicles. Androgen accumulation is not as pronounced in the multilayered follicle but progesterone is still elevated. Together with the growth and survival data, the steroid profiles suggest that the permissive environment is one that is less stiff.

EXAMPLE 6 Oocyte quality

The quality of oocytes retrieved from follicles cultured within alginate hydrogels was measured by their ability to resume meiosis. Oocytes isolated from follicles cultured in 3% alginate demonstrated the lowest rate of meiotic resumption: 31.3% of two-layered secondary follicles and 46.4% of multilayered secondary follicles (Figure 7). Increased percentages of oocytes able to resume meiosis, as indicated by germinal vesicle breakdown (GVBD), were observed in the lower shear moduli alginate conditions. Irradiated alginate culture yielded the highest percentage of GVBD oocytes from two- layered secondary follicles (65.7%), while 0.7% alginate yielded the highest percentage of GVBD oocytes from multilayered secondary follicles (90.8%).

It is understood that the disclosed invention is not limited to the particular methodology and protocols as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art, from reading the present disclosure, will be able to ascertain and will be enabled to practice many additional features and embodiments of the invention, and it is to be understood that such features and embodiments, including similar to and equivalents to those specifically described herein are within the scope of the disclosure and of the claims corresponding thereto.

Publications cited herein and the material for which they are cited are specifically incorporated by reference. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Claims

What is claimed is:

1. A natural or synthetic alginate hydrogel composition for maturing and/or developing an encapsulated preantral follicle, wherein the hydrogel is produced by (a) treating an alginate solution with gamma radiation; and (b) crosslinking the treated alginate solution.

2. The alginate hydrogel composition of claim 1 , wherein the alginate solution is exposed to a dose of gamma radiation of between about 0.25 and 5 Mrad.

3. The alginate hydrogel composition of claim 1, wherein the alginate solution treated with gamma radiation comprises between about 55% and 65% guluronic acid.

4. An alginate hydrogel composition for maturing and/or developing an encapsulated preantral follicle, wherein the hydrogel is produced by oxidizing an alginate solution to a theoretical extent of between about 0.25% and 5%, prior to encapsulating the preantral follicle and subsequently crosslinking the solution.

5. The hydrogel composition of claim 4, wherein the alginate solution is oxidized by treatment with an oxidizing agent selected from the group consisting of sodium periodate, Ammonium Cerium (IV) Nitrate, Bleach, N-Bromosuccinimide, ^~N-tert - Butylbenzenesulfϊnimidoyl chloride, te/t-Butyl hydroperoxide, CAN, Cerium Ammonium Nitrate, 3-Chloroperoxybenzoic acid, Chromium Compounds, CMHP, Copper Compounds, Cumene hydroperoxide, Dess-Martin Periodinane, Dimethyl sulfoxide, Ferric Nitrate, Formic Acid, Hydrogen peroxide, Hydrogen peroxide urea adduct, Hypervalent iodine compounds, IBX, Iodine, lodosobenzene diacetate, lodosylbenzene, 2- Iodoxybenzoicacid, Iron(III), (V) and (IV), Manganese Compounds, Manganese (IV) oxide, MCPBA, meto-Chloroperbenzoic acid, JV-Methylmorpholine-iV-Oxide, Molybdenum Compounds, N-Bromosuccinimide, N-Methylmorpholine-iV-Oxide, NMO, N-tert -Butylbenzenesulfmimidoyl chloride, Osmium tetroxide, Oxalyl chloride, Oxone, Oxygen, Ozone, Peracetic acid, Periodic acid, Peroxy acids, Potassium Permanganate, Pivaldehyde, Potassium peroxomonosulfate, Ruthenium(III) and (IV), Sodium hypochlorite, Sodium periodate, TEMPO, N-tert -Butylbenzenesulfmimidoyl chloride, tert-butyl hydroperoxide, Tetrabutylammonium peroxydisulfate, 2,2,6,6- Tetramethylpiperidinyloxy, Triacetoxyperiodinane, Trifluoroacetic peracid,

Trimethylacetaldehyde, UHP, Urea hydrogen peroxide adduct, and Vanadium compounds.

6. The composition of claim 1, wherein the gamma irradiated alginate is mixed with a 50% solution of untreated alginate prior to step (b) of claim 1.

7. An alginate hydrogel composition for maturing and/or developing an encapsulated cell, comprising a solution crosslinked/prepared from an alginate solution comprising between about 55% and 65% guluronic acid, wherein the hydrogel has a shear modulus of between about 100 Pa and 20 kPa.

8. The composition of claim 7, wherein the solution is gamma irradiated.

9. The composition of claim 7, wherein the solution is treated with an oxidizing agent.

10. An in vitro method for maturing a preantral follicle comprising:

(A) irradiating a non-crosslinked alginate solution with gamma radiation;

(B) suspending a preantral follicle into irradiated non-crosslinked solution;

(C) crosslinking the suspension, thereby forming a preantral follicle- single three dimensional gel matrix, wherein the follicle is encapsulated within the matrix, and wherein the gel matrix has a shear modulus of between about 100 Pa and 20 kPa;

(D) culturing the preantral follicle in the three dimensional matrix, wherein the preantral follicle forms an antral cavity; and

(E) releasing the antral follicle from the three dimensional gel matrix.

11. The alginate hydrogel of claim 1 , wherein the hydrogel has a shear modulus of between about 100 Pa and 20 kPa.

12. The alginate hydrogel of claim 4, wherein the hydrogel has a shear modulus of between about 100 Pa and 20 kPa.