WO2013025763A2 - Tissue engineering using injectable, oxidized alginate hydrogels - Google Patents

Abstract

Disclosed herein is a cell implantation pre-matrix composition comprising oxidized high molecular weight alginate and oxidized low molecular weight alginate, for combination with cells and polymerization into a hydrogel. Also disclosed are methods of generating a tissue at a site in a subject by the administration of the hydrogel described herein. The generation of fat tissue in a subject by the methods described herein is envisioned.

Description

TISSUE ENGINEERING USING INJECTABLE, OXIDIZED ALGINATE

HYDROGELS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional

Application Nos. 61/523,506 filed August 15, 2011 and 61/549,012 filed October 19, 2011, the contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 13, 2012, is named 286732PC.txt and is 5,724 bytes in size.

FIELD

[0003] The invention relates to the field of tissue generation.

BACKGROUND

[0004] Contour defects due to loss of soft tissue, mostly subcutaneous adipose tissue, are associated with trauma, tumor resection and congenital abnormalities. These affect patients not only cosmetically, but also affect the emotional well being of patients and may impair functions such as range of motion. Currently, several surgical approaches, including the use of autologous tissue flaps, free fat grafting, and the implantation of commercially available prosthetic materials are used to restore or replace a volume of adipose tissue¹. Current treatment modalities for soft tissue augmentation which use autologous grafting and commercially available fillers present a number of challenges and limitations, such as donor site morbidity and volume loss over time.

[0005] Injectable soft tissue fillers are in high demand because there is a shorter recovery time, results are immediate, and injection is both safer and more cost-effective than surgical implantation. Autologous fat is readily available, easy to harvest, and safe. However, consistent results are lacking because a majority of the injected fat tissue is resorbed². Fat graft survival is dependent on the number of viable adipocytes at the time of transplantation, host physiology, and the recipient site environment³.

[0006] Hyaluronic acid, collagen, polymethyl methacrylate spheres, and calcium hydroxyl apatite, and poly-L -lactic acid have all been used as tissue surrogates, due to the limitations of autologous solutions⁴'⁵. But these commercially available artificial fillers also present potential limitations, including foreign body reaction, fibrous capsule contraction, distortion, suboptimal mechanical properties, migration, and long-term resorption¹.

SUMMARY

[0007] One aspect of the present invention relates to a cell implantation pre-matrix composition comprising, a solution of oxidized high molecular weight (HMW) alginate and oxidized low molecular weight (LMW) alginate, wherein a) the oxidized HMW alginate and oxidized LMW alginate are present at a ratio of from about 1:3 to about 1 :4; b) the oxidized HMW alginate and oxidized LMW alginate are present at a weight/volume concentration of from about 1% to about 4%; c) the oxidized HMW alginate and oxidized LMW alginate are oxidized at about 2% of their sugar residues; and d) one or both of the high molecular weight alginate and the low molecular weight alginate are coupled to an adhesion peptide at a yield of about 2 peptides per alginate polymer chain. In one embodiment, the adhesion peptide comprises the amino acid sequence G4RGDASSKY (SEQ ID NO:2). In one embodiment, the composition further comprises adipogenic growth factors. In one embodiment, the composition further comprises one or more angiogenic factors. In one embodiment, the angiogenic factor is vascular endothelial growth factor. In one embodiment, the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration of about 4%. In one embodiment, the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration from about 1 % to about 3 %. In one embodiment, the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration of about 2%. In one embodiment, the composition further comprises cells. In one embodiment, the cells are selected from the group consisting of preadipocytes, adult mesenchymal stem cells, human adipose stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof. In one embodiment, the cells are primary cells. In one embodiment, the cells are predifferentiated cells. In one embodiment, the predifferentiated cells are adipocytes. In one embodiment, the composition further comprises a cross- linking agent or precursor thereof. In one embodiment, the composition is cross-linked to produce a hydrogel.

[0008] Another aspect of the invention relates to a kit comprising the cell implantation pre- matrix composition as described herein, and directions for use. In one embodiment, the kit further comprises one or more of an adipogenic growth factor and an angiogenesis factor.

[0009] Another aspect of the invention relates to a method of generating adipose tissue at a specific site in a subject. The method comprises administering to the specific site cells encapsulated in a hydrogel, under conditions suitable for generating new adipose tissue, wherein the hydrogel is generated from crosslinking the cell implantation matrix described herein. In one embodiment, administering is performed by a method selected from the group consisting of subcutaneous injection, submuscular injection, intramuscular injection, subfascial injection. In one embodiment, the cells are primary cells. In one embodiment, the cells are autologous. In one embodiment, the cells are selected from the group consisting of adipocytes, preadipocytes, adult mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof. In one embodiment, the specific site is in a region selected from the group consisting on the face, the breast, the buttocks, the hand, and the penis. In one embodiment, the specific site of the face is the lip, nasolabial fold, temple, lower eye lid, or upper eye lid. In one embodiment, the specific site is a scar of the subject. In one embodiment, the scar is a depression scar. In one embodiment, the scar is a post-burn contracture. In one embodiment, the scar is from post-irradiation soft tissue atrophy. In one embodiment, the scar is a post-traumatic soft tissue deficiency. In one embodiment, the specific site is a congenital anomaly. In one embodiment, the congenital anomaly is hemifacial microsomia or facial asymmetry.

Definitions

[0010] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic and/or aesthetic treatments for a deficit of soft tissue, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition associated with a deficit of soft tissue. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a deficit of soft tissue. Treatment is generally "effective" if one or more symptoms or clinical markers of a soft tissue deficit are reduced. Alternatively, treatment is

"effective" if the progression of the soft tissue deficit is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers of a soft tissue deficit, but also a cessation or at least slowing of progress or worsening of symptoms of a soft tissue deficit that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of the deficit, delay or slowing of deficit, and amelioration or palliation of the deficit.

[0011] The terms "decrease," "reduce," "reduced", "reduction" , "decrease," are all used herein generally to mean a decrease by a statistically significant amount relative to a reference. However, for avoidance of doubt, "reduce," "reduction" or "decrease" typically means a decrease by at least about 5%-10% as compared to the absence of the treatment and can include, for example, a decrease by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% decrease or more, i.e. absent level, as compared to the absence of the treatment, or any decrease between 10-99% as compared to the absence of the treatment.

[0012] The term "isolated" as used herein in reference to cells refers to a cell that is

mechanically separated from another group of cells in which it is found in nature. Such a cell that is isolated from a natural source is referred to herein as a primary cell. Methods for isolating one or more cells from another group of cells are well known in the art. See, e.g., Culture of Animal Cells: a manual of basic techniques (3rd edition), 1994, R. I. Freshney (ed.), Wiley-Liss, Inc.; Cells:a laboratory manual (vol. 1), 1998, D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring Harbor Laboratory Press; sad Animal Cells: culture and media, 1994, D. C. Darling, S. J. Morgan, John Wiley and Sons, Ltd.

[0013] As used herein, the phrase "therapeutically effective amount", "effective amount" or

"effective dose" when used to refer to compositions delivered to a subject refers to an amount that provides a therapeutic or aesthetic benefit in the treatment, prevention, or management of a tissue (e.g., soft tissue) deficit. Such an amount provides a statistically significant decrease in at least one symptom, sign, or marker of a tissue deficit. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.

[0014] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0015] As used herein, a "subject" means a human or animal. In one embodiment, the animal is a vertebrate such as a primate, rodent, or domestic animal. The terms, "patient", "individual" and "subject" are used interchangeably herein.

[0016] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, rodent (e.g., mouse, rat, rabbit, guinea pig, etc.), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), bovine (e.g., cow), but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of soft tissue deficits. In addition, the methods described herein can be used to treat domesticated animals and/or pets. A subject can be male or female.

[0017] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) difference, above or below a reference value.

[0018] As used herein the term "comprising" or "comprises" is used in reference to

compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0019] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0020] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 depicts the morphology of adipogenic differentiating human adipose-derived stem cells (hADSCs) after treatment with induction medium for 10 days. A phase contrast photomicrograph of the intracellular accumulation of small lipid droplets in differentiated hADSCs is shown. All scale bars =100μπι. [0022] Figure 2 depicts a gross image of newly developed tissue at 10 weeks after implantation demonstrating pale yellowish, semi-transparent, soft tissue structure.

[0023] Figures 3A-3C depict histological and immunohistochemical analyses of the newly formed tissue 10 weeks after subcutaneous injection as compared to native adipose tissue. Figure 3A depicts H&E staining (left panel - new tissue/right panel -inguinal fat tissue). Figure 3B depicts ORO staining (left panel -new tissue/right panel -inguinal fat tissue). Figure 3C depicts Western blot results demonstrating expression of PPAR-γ (53kDa), C/EBPa (42kDa) and adiponectin (27kDa) in the newly generated tissue (left), compared with native inguinal fat tissue (right), β-actin was used as loading control.

[0024] Figure 4 depicts a scanning electron micrograph of newly developed tissue at 10 weeks after implantation showing the adipocytes surrounded by extracellular matrix fibers.

[0025] Figures 5A-5B depict charts of the volume of alginate hydrogels comprising pre- differentiated adipose cells (Figure 5A) or human adipose-derived stem cells (hADSC's) (Figure 5B) in a 2% (1 :3) gel at the time of implantation (initial) and 10 weeks after injection (final).

[0026] Figures 6A-6B depict the performance of hydrogels of varying compositions with hADSCs. Figure 6A depicts a chart of initial and final volume in 2%, 1 :4 (HMW:LMW) vehicle (n=9). There was statistical significance between initial and final group. (Wilcoxon signed-rank test,

Figure 6B depicts a chart of initial and final cell volume in 1%, 1 :3 (HMW:LMW) vehicle (n=9). There was statistical significance between initial and final group. (Wilcoxon signed-rank test, p=0.008)

[0027] Figures 7A-7B depict the effect of hydrogel administration on epidermal and dermal thickness. Figure 7A depicts a chart of epidermal thickness (n=12). There was no statistical significance between control and experimental group, (paired t-test, /?=0.241). Figure 7B depicts a chart of dermal thickness (n=12). There was statistical significance between control and experimental group, (paired t-test, /?<0.001)

[0028] Figures 8A-8B depict Hematoxylin and eosin staining of tissue after implantation of an alginate hydrogel with hADSCs (Figure 8B) or a control, mock implantation (Figure 8A).

[0029] Figures 9A-9B depict Masson's trichrome staining of tissue after implantation of an alginate hydrogel with hADSCs (Figure 9B) or a control, mock implantation (Figure 9 A),

[0030] Figures 10A-10B depict phase contrast photomicrographic images of picosirius red staining of tissue after implantation of an alginate hydrogel with hADSCs (Figure 10B) or a control, mock implantation (Figure 10A),

DETAILED DESCRIPTION OF THE INVENTION

[0031] Aspects of the invention relate to the discovery of a formulation of oxidized high molecular weight alginate and low molecular weight alginate used to generate a matrix for encapsulation of cells implanted into a subject. The matrix results from the polymerization of the alginates within the formulation into a hydrogel. The matrix (or hydrogel) is used to implant cells into the body of a subject in order to generate new tissue in the subject from the implanted cells. The hydrogel serves as a delivery vehicle for the cells (e.g., autologous cells) incorporated therein, and once delivered to the intended site prevents dissemination and absorption of the cells by the body. The matrix provides one or more of structure, shape, and the proper 3-dimensional positioning of the cells for tissue generation and vascular ingrowth. Upon implantation, the hydrogel material degrades at a rate that allows generation of new tissue from the implanted cells. Tissue generated from the use of the disclosed matrix exhibits superior properties to engineered tissue known in the art with respect to graft volume stability and graft location stability. When seeded with adipose cells and/or adipose precursor cells, tissue is formed that is pale yellow, semi-transparent, soft adipose-like tissue characterized by the presence of adipose cells, expression of PPARy, adiponectin and C/EBP alpha/beta, and neovascularization. The generated tissue exhibits typical adipose tissue morphology, having increased dermal thickness at the graft site, increased collagen deposition at the graft site, and the presence of new, thinner collagen depositions in the dermal region.

[0032] One aspect of the invention relates to a composition comprising components of the matrix prior to polymerization of the alginates therein into a hydrogel. This composition is referred to herein as a pre-matrix composition. At minimum, the pre-matrix composition comprises the oxidized high molecular weight alginate and oxidized low molecular weight alginate, as described herein that will polymerize to form the hydrogel used to encapsulate the cells for implantation. The pre-matrix composition can optionally further comprise additional components that are to be used to generate the or included in the final hydrogel product (e.g., growth factors, angiogenic factors, cross-linkers, etc).

[0033] Various forms of the pre-matrix composition are envisioned that are convenient for transport, storage and use. One such form is an aqueous solution ready for the addition of other components (e.g., cells). Various concentrations of the aqueous solution are envisioned relative to the final concentration in which cross-linking occurs (e.g., lx, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, and lOx concentrations). The aqueous solution can conveniently be of a concentration convenient for the addition of other components by the practitioner just prior to use, such as cells (e.g., a 2X

concentration, to be mixed with an equal volume of cell solution to produce a final IX composition ready for use). Other possible forms of the pre-matrix composition include, without limitation, the form of a solid, semi-solid (e.g., gel or paste) or powder (e.g., from lyophilization). Such forms facilitate storage and transport. Such non-aqueous forms of the pre-matrix composition can be provided in appropriate concentrations and/or amounts that are ready for rapid use by simple solubilization (and possibly sterilization) and addition of remaining useful components.

[0034] The pre-matrix compositions described herein are suitable for cross-linking to form a hydrogel, following any necessary manipulation by the skilled artisan (e.g., solubilization, dilution, etc.). A "hydrogel" is a water-containing polymer network formed by polymers which expand in volume upon hydration. All stages of polymerization of the pre-matrix composition are encompassed by the invention. Hydrogels formed from cross-linking of the pre-matrix composition described herein are also encompassed by the invention. The pre-matrix composition may undergo partial crosslinking (with the remaining crosslinking to occur upon use), or be fully cross-linked to form the final hydrogel product. Typically, cells and other hydrogel components are added to the pre-matrix composition prior completion of the cross-linking reaction in order to achieve adequate dissemination throughout the final hydrogel product. Hydrogels described herein can slowly dissolve or erode with time.

Alginate Hydrogels

[0035] As used herein, "alginate" refers to a salt of alginic acid, e.g. the calcium, magnesium, sodium, potassium, or propylene glycol. Alginic acid is a polyuronic acid made of two uronic acids: D-mannuronic acid and L-guluronic acid. Alginates occur naturally in various algae (such as Laminaria japonic a) and bacteria. The ratio of mannuronic acid and guluronic acid varies with factors such as seaweed species, plant age, and part of the seaweed (e.g., stem, leaf). Alginic acid is substantially insoluble in water. It forms water-soluble salts with alkali metals, such as sodium, potassium, and, lithium; magnesium; ammonium; and the substituted ammonium cations derived from lower amines, such as methyl amine, ethanol amine, diethanol amine, and triethanol amine. Alginates are hydrophilic colloids.

[0036] Alginates can also be synthetic and semisynthetic. The term semisynthetic alginate refers to a naturally occurring alginate which has been transformed chemically or biologically by man. For example, a naturally occurring alginate may be oxidized or acetylated. Acetylation under Schotten Bauman conditions will produce an acetylated semisynthetic alginate, or epimerized in vitro using a mannuronan C-5 epimerase to produce a different semisynthetic alginate. The properties of alginates are described further in Gaserod O, Smidsrod O, Skjak-Braek G., 1998, Biomaterials 19(20): 1815-25, which is incorporated by reference herein in its entirety.

[0037] Alginates can be characterized by the ratio of mannuronic acid (M) and guluronic acid

(G). Increasing the proportion of G units results in a more viscous material. The M:G ratio can be altered to affect the rate of hydrogel formation or the final characteristics of the hydrogel. In one embodiment, the M:G ratio is from 30:70 to 50:50. In one embodiment, the M:G ratio is about 40:60.

[0038] Alginates can be characterized by the viscosity which they produce when in solution.

The present invention encompasses formulations that incorporate alginate categorized by specific degrees of viscosity as described herein.

[0039] Viscosity of an aqueous solution of alginates is influenced by the concentration of the alginate and the chain length (i.e. degree of polymerization), and thus the molecular weight, of the individual alginate molecules. The viscosity of alginate solutions is also influenced, for example, by the shear rate of the solution, temperature, acid conditions, and M:G ratio of the alginate molecule.

[0040] Typically alginate molecules are categorized in the art as either low viscosity alginates

(e.g. LV alginates), medium viscosity alginates (e.g. MV alginates) or high viscosity alginates (e.g. HV alginates). Additional categories such as ultra high viscosity (e.g. UHV alginates) or very low viscosity alginates (e.g. VLV alginates) are sometimes used to describe certain alginate molecules.

[0041] The category of viscosity of an alginate is determined using a 1% weight/weight solution of the alginate at 20^°C. For example, an alginate is a very low viscosity alginate if a 1% weight/weight solution of that alginate at 20^°C has a viscosity of less than 20 mPa _* s. An alginate is a low viscosity alginate if a 1 % weight/weight solution of that alginate at 20 C has a viscosity of 20 to 200 mPa _* s. An alginate is a medium viscosity alginate if a 1% weight/weight solution of that alginate at 20 C has a viscosity of approximately 200 to 600 mPa _* s. An alginate is a high viscosity alginate if a 1% weight/weight solution of that alginate is "drip-free" at 20 C, or a 1% weight/weight solution of that alginate at 20 C has a viscosity of greater than 600 mPa _* s.

[0042] Alginates of the various viscosities known in the art are envisioned for use in the pre- matrix composition and hydrogels described herein. In one embodiment, the alginate used in the methods and compositions described herein is a medium viscosity alginate (e.g. a MV alginate).

[0043] Alginates are also categorized by the ratio of the specific uronic acids contained in the alginate molecule (e.g. mannuronic acid (M) and guluronic acid (G)). Increasing the proportion of G units results in a more viscous material. The M:G ratio affects the rate of hydrogel formation and/or the final characteristics of the hydrogel. Alginates are typically referred to as "M" alginates when they have at least 50% mannuronic acid and no more than 50% guluronic acid. Alginates are typically referred to as "G" alginates when they have at least 60% guluronic acid and no more than 40% mannuronic acid. In one embodiment, the pre -matrix composition described herein comprises an alginate with an M:G ratio from 30:70 to 50:50 (e.g. approximately 40:60).

[0044] The viscosity and subunit descriptors described above can be combined combined to categorize the alginates. A MVG alginate is a medium viscosity alginate having at least 60% guluronic acid; a MVM alginate is a medium viscosity alginate having a least 50% mannuronic acid; a HVM alginate is a high viscosity alginate having at least 50% mannuronic acid; a HVG alginate is a high viscosity alginate having at least 60% guluronic acid; a LVM alginate is a low viscosity alginate having at least 50% mannuronic acid and a LVG alginate is a low viscosity alginate having at least 60% guluronic acid.

[0045] In one embodiment, the alginate for use in the methods and compositions described herein is a G alginate. In one embodiment, the alginate is a M alginate. In one embodiment, the alginate is a MVG alginate. In one embodiment, the alginate is a MVM alginate.

[0046] Commercial alginate preparations are available that are suitable for use in the pre- matrix compositions and hydrogels described herein. In one embodiment, the alginate is PRONOVA UP MVG alginate (UP references an ultra-pure, sterile alginate, MV references a medium viscosity alginate (i.e., >200 mPa _* s as a 1% w/w solution at 20 ^°C), and G references an alginate molecule having at least 60% G residues; Cat No. 4200006; NovaMatrix FMC Corp., Oslo, Norway) with a M:G ratio of approximately 40:60. [0047] Water-insoluble alginate salts, in which the principal cation is calcium, are found in the fronds and stems of seaweeds of the class Phaeophyceae, examples of which are Fucus vesiculosus, Fucus spiralis, Ascophyllum nodosum, Macrocystis pyrifera, Alaria esculenta, Eclonia maxima, Lessonia nigrescens, Lessonia trabeculata, Laminaria japonica, Durvillea antarctica, Laminaria hyperborea, Laminaria longicruris, Laminaria digitata, Laminaria saccharina, Laminaria cloustoni, and Saragassum sp. Methods for the recovery of alginic acid and its water-soluble salts, especially sodium alginate, from natural sources are well known, and are described, for example, in Green, U.S. Pat. No. 2,036,934, and Le Gloahec, U.S. Pat. No. 2,128,551 which are incorporated by reference herein in their entirety. Alginates of various forms are commercially available, for example, under the PRONOVA name (Catalog Nos: 4202106, 4202006, 4202306, and 4202206 NovaMatrix, FMC Corp., Oslo, Norway).

Molecular Weight

[0048] Alginates useful for the compositions described herein are "high molecular weight" or

"low molecular weight" alginates. A high molecular weight (HMW) alginate is an alginate having a molecular weight equal to or greater than 90,000 g/mol. In one embodiment, the high molecular weight alginate is from 100,000 g/mol-500,000 g/mol. In one embodiment, the high molecular weight alginate is from 105,000 g/mol-400,000 g/mol. In one embodiment, the high molecular weight alginate is from 110,000 g/mol-300,000 g/mol. In one embodiment, the high molecular weight alginate is be from 115,000 g/mol- 130,000 g/mol. In one embodiment, the high molecular weight alginate is from 250,000 g/mol-290,000 g/mol.

[0049] A low molecular weight (LMW) alginate is an alginate having a molecular weight less than 90,000 g/mol. In one embodiment, the low molecular weight alginate is from 10,000 g/mol- 85,000 g/mol. In one embodiment, the low molecular weight alginate is from 30,000 g/mol-80,000 g/mol. In one embodiment, the low molecular weight alginate is from 40,000 g/mol-80,000 g/mol. In one embodiment, the low molecular weight alginate is from 50,000 g/mol-70,000 g/mol.

[0050] In one embodiment, the LMW alginate has a molecular weight of approximately 60,000 g/mol and the HMW alginate has a molecular weight of approximately 120,000 g/mol.

[0051] LMW alginates can be generated from HMW alginates. By way of non-limiting example, the molecular weight of alginates can be reduced by γ-irradiating alginates with cobalt-60. For example 5 Mrad of radiation from a cobalt-60 source can be used to create alginates of approximately 60,000 g/mol molecular weight from alginates of approximately 120,000 g/mol. LMW alginates may also be generated by acid hydrolysis, thermal depolymerization or enzymatic degradation. Non-limiting examples of such techniques are disclosed in U.S. Patent Nos. 6,121,441 ; 6,511,650 and 6,747,015 and U.S Patent Publication 2008/0085295, which are incorporated by reference herein in their entirety.

[0052] The molecular weight of an alginate can be determined by any method in the art. By way of non-limiting example, the molecular weight can be determined by size exclusion chromatography. Alginates can be dissolved in a 0.1 M NaN0₃ buffer (pH 6.3) mobile phase and examined using a SEC system (Viscotek) equipped with a laser refractometer 9LR 40), a differential viscometer (T60) and a right angle laser light scattering detector (RALLS) (SEC-MALS). TSK-gel columns can be used, for example, a G40000PW_XL and a G3000PW_XL.

Ratios

[0053] The pre-matrix composition described herein and hydrogels created therefrom is comprised of both HMW and LMW alginates. The ratio of HMW:LMW alginate influences the characteristics of the hydrogel. In general, higher relative levels of HMW alginate result in a hydrogel composition which is more resistant to degradation or erosion. In one embodiment, the pre- matrix/hydrogel comprises a ratio of HMW:LMW alginate, by weight in the range of from 1 :2 to 1 :5. In one embodiment, the ratio of HMW:LMW alginate is in the range of from 1 :2.5 to 1 :4.5. In one embodiment, the ratio of HMW:LMW alginate is from 1 :3 to 1 :4. In one embodiment, the ratio os HMW: LMW is 1 :3 or 1 :4.

Oxidation of Alginates

[0054] The alginates (HMW and/or LMW) in the pre-matrix composition described herein are partially oxidized at their sugar residues. Partially oxidized alginates degrade faster than alginates which have not been partially oxidized and form hydrogels with reduced stiffness. In one

embodiment, the partially oxidized alginate has a fraction of oxidized sugar residues of at least 0.015, e.g., 0.015 or greater, or 0.02 or greater, or 0.025 or greater, or 0.03 or greater, or 0.04 or greater, or 0.05 or greater. In one or more of these embodiments, the fraction of oxidized sugar residues is less than 0.5. In one embodiment, the fraction of oxidized sugar residues is 0.05. In one embodiment, the partially oxidized alginate has a percentage of oxidized sugar residues in the range of

approximately .02% to 50%. In one embodiment, the percentage of oxidized sugar residues is in the range of about .05% to 30%. In one embodiment, the percentage of oxidized sugar residues is in the range of about .07% to 10%. In one embodiment, the percentage of oxidized sugar residues is in the range of about 1% to 5%. In one embodiment, the percentage of oxidized sugar residues is about 2%. In one embodiment, the percentage of oxidized sugar residues is about 5%.

[0055] Alginates can be (partially) oxidized by a variety of methods known in the art. One such method is by creating hydrolytically labile acetal-like groups. By way of non-limiting example, this oxidation can be performed by reacting the alginates with sodium periodate. A 1% solution of alginate in distilled water can be mixed with a 0.25M aqueous solution of sodium periodate. The reaction can be allowed to proceed at room temperature for 1 hour or more, e.g., 2 hours, 6 hours, 12 hours, 24 hours, or 48 hours or more, preferably about 24 hours. Unreacted sodium periodate can be neutralized by the addition of ethylene glycol. The partially oxidized alginate can be purified by filtration or by sodium chloride/ethanol precipitation. The partially oxidized alginate can also be purified by dialysis, for example, by using the Spectra/Por® MWCO 1000 for a three day dialysis. Preparation of oxidized alginates is described in Bouhadir et al (Biotechnol. Prog. 2001 17, 945-950), which is incorporated herein by reference in its entirety.

[0056] Alginates can also be oxidized by reacting them with dinitrogen tetroxide (see U.S.

Patent No. 6,440,940; which is incorporated by reference herein in its entirety).

[0057] The degree of oxidation can be measured by methods known to those of skill in the art, for example, by measuring the number of aldehyde groups using t-butyl carbazate (see e.g. Bouhadir,

Polymer 1999 40, 3575-84).

Weight/Volume

[0058] The pre-matrix composition described herein is used to create an alginate hydrogel.

The pre-matrix composition in aqueous solution can be characterized by the percentage

( weight/volume) of alginate contained therein, e.g., a 1% alginate hydrogel is lg of alginate in 100 mL of solvent. An aqueous pre-matrix composition suitable for polymerization into a hydrogel described herein has an alginate weight volume concentration that falls within the range of from about 0.5% to 10%. In one embodiment, the pre-matrix composition has an alginate weight volume concentration that falls within the range of from about 1% to about 5%. In one embodiment, the pre- matrix composition has an alginate weight volume concentration that falls within the range of from about 1% to 3%. In one embodiment, the pre-matrix composition has an algimate weight volume concentration that falls within the range of from about 1.5% to 2.5%. In one embodiment, the alginate weight volume concentration is about 1%, 1.5%, 2%, 2.5 %, 3%, 3.5%, 4%, 4.5%, or 5%. In one embodiment the alginate weight olume concentration of about 2%. The pre-matrix composition when appropriately crosslinked will result in a hydrogel of the same alginate weight volume composition.

[0059] In one embodiment, the pre-matrix composition is at a higher concentration (as measured by w/v) of alginate than the final hydrogel which will be placed in a subject. Such a formulation is convenient for rapid mixture with additional components (e.g. with cells in media) to the final weight volume concentration desired for polymerization. In a concentrated form, the pre- matrix composition can be at least about 1%, e.g., about 1% or greater, or about 2% or greater, or about 3% or greater, or about 4% or greater, or about 5% or greater, or about 6% or greater. One convenient formulation is a pre-matrix composition that is about 4% (w/v) alginate solution. Such a formulation is conveniently diluted 1 : 1 to produce a a 2% (w/v) alginate solution by the addition of an equal volume of another substance (e.g., cells in media). Appropriate solvents for the dilution of concentrated pre-matrix composition include without limitation, water, an isotonic solution, and any pharmaceutically acceptable solvent which is comprised substantially of water e.g. Dulbecco's modified Eagle's medium (DMEM).

Adhesion Peptide

[0060] The cell implantation pre-matrix composition further comprises a factor which facilitates cell adhesion to the hydrogel that is ultimately produced. Various factors are known in the art and can be incorporated into the pre-matrix composition. One way to facilitate cell adhesion is with a cell adhesion peptide sequence. In one embodiment, the cell implantation pre-matrix composition comprises an adhesion peptide sequence (e.g., attached to the alginate). As used herein, an "adhesion peptide sequence" refers to a sequence of amino acids forming a peptide which, when present on a substance increases the adhesion of cells to the substance. When an adhesion peptide sequence is used appropriately in the pre-matrix composition of the present invention, adhesion of cells to the hydrogel will be increased by at least about 20%, 50%, 75%, 100%, 150%, 200%, 300%, 500%, 1000% or more. In one embodiment, sufficient adhesion peptide is incorporated into the pre- matrix composition to increase the adhesion of cells to the hydrogel by at least 2 fold over adhesion to the same hydrogel in the absence of the adhesion peptide. In one embodiment, sufficient adhesion peptide is incorporated into the pre-matrix composition to increase the adhesion of cells to the hydrogel by at least about 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or more fold. In one embodiment, sufficient adhesion peptide is incorporated into the pre-matrix composition to increase the adhesion of cells to the hydrogel by at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 or more fold.

[0061] In one embodiment, the adhesion peptide sequence comprises the amino acid sequence

"RGD" (SEQ ID NO: 01). In one embodiment, the adhesion peptide sequence comprises

G4RGDASSKY (SEQ ID NO: 02). In one embodiment, the adhesion peptide sequence comprises G4RGDASSKY-OH (SEQ ID NO: 03). Methods of making peptides are well known to those of skill in the art. For example, peptides can be produced recombinantly (see Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001)) or synthesized. Peptide synthesis services are available from numerous companies, including Commonwealth Biotechnologies, Inc. of Richmond, Va., USA and Peptides International, Louisville, KY).

[0062] In one embodiment the adhesion peptide sequence comprises YIGSR (SEQ ID NO:

04), IKVAV (SEQ ID NO: 05), REDV (SEQ ID NO: 06), DGEA(SEQ ID NO: 07), VGVAPG (SEQ ID NO: 08), GRGDS (SEQ ID NO: 09), LDV (SEQ ID NO; 10), RGDV (SEQ ID NO: 11), PDSGR (SEQ ID NO: 12), RYWLPR (SEQ ID NO: 13), LGTIPG (SEQ ID NO: 14), LAG (SEQ ID NO: 15), RGDS (SEQ ID NO: 16), RGDF (SEQ ID NO: 17), HHLGGALQAGDV (SEQ ID NO: 18), VTCG (SEQ ID NO: 19), SDGD (SEQ ID NO: 20), GREDVY (SEQ ID NO: 21), GRGDY (SEQ ID NO: 22), GRGDSP (SEQ ID NO: 23), VAPG (SEQ ID NO: 24), GGGGRGDSP (SEQ ID NO: 25),

GGGGRGDY (SEQ ID NO 26), or FTLCFD (SEQ ID NO: 27). Cell adhesion peptide sequences may be derived from EGF, VEGF, b-FGF, FGF, TGF, TGF-β, proteoglycans or fragments or derivatives thereof. Suitable cell adhesion peptide sequences comprising RGD (SEQ ID NO: 1) include, but are not limited, to Novatach RGD (SEQ ID NO: l) (NovaMatrix, FMC BioPolymer, Oslo, Norway) and those disclosed in U.S. Pat. No. 6,642,363; which is hereby incorporated by reference in its entirety. [0063] The adhesion peptide sequence can be linked (e.g., chemically coupled) to the alginate of the pre-matrix composition (e.g., prior to the formation of the pre-matrix composition or the hydrogel). Methods of coupling peptides to polymer backbones are well known to those of skill in the art and can be adapted for the present invention. In one embodiment, the adhesion peptide sequence is coupled to the polymer using carbodiimide chemistry. By way of non-limiting example, the adhesion peptide sequence is coupled to the alginate polymer using a 1 % alginate solution in a 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing 0.5 M NaCl. N- hydroxysulfosuccinimide (Sulfo-NHS) is used as a co-reactant greatly increasing EDC efficiencies in a similar manner to HOBt. Sulfo-NHS is added to the reaction solution followed by the peptide and the EDC. The ratio of uremic acid: EDC :sulfo-NHS can be constant, while only the peptide available for reaction is varied. This method of coupling typically results in 65-75% efficiency relative to available peptide. The solution can be allowed to react for 14-18 hours, at which time hydroxyl amine can be added to quench any unreacted activated sulfo-NHS -esters and reestablishing carboxylates. The solution can be extensively dialyzed against water in 3500 MWCO dialysis tubing. The reaction can also be performed using diH20 in place of MES, which will result in an approximately 10-fold lower efficiency. Chemical techniques for coupling peptides to the alginate backbones may also be found in U.S. Pat. No. 6,642,363 which is incorporated by reference herein in its entirety.

[0064] In one embodiment, the adhesion peptide sequence is coupled to the HMW alginate. In one embodiment, the adhesion peptide sequence is coupled to the LMW alginate. In one embodiment, the adhesion peptide sequence is coupled to both HMW and LMW alginates. Varying degrees of coupling with respect to the HMW and LMW alginates are contemplated. The HMW and LMW alginates may have differing amounts of coupling as well in the same composition or hydrogel.

[0065] In one embodiment, the adhesion peptide sequence is present in the cell implantation pre-matrix composition or hydrogels created therefrom at a concentration of 0.5 to 10 peptides per polymer chain (one or both of HMW and LMW polymers). In one embodiment, the adhesion peptide sequence is present at a concentration of 1 to 5 peptides per polymer chain (one or both of HMW and LMW polymers). In one embodiment, the adhesion peptide sequence is present at a concentration of 1.5 to 2.5 peptides per polymer chain (one or both of HMW and LMW polymers).

Storage and Hydrogel Formation

[0066] In one embodiment, when the alginate polymers of the desired molecular weights, ratios and modifications (e.g., adhesion peptide sequences) are used, the cell implantation pre-matrix composition is purified, sterilized, and/or prepared for storage prior to use.

[0067] Purification of the pre-matrix composition or components thereof can be accomplished by any method in the art. In one embodiment, purification is performed by filtration, precipitation, and/or dialysis as described herein. The pre-matrix composition or components thereof can be sterilized by any method known to those of skill in the art, including, for example, gamma radiation, E-beam, ethylene oxide, autoclaving, NOx gases, hydrogen gas plasma sterilization, or contacting the polymer with alcohol prior to rehydrating. In one embodiment, sterilization can be performed by, for example, filter sterilization using a 0.22 μπι filter.

[0068] In one embodiment, cell implantation pre -matrix compositions or components thereof are treated with activated charcoal prior to sterilization to further enhance purification and shelf-life.

[0069] In one embodiment, the cell implantation pre-matrix composition and/or the

components thereof has an endotoxin level equal to or less than 1 ,500 EU/g, 1 ,000 EU/g, 500 EU/g, 150 EU/g, 100 EU/g, 75 EU/g, 50 EU/g, or 35 EU/g. In one embodiment, they have an endotoxin level equal to or less than 1,500 EU/g, 1,000 EU/g, 500 EU/g, 150 EU/g, 100 EU/g, 75 EU/g, 50 EU/g, or 35 EU/g.

[0070] The pre-matrix composition can be stored as an aqueous solutions or in dry form such as freeze-dried, i.e. lyophilized. Pre-matrix compositions of differing characteristics or properties (e.g., those described herein) can be mixed before purification, before sterilization, before being freeze-dried, before being stored, or after being stored in order to obtain a final cell implantation pre- matrix composition with the desired properties.

[0071] Prior to administration to a subject, the pre-matrix composition can be formed into a hydrogel by polymerization of the alginate contained therein. Pre-matrix compositions can be diluted in a solvent to the desired concentration and an effective amount of a cross-linking agent or a solution comprising a cross-linking agent can be added to the pre-matrix composition induce hydrogel formation. In one embodiment, the pre-matrix composition is placed in a mold or container with the desired shape and/or volume for polymerization. As the term is used herein, an effective amount of the cross-linking agent is sufficient to polymerize the pre-matrix composition into a hydrogel for encapsulation of the cells distributed therein in the desired time frame. One such time frame is within minutes of addition of the cross-linking agent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes).

[0072] As used herein, a "cross-linking agent" can be any agent (e.g. ion, salt, acid, amino acid, or molecule) which polymerized the alginates to produce a hydrogel from the pre-matrix composition described herein. Cross-linking can occur ionically or covalently. Examples of cross-linking agents include, but are not limited to, calcium chloride, calcium sulfate, zinc chloride, sodium sulfate, calcium ions, barium ions, strontium ions, zinc ions, copper(+2) ions, aluminum ion, lysine, L-poly- lysine, polyethylene glycol, glutaraldehyde, dycyclohexylcarbodiimide, hexamethylene diisocyanate, adipic acid hydrazide (AAD) (optionally in a buffer comprising 2-(N-morpholino) ethanesulfonic acid (MES) beffer with 1-hydroxybenzotriazole (HOBt), and l-ethyl-3-[3-(dimethylamino)propyl] carbodiimde (EDC)). In one embodiment, cross-linking agents are used that can form ionic crosslinks and then covalent cross-links over an extended period of time (e.g. hours), such as calcium chloride or calcium sulfate. This allows the hydrogel to slowly increase in viscosity, providing a material which is more easily manipulated immediately after addition of the cross-linker (e.g. for injection) and which becomes more viscous over the course of several hours. Alternatively, AAD is known to form cross-links slowly and thus can be utilized in a similar manner. In one embodiment, two or more cross-linking agents are used.

[0073] In one embodiment, the cross linking agent is calcium sulfate which is added in the form of an aqueous slurry (e.g., 0.21g CaS0₄/mL distilled H₂0) at a ratio of 25: 1. In one embodiment, the cross-linking agent is added and polymerization is achieved in the tool (e.g., syringe) that will be used to deposit the mixture to a site on the subject.

[0074] In one embodiment, the pre-matrix composition comprises a cross-linking agent in a form that is inert but can be activated, examples of which are known in the art and described herein. In one embodiment, the cross-linking agent is temperature sensitive, and as such, can be incorporated into the pre-matrix composition and inactive if the composition is stored at a non-permissive temperature (e.g., at 4°C). In one embodiment, the cross-linking agent is in a time -release formulation, such that the agent is released into the polymer solution at a desired rate, thus causing hydrogel formation at a desired rate. Any time -release or controlled-dose formation known to those of skill in the art can be used.

[0075] In one embodiment, the cross-linking agent is calcium ions in the form of insoluble calcium alginate. This "self-gelling" method of hydrogel formation is described in U.S. Patent Application 2008/0085295, which is incorporated by reference herein in its entirety.

[0076] The amount of cross-linking agent can be determined by the skilled practitioner. In one embodiment, the ratio between the cross-linking agent and the alginate sugar residues is from about 1.0:0.45 to 1.0:0.05. In one embodiment, the ratio between the cross-linking agent and the alginate sugar residues is from 1.0:0.07 to 1.0:0.3. In one embodiment, the ratio between the cross-linking agent and the alginate sugar residues is from 1.0:0.10 to 1.0:0.3. In one embodiment, the ratio between the cross-linking agent and the alginate sugar residues is from 1.0:0.15 to 1.0:0.3. Cross- linking of alginate hydrogels is further described in U.S. Patent Nos. 5,144,016 and 6,642,363 and U.S. Patent Publication 2008/0085295, which are incorporated by reference herein in their entirety.

[0077] In one embodiment, cells (described herein) are added to the pre-matrix composition prior to addition of the cross-liking agent or prior to any substantial cross-linking of the alginate polymers in the composition. In one embodiment, cells are added to the cell implantation pre-matrix composition during or after the cross-linking agent has been added but prior to the cross-linking process reaching completion. It may further be possible to add the cells to an incompletely or completely cross-linked hydrogel.

Growth and Differentiation Factors

[0078] In one embodiment, the pre-matrix composition or hydrogel created therefrom can comprise one or more factors to facilitate tissue generation, described herein (e.g, growth factors, angiogenic factors, differentation factors, etc.). One such type of factor is a growth factor. Many growth factors are known in the art that will increases the growth of the cells implanted in the context of the hydrogel created from the pre-matrix composition described herein, and/or increase the growth of the cells and/or tissue surrounding the hydrogel composition in the subject, and/or the growth of the cells and/or tissue which may displace the hydrogel composition and form new tissue

[0079] As used herein, the term "angiogenic factor," also known in the art as "angogenic growth factor" refers to a factor that directly or indirectly promotes new blood vessel formation. Non- limiting examples of angiogenic factors include, angiogenin, hypoxia-inducible factor (HIF-1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factor (FGF), acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), platelet-derived growth factor, transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-β), vascular permeability factor (VPF), tumor necrosis factor alpha (TNF-a), interleukin-3 (IL-2), interleukin-8 (IL-8), platelet-derived endothelial growth factor (PD-EGF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), scatter factor (SF), pleitrophin, proliferin, follistatin, placental growth factor (PIGF), midkine, platelet-derived growth factor-BB (PDGF), E-cadherin,fibrinogen, fibronectin, heparanase, hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), IGF, BP-3, PDGF, pigment epithelium-derived factor (PEDF), vitronection, leptin, trefoil peptides (TFFs), CYR61 (CCNl), NOV (CCN3), leptin, platelet-derived endothelial cell growth factor (PDECGF), progranulin, c-Myc, granulocyte colony-stimulating factor (G-CSF), stromal derived factor 1 (SDF-1), scatter factor (SF), osteopontin, stem cell factor (SCF), matrix metalloproteinases (MMPs),tllrombospondin-l (TSP-1) CCL2 (MCP-1), CCL5 (RANTES), fractalkine, dexamethasone, 3-isobutyl-lmethylxanthine (IB MX), indomethacin, pantothenate, steroids, fatty acids and derivatives, fragments or modifications thereof. Those of skill in the art will recognize that the many such factors can exhibit a variety of growth and differentiation properties depending upon the environment and types of cells to which they are exposed. As such, an angiogenic factor may also function as a growth factor and/or a differentiation factor for the appropriate tissue type.

[0080] In one embodiment, the factor promotes the appropriate generation and/or accumulation of extracellular matrix components that are typically present in the specific tissue being generated (e.g., collagen, fibronectin, etc.). In one embodiment, the factor is specific for growth and differentiation of the tissue being generated. In one embodiment, the factor is adipogenic growth factor.

[0081] In one embodiment, the factor directly or indirectly promotes adipose formation of the appropriate precursor cells. Such a factor is referred to herein as an adipogenic growth factor. Non- limiting examples of adipogenic growth factors include cAMP response element binding protein (CREB), C/ΕΒΡβ, C/EBPa, PPARy, insulin, insulin-like growth factor (IGF-1), sterol response element-binding protein 1 (SREBP1), BMP2, TGF-beta, FGF-1, FGF-2, basic FGF, IGF-1, EGF, as well as and derivatives, fragments or modifications thereof. Cells

[0082] In one embodiment, the pre-matrix composition described herein, or hydrogels created therefrom, comprises an effective concentration of cells. An effective concentration of cells refers to an amount of cells sufficient to produce a concentration of cells within the final hydrogel product to promote tissue generation upon delivery. Envisioned concentrations of cells for incorporation into the final hydrogel product range from 10³ cells/ml to 10⁸ cells/ml. In one embodiment, from about 10⁴ to 10⁷ cells/mL are incorporated (e.g., about 10⁴, 10⁵, 10⁶, 10⁷ cells/ml are incorporated). In one embodiment, from about 10⁵ to 10⁶ cells/ml are incorporated. In one embodiment, from about 10⁶ to about 5xl0⁶ cells/mL are incorporated. In one embodiment, about 2 x 10⁶ cells/mL are incorporated.

[0083] The cells may be described by virtue of the subject on whom the composition or hydrogel is intended for use. In one embodiment, the cells are autologous. In one embodiment, the cells are allogenic. In one embodiment, the cells are xenogenic.

[0084] The cells can be of any species suitable for use as a subject in the methods described herein. The cells can be animal cells, such as a mammal, (e.g., primate, rodent, or domestic animal) Examples of such mammals are a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The cells can be of male or female origin. The cells may be primary cells obtained form an adult or an immature animal (e.g., embryo, fetus, infant, child).

[0085] In one embodiment, the cells are stem cells that have the potential to differentiate into the desired tissue type. For example, when generating adipose/fat tissue, one may use adipose- derived stem cells (ADSCs). The use of a combination of different cell types (e.g, cells of different origin and/or cells in different stages of differentiation) in a pre-matrix composition/hydrogel described herein is also envisioned.

[0086] In one embodiment, the pre-matrix composition comprises human ADSCs. ADSCs are also referred to in the art as, variously, preadipocytes, stromal cells, processed lipoaspirate cells, multipotent adipose-derived stem cells, and adipose-derived adult stem ceils. As used herein, "adipose-derived stern cells'^' refers to multipotent stern cells isolated from adipose tissue which have osteogenic, adipogenic, myogenic, chrondrogenic, and neurogenic differentiation potential. ADSCs are a subpopulation of mesenchymal stem, ceils but can be differentiated from the general population of mesenchymal stem ceils (MSCs) by the expression of CD49d and the absence of CD 106 expression.

[0087] ADSCs can be derived from adipose tissue, which can be harvested by direct excision or more commonly from lipoaspirate, the discarded tissue following liposuction surgery. The term "adipose tissue" refers to loose connective tissue composed of multiple cell types including adipose cells, adipocytes and microvascular cells. Adipose tissue includes stem and progenitor cells and endothelial precursor cells. Accordingly, adipose tissue refers to fat including the connective tissue that stores the fat. The tissue can be washed and red blood cells removed. Digestion with collagenase can be performed and the tissue is centrifuged to obtain a cell pellet, known as the stromal vascular fraction (SVF). The SVF can contain, in addition to ADSCs, mesenchymal stem cells (MSCs) and endothelial ceils. ADSCs ca be purified from the SVF by, for example, prolonged culture of SVF, relying on the ability of ADSCs to oulcompete other cell populations under the culture conditions over time. The number of stem ceils present can be increased by subjecting the SVF to a 24-hour adhesion period before washing away nonadherent cells; the fraction of stem ceils can be further increased by a forceful washing step at 1 hour into the 24-hour adhesion period. Alternatively, ceil sorting (e.g. FACS) based on ceil surface markers expressed by ADSC may allow purification of ADSCs from the SVF (see Miranville et al. Vascular Medicine 2004 110:349-355; Locke et al. Stem Cells 2011 29:404-411; Zuk et al. Molecular Biology of the Cell 2002 13:4279-4295; which are incorporated by reference herein in their entirety).

[0088] In one embodiment, the cells are preadipocytes. The term is used in the art to refer to cells present in adipose tissue which do not display a differentiated adipose phenotype, which phenotype is described herein below. Preadipocytes include the population of cells and cell types which comprise the SVF of adipose tissue. Preadipocytes can be isolated from adipose tissue according to the methods described herein. Preadipocytes can also be obtained by co-culturing macrophages and adipose cells, both obtained from adipose tissue, as described in U.S. Patent Publication 2009/0317367, which is incorporated by reference herein in its entirety.

[0089] In one embodiment, the cells incorporated into the hydrogel are adult mesenchymal stem cells. Mesenchymal stem cells are refers to multipotent stem cells that can be differentiated into a variety of cell types including osteoblast, chondrocytes (cartilage cells), adipocyte (fat cells), etc. Methods of isolating and identifying mesenchymal stem cells are known in the art and can include isolating mesenchymal stem cells from adipose tissue (see U.S. Patent No. 5,486,359 U.S. Patent Publication 2009/0148419; 2011/0171726; which are incorporated by reference herein in their entirety).

[0090] In one embodiment, the cells are embryonic stem cells. Embryonic stem cells are totipotent and derived from tissue formed after fertilization but before the end of gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Embryonic stem cells can be obtained directly from suitable tissue, including, but not limited to human tissue, or from established embryonic cell lines. In one embodiment, embryonic stem cells are obtained as described by Thomson et al. (U.S. Pat. Nos. 5,843,780 and 6,200,806; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133 ff, 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995 which are incorporated by reference herein in their entirety).

[0091] In one embodiment, the cells are pluripotent stem cells (e.g., isolated from a subject as pluripotent, or "induced pluripotent stem cell" (iPSC)) . The term iPSC refers to pluripotent cells derived from differentiated cells. For example, iPSCs can be obtained by overexpression of transcription factors such as Oct4, Sox2, c-Myc and Klf4 according to the methods described in Takahashi et al. (Cell, 126: 663-676, 2006). Other methods for producing iPSCs are described, for example, in Takahashi et al. Cell, 131 : 861-872, 2007 and Nakagawa et al. Nat. Biotechnol. 26: 101- 106, 2008; which are incorporated by reference herein in their entirety.

[0092] In one embodiment, the cells are adipocyte precursor cells. As used in the art, the term

"adipocyte precursor cells" refers to any cell type or mixture of cell types which are capable of differentiating into mature adipocytes. By way of non-limiting example, adipocyte precursor cells can include preadipocytes, adult mesenchymal stem cells, embryonic stem cells, adipose stem cells, or induced pluripotent stem cells.

[0093] In one embodiment, the pre-matrix composition and hydrogel created therefrom comprises a mixture of one or more cell types. In one embodiment, the pre-matrix composition or hydrogel described herein is created by introducing into the pre-matrix composition and/or hydrogel a population of cells that is substantially one cell type (e.g., at least about 90%, 95% 98%, 99% one cell type) as determined by methods known in the art. In one embodiment, multiple cell types are introduced, e.g., two or more populations of cells, each population comprising substantially one cell type. In one embodiment, the plurality of cell types are added to the pre-matrix composition and/or hydrogel, by adding an mixed population of cell types such as that which is present when cells are isolated from a subject and not further or completely purified by cell type. A population comprising a plurality of cell types has at least two cell types which are present at greater than 5% of the total number of cells, e.g. 80% of a first type of cells and 20% of a second type of cells. Other ratios are envisioned (e.g., about 95:5, 90: 10, 85: 15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, or 50:50, etc.).

[0094] The cells can be obtained from a subject, e.g., primary cells, or can be obtained from storage or propogation in tissue culture, or a mixture thereof. In one embodiment, the pre-matrix composition and/or hydrogel described herein comprises primary cells. As used herein, "primary cells" are those cells which have not undergone significant passaging or subculture in vitro post isolation from a donor subject.

[0095] A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. As used herein "passage" refers to the process of subculturing cells. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated PO. Following the first subculture, the cells are described as a secondary culture (PI or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passages depends on many factors, including but not limited to the seeding density, substrate, medium, growth conditions, and time between passaging. In one embodiment, the pre-matrix composition and/or hydrogel described herein comprises cells which have been passaged less than 5 times, e.g. 4, 3, 2, 1, times or not at all). In one embodiment, the cells have been passaged 10 or fewer times (e.g., 5, 6, 7, 8, 9, or 10 or more times). Cells which have experiences more passaging may also be useful in the present invention (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). Greater than 20 passages is also envisioned. In one embodiment, the passaged cells still exhibit viability and the desired phenotypes and differentiation status.

[0096] In one embodiment, the pre-matrix composition and/or hydrogel described herein comprise predifferentiated cells. As used herein, "predifferentiated cells" refers to cells which have been isolated in a less than fully differentiated state (e.g., in vivo) and treated in vitro to induce differentiation. The stage of differentiation of a population of cells can be described, for example, in terms of the percentage of cells that exhibit a specific stage of differentiation (e.g., fully differentated, predifferentiated, etc.). Differentation can be determined by methods known in the art, for example, by morphology, detection of differentiation specific molecular markers, etc. In one embodiment, at least 5% or more of the population exhibits the desired stage of differentiation prior to addition in the pre-matrix composition and/or hydrogel described herein (e.g. at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% of the cells) 98% or more of the population prior to the cells being administered to a subject.

[0097] In one embodiment, predifferentiated cells are differentiated into adipogenic cells (e.g. they have undergone adipogenesis) prior to use. Adipogenesis can be induced, for example, by culturing the appropriate precursor cells (e.g., stem cells or pre-adipocytes) in adipogenic media. Non-limiting examples of adipogeneic media include media containing steroids, a cyclic AMP inducer, and fatty acids. A further example of adipogenic media is DMEM/F-12 with 3% FBS, 33 μΜ biotin, 17 μΜ pantothenate, 1 μΜ bovine insulin, 1 μΜ dexamethasone, 0.5 mM

isobutylmethylxanthine (IB MX), 5 μΜ rosiglitazone, and 100 U penicillin/ 100 μg streptomycin/0.25 μg fungizone (described further in U.S. Patent No. 6,322,784; 7,001,746; U.S. Patent Publication Nos. 2005/0158706; and Mitchella et al. Stem cells 2006 24:376-385; Zuk et al. Tissue Eng 2001 7:211- 228; and Gimble et al., Cytotherapy 2003 5:362-9, which are incorporated by reference herein in their entirety). The generated or obtained adipocytes can be maintained in culture using any adipocyte maintenance media known to those of skill in the art. By way of non-limiting example, one adipocyte maintenance media is DMEM/F-12 with 3% FBS, 33 μΜ biotin, 17 μΜ pantothenate, 1 μΜ bovine insulin, 1 μΜ dexamethasone, and 100 U penicillin/ 100 μg streptomycin/0.25 μg fungizone.

[0098] In one embodiment, the predifferentiated population is at least about 10% of the cells,

(e.g. at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more predifferentiated, as determined by one or more standard method known in the art). Such populations can be determined by their display of one or more molecular markers or hallmarks of morphology of the desired cell type (e.g., adipose cells).

[0099] Molecular markers of adipose cells include, but are not limited to expression of lipoprotein lipase (LPL; NCBI Gene ID No. 4023), stearoyl-CoA-desaturase (SCD1 ; NCBI Gene ID No. 6319), glucose transporter (GLUT4; NCBI Gene ID No. 6517) and fatty acid binding protein 4 (FABP4, NCBI Gene ID No. 2167) (See U.S. Patent No. 6,322,784 which is incorporated herein by reference in its entirety). Expression of such markers can be detected by methods known to those of skill in the art, including, for example, RT-PCR, real time RT-PCR, or microarray analysis.

[00100] Characteristics of adipose cell morphology include, but are not limited to the formation of cytoplasmic lipid droplets. Methods of detecting these morphologic characteristics are known to those of skill in the art. By way of non-limiting example, the development of cytoplasmic lipid droplets can be monitored by including oil red O or Nile red in the medium. These dyes will stain the lipid droplets red, readily indentifying adipocytes from undifferentiated precursor cells.

Cell Harvest

[00101] In one embodiment, the cells incorporated in the hydrogel are autologous cells. In one embodiment, the cells are autologous to the subject receiving a treatment according to the methods described herein. In one embodiment, the cells are obtained from the subject. In one embodiment, the cells are obtained from the subject's adipose tissue. Such adipose tissue can be, e.g. gluteal adipose tissue or subcutaneous abdominal adipose tissue. Adipose tissue can be harvested, for example, during plastic surgery procedures including lipoaspiration or lipectomy. Adipose tissue can be harvested from one location in the subject or from multiple locations in the subject.

[00102] The site of adipose tissue harvesting can be determined, for example, by examination of the patient by a qualified medical professional. The examination may be performed at bedside or in an operating suite with hemodynamic monitoring appropriate to the patient's clinical status. Preferred harvest site(s) have potential space(s) limited by normal anatomical structures, no major vascular or visceral structures at risk for damage and ease of access. While virgin harvest sites are typically preferred, a previous harvest site does not preclude additional adipose tissue harvest. These preferred sites include but are not limited to the following: lateral and medial thigh regions of bilateral lower extremities, anterior abdominal wall pannus, and bilateral flank regions. These procedures may frequently be performed concomitantly with liposculpture. The site of adipose tissue collection may also be determined by the patient's aesthetic expectations as well as the safety profile as determined by the physician.

[00103] Lipoaspiration for the collection of adipose tissue can be done by any method known in the art. Adipose tissue can also be harvested by, for example injecting the subject subcutaneously, with a standard tumescent fluid solution, which may or may not contain a combination of lidocaine, saline, and/or epinephrine in different standardized dosing regimens. Using an 11 -blade scalpel (or other standard blade), a small puncture wound is made in order to transverse the dermis near the harvest area. The blade is rotated, such as being turned 360 degrees, to complete the wound. A blunt tip 14-guage (or appropriately sized) cannula may then be inserted into the subcutaneous adipose tissue plane. The cannula may be connected to a power assisted suction device or to a syringe for manual aspiration. The cannula is then moved throughout the plane to disrupt the connective tissue architecture. The volume of aspirate obtained may range from about 0 cc to about 1500 cc. Adipose tissue in the form of liposuction aspirates can be collected into a device designed for and dedicated to the purpose of collecting the liposuction aspirates for storage or it can be collected into the usual devices used for collection of liposuction aspirates by personnel performing liposuction procedures. The collection of liposuction aspirates is preferably made under sterile conditions. During the collection of the liposuction aspirates the pressure at the pump should not be higher than 15 mm Hg in order to maintain the viability of the aspirated cells.

[00104] In one embodiment, these plastic surgery methods can be used to both a) remove adipose tissue from the subject for aesthetic results effected by reduced adipose tissue in the harvesting location(s) and b) to provide cells for use in the methods and compositions described herein. By way of non-limiting example, the subject may desire to reduce the amount of

subcutaneous abdominal adipose tissue (i.e. a "tummy tuck") and to be treated according to the methods described herein to reduce the appearance of facial wrinkles. The adipose tissue removed from the abdominal area can be used as a source of cells for use in the methods described herein for the reduction of the appearance of facial wrinkles.

[00105] In one embodiment, the adipose tissue is preserved prior to isolation of cells. For example, in one embodiment, the adipose tissue is cryopreserved (see U.S. Patent Publications 2011/0008300 and 2007/0212336; which are incorporated by reference herein in their entirety). In one embodiment, cells are isolated from the adipose tissue and isolated cells are cyropreserved. In one embodiment, the cells are differentiated prior to cryopreservation. In one embodiment, the cells are differentiated when they are removed from cryopreservation. Cells can be cryopreserved by any method known in the art. See for example, U.S. Patent Nos.7, 811,819 and U.S. Patent Publications 2005/0106554; 2008/0220520; 2003/0054331; 2010/0190248; which are incorporated by reference herein in their entirety.

[00106] In one embodiment, the cells are obtained from non-adipose tissue of the subject. By way of non-limiting example, autologous induced pluripotent stem cells can be obtained from fibroblasts and numerous other cell types.

Delivery to a Subject

[00107] The pre-matrix composition is used to encapsulate cells in a hydrogel for placement in the body of a subject at a site where tissue generation is desired. The polymerization state of the hydrogel material that is delivered can be fully polymerized, partially polymerized or pre -polymerized, the choice of which will depend upon factors such as the specific procedure performed. In one embodiment, the hydrogel is fully polymerized at the time of delivery. In one embodiment the hydrogel is either partially polymerized or in a pre -polymerization state, at the time of delivery, to become fully polymerized within the delivery site. A "pre -polymerization state" as used herein, refers to a pre-matrix composition formulation that includes all necessary components to undergo the appropriate polymerization, but has not yet polymerized (e.g., due to reduced temperature, or insufficient time having passed for polymerization to have occurred).

[00108] The term "hydrogel material" is used herein in conjunction with delivery/administration to a subject to refer to the pre-matrix composition complete with all components required (including cells) to form a polymerized hydrogel, existing in any polymerization state, the appropriate polymerization state being determined by the skilled practitioner for the specific procedure being performed.

[00109] Another aspect of the present invention relates to a method of generating tissue (e.g., adipose tissue) at a specific site in a subject. The method comprises delivery/administering to the specific site the hydrogel material prepared as described herein under conditions suitable for generating new tissue. Examples of suitable method of delivery and sites for delivery are described herein. When used to generate adipose tissue in a subject, methods known in the art for delivery of fat cells to a subject can be applied for delivery of the hydrogel material described herein. Methods known in the art for delivery of dermal fillers may also be applied for delivery of the hydrogel materials described herein.

Modes of Delivery

[00110] Various methods known in the art can be used to generate tissue (e.g., adipose tissue) at a specific site in a subject using the hydrogel material described herein. The specific method will vary with respect to a variety of factors, such as, the characteristics of the individual subject, the desired tissue, and the environment in which the tissue will be generated. Determination of the appropriate delivery method for each location in a subject can be performed by the skilled practitioner. Delivering or administering the hydrogel material described herein by any method available to the skilled practitioner, such as any plastic surgery technique, for delivery of cells or fat transfer or implantation of substances to generate or simulate adipose tissue, (e.g, subcutaneous injection, submuscular injection, intramuscular injection, and subfascial injection), is envisioned.

[00111] Administration may involve the use of needles, catheters and syringes suitable for injection, grafting cannula or surgical implantation. For example, in soft tissue augmentation procedures, the route of delivery may include open delivery through a standard blunt tip cannula (e.g. 14 gauge) inserted into the soft tissue through an appropriately placed incision.

[00112] As used herein, the terms "deliver", "delivery", "delivering", etc. are used

interchangeably with "administer," "administration" and "administering", with respect to placement of the hydrogel material into a subject. As used herein, the term "administer" or "deliver" refers to the placement of a composition (e.g., the hydrogel material) into a subject by a method or route which results in at least partial localization of the composition or components therein, at a desired site.

[00113] The hydrogel material described herein may be administered by any appropriate route known in the art including, without limitation, injection, implantation, via plastic or reconstructive surgery methods, microinjection, and direct application. "Injection" includes, without limitation, intramuscular, intradermal, subdermal, and subcutaneous.

[00114] The use of a combination of delivery means, and sitesplaces of delivery are

contemplated to achieve the desired clinical effect.

[00115] In one embodiment, the hydrogel material described herein is administered to a subject under conditions suitable for generating new adipose tissue. Such conditions include the presence of the required components (e.g., preadipocytes) and delivery to the appropriate site within a subject for the generation of new adipose tissue at that site.

Rate of Dissolution

[00116] The alginate portion of the hydrogel degrades, partially or wholly, after administration to the subject, leaving at least a portion of the cells contained therein deposited in the region of administration. The total volume of the administered material may decrease following administration, but a substantial volume will remain. This may be due to retention and/or proliferation of the administered cells, and/or replacement of the alginate hydrogel with extracellular matrix components, and/or recruitment of other cells from the body into the site. Some of the decrease in volume is attributed to the decrease in the hydrogel polymer surrounding the cells. In one embodiment, the volume of the non-cellular portion of the hydrogel material administered decreases within a few weeks of administration (e.g., 4 weeks, 5, 6, 7, 8, 9, or 10 weeks). In one embodiment, the non- cellular portion of the administered volume is decreased by at least 50% as compared to the original volume within a few weeks of administration, e.g. at least 60% decrease in volume, at least 70% decrease in volume, at least 80% decrease in volume, at least 90% decrease in volume, at least 95% decrease in volume, at least 98% decrease in volume, or at least 99% decrease in volume within a few weeks of administration. Generally, the components of the hydrogel (e.g., hydrogel and/or the cells distributed therein) do not significantly migrate within the subject, nor do the hydrogel or the cells invade surrounding tissue.

[00117] New tissue forms as the alginate polymers are eliminated from the site to result in a significant retention of total volume/mass at the site. Without being bound by theory, a significant portion of the cells are thought to become a component of tissue generated at the delivery site, rather than migrating away from the site and/or being absorbed by the body. The cells administered in the hydrogel contribute to the generation of and/or become established as new tissue. They can proliferate and/or differentiate and/or recruit additional cells from the surrounding tissue in the formation of new tissue. Some reduction in originally deposited volume (e.g., cell number) may occur. A reduction in total volume of the delivered composition may occur, but a significant portion is retained in the subject to contribute to or be replaced by the new tissue generated therefrom. In one embodiment, the reduction in administered volume after a specified time period (e.g., a few weeks, 1- 30 days, 2-6 months, etc.) is less than 50% of that originally delivered (e.g., less than 50%, 40%, 30%, 20%, or 10% reduction in total volume). In one embodiment, the reduction in cells, after a specified time period, (e.g., 1- 30 days, 2-6 months, etc.) is less than 50% of that delivered (e.g., less than 50%, 40%, 30%, 20%, or 10% reduction in total cells).

[00118] In one embodiment, the total volume of the hydrogel material which is administered to the patient decreases following administration. In one embodiment, by 4 weeks after administration, the cells administered as a component of the hydrogel form or promote the formation of new tissue equal to at least 30% of the original volume of the hydrogel. In one embodiment, the volume of the new tissue is at least 40% of the original volume of the hydrogel. In one embodiment, the volume of the new tissue is at least 50% of the original volume of the hydrogel. In one embodiment, the volume of the new tissue is at least 60% of the original volume of the hydrogel. In one embodiment, the volume of the new tissue is at least 80% of the original volume of the hydrogel. In one embodiment, the volume of the new tissue is at least 90% of the original volume of the hydrogel.

[00119] The volume of the new tissue generated within 4 weeks of administration is substantially stable and remains in the subject over an extended period of time, e.g. does not change by more than 20%, e.g., does not change by more than 15%, by more than 10%, or by more than 5% within an established time frame (e.g., 2, 3, 4, 5, 6, or 12 months, 1.5 years, 2, 3, 4, or 5 years, etc.).

[00120] Growth of a cell and/or tissue can be measured by cell number, cell and/or tissue volume, or the incidence and extent of angiogenesis. An increase in growth is indicated by a greater value of at least one parameter of growth as compared to that parameter in an appropriate control, e.g. 5% greater or more, 10% greater or more, 20% greater or more, 50% greater or more, 100% greater or more, 150% greater or more, 200% greater or more, or 300% greater or more.

Additional agents

[00121] The pre-matrix compositions, and/or the hydrogel described herein, may further comprise additional agents which facilitate development or retention of the tissue generated in the subject, or the additional agent may be administered/delivered separately from the hydrogel material, but by administration to the same location (e.g., at the same time or at another time). By way of non- limiting example, such additional agents can include antibiotics, fibrosis-inhibiting agents (see U.S. Patent Publication No. 2009/0214652), immunosuppressive agents (particularly when the cells are not autologous), amino acids, vitamins and steroids. Non-limiting examples of immunosuppressive agents include cyclosporine A, myophenylate mofetil, rapamicin, and anti-thymocyte globulin

Administration Amounts

[00122] The amount of hydrogel material administered to the subject will vary depending upon the specific circumstances of use (e.g., the specific procedure the subject is to undergo and the desired aesthetic results). The amount of hydrogel material to be administered is also dependent upon the specific formulation of the hydrogel produced and the expected properties thereof with respect to expected volume/mass retention upon generation of resulting tissue. By way of non-limiting example, a particular composition is described in the Examples herein where the new tissue (adipose tissue) formed in the subject is approximately 50% of the initial hydrogel material volume. For such a formulation, a procedure where 100 mL of new tissue is desired, approximately 200 mL of hydrogel material should be administered. Such determinations are to be made by the skilled practitioner.

[00123] In one embodiment, the hydrogel material is administered (e.g., injected or implanted) in a single dose at one location to produce the desired results. In one embodiment, the hydrogel material is administered in several doses, at several different locations, to produce the desired results. In one embodiment, administration is repeated at an interval of time, e.g. 2 day intervals, 1 week intervals, 2 week intervals, 1 month intervals, 2 month intervals, 3 month intervals, etc until the desired result is achieved.

Clinical Applications

[00124] Numerous defects, disorders or aesthetic concerns may be treated with the methods and compositions described herein. The methods and compositions described herein can be used to treat any condition which will be improved by an increase in the desired tissue (e.g., adipose tissue) at a particular location. Additionally, the methods and compositions described herein can be used to effect aesthetic changes via an increase in tissue (e.g., adipose tissue), e.g. augmentation, tone improvements, etc. Described below are some clinical applications in which the methods and compositions described herein may be employed. Various combinations of the delivery methods provided by example are envisioned for use in the present invention.

[00125] The methods described herein result in the generation of vascularized tissue (e.g., adipose tissue) at the implant site.

[00126] Subject monitoring prior to, during, and after the administration can be used to determine successful tissue generation. Methods of monitoring include, but are not limited to, the following: coagulation studies, oxygen saturation, hemodynamic monitoring, and wound status. Additional monitoring will be specific to the desired clinical effect. The efficacy of the treatment can be assessed by monitoring, for example, tissue shape, tissue function, tissue contour, stability of such changes, and patient satisfaction and quality of life.

RECONSTRUCTION

[00127] In one embodiment, the methods and compositions described herein are employed to reconstruct tissue that has been damaged or removed as a result of trauma, surgery, chemotherapy and/or radiation therapy. In one embodiment, the methods and compositions described herein are employed to treat degenerative or congenital diseases which result in misformed or deficient tissue.

[00128] In one embodiment, the methods and compositions described herein are employed to protect non-adipose tissues (e.g. protection of a nerve root following surgery) or to provide padding of body prominences that lack sufficient buffers against pressure. By way of non-limiting example, when adipose tissue padding is lacking, the overlying skin may be adherent to the bone, leading to discomfort and even pain, which occurs, for example, when a heel spur or bony projection occurs on the plantar region of the heel bone (also known as the calcaneous). In this case, the hydrogel material described herein may provide the interposition of the necessary padding between the bone and the skin. The hydrogel material can be delivered by injection between the bone and the skin or by placing a hydrogel composition of the necessary dimensions and thickness between the bone and skin via an incision.

Congenital anomaly

[00129] A number of congenital anomalies can result in an insufficient amount of soft tissue which can be treated according to the methods and compositions described herein. Examples of congenital anomalies which result in a insufficient amount of soft tissue include, but are not limited to, hemifacial microsomia, short palate, fat atrophy as a result of lupus erthymatosis, morphea zoniform, and ozena.

[00130] Hydrogel compositions described herein can also be used to treat congenital anomalies as described above. Methods of delivering autologous cells(e.g., fat cells) for tissue augmentation, such as injection or direct application, are known in the art and can be adapted for delivering the hydrogel material described herein. One example of a method of delivering the hydrogel material for the treatment of hemifacial microsomia is as follows: the hydrogel material is delivered via a syringe with a blunt needle having an outside diameter or 1.5 or 3 mm. Hydrogel material is administered in small quantities, depositing it radially from the distal to the proximal. The hydrogel material is administered to the subcutaneous tissue and under the superficial musculoaponeurotic system, in multiple tunnels and planes. (Li et al. (2010) "Correction of Hemifacial Atrophy with Fat Transfer" in Shiffman (Ed) Autologous Fat Transfer (331-339) Springer-Verlag: Berlin; which is incorporated by reference herein in its entirety).

[00131] In addition to the treatment of congenital anomalies, the hydrogel compositions described herein can be used in methods for treatment of various forms of scarring in a subject. As used herein, a scar is something that results from an occurrence to the subject following birth, and is separate from a congenital anomaly. Various examples of types of scars are described herein, including, without limitation, a depression scar, post-burn contracture, post-irradiation soft tissue atrophy, and post-traumatic soft tissue deficiency.

Following cancer ablation

[00132] Removal of tissue as part of a treatment or therapy for cancer can leave scars, depressed scars (e.g. as after large skin cancer excision for melanoma), and depressions or deformations in areas such as the breast or face. Methods of treating tissue deficits in each of these areas are described elsewhere herein.

Post-irradiation soft tissue atrophy

[00133] Radiation exposure, e.g. as a cancer treatment or accidental exposure, can result in atrophy of soft tissue. The resulting tissue deficits can be treated by administering a hydrogel material described herein. Methods of administration of autologous cells (e.g., fat cells) for such a treatment, such as by injection or surgical implantation, are known in the art and can be adapted for delivery of the hydrogel materials described herein. Preferred methods of administration will vary depending upon the part of the body that is to be treated. Described elsewhere herein are methods of administering hydrogel material to various parts of the body (See: Tholen et al. (2010) "Recontouring Postradiation Thigh Defect with Autologous Fat Grafting" in Shiffman (Ed) Autologous Fat Transfer (341-346) Springer- Verlag: Berlin; which is incorporated by reference herein in its entirety).

Post-burn scar contracture

[00134] The scar formed after a burn injury may contract, lack elasticity, and/or form a depression. Methods of administration of autologous cells (e.g., fat cells) to a burn scar for such a treatment, such as by injection, are known in the art and can be adapted for delivery of the hydrogel materials described herein. One example of a method of delivering a hydrogel material to an area affected by a burn scar is as follows: the hydrogel material is administered to the dermal-hypodermal junction in the scarred area, using a sharp 01.-0.2 mm cannula. Administrations can be repeated twice at 3 month intervals as needed (Klinger et al. Aesth Plast Surg 2008 32:465-9; which is incorporated by reference herein in its entirety).

Post-traumatic soft tissue deficiency

[00135] Trauma, e.g. cuts, burns, etc, as well as treatments employed to treat a trauma (e.g. surgical removal of irreparably damaged tissue) can result in deficiency of soft tissue. The resulting tissue deficits can be treated by administering the hydrogel material. Methods of administration of autologous fat cells to treat post-traumatic soft tissue deficiency, including injection or surgical implantation, are known in the art and can be adapted for delivery of the hydrogel materials for such treatment. Preferred methods of administration will vary depending upon the part of the body that is to be treated. Described elsewhere herein are methods of administering hydrogel material to various parts of the body.

Soft tissue padding around joint area

[00136] Trauma or disease can result in an insufficient amount of soft tissue near a joint, causing pain and limiting use of the joint. Hydrogel compositions described herein can also be used to prevent fibrosis or heterotopic bone growth. Methods of delivering autologous fat cells to a joint area in need of an increase in soft tissue padding, including via injection or direct application, are well known to those of ordinary skill in the art and can be adapted by the skilled practitioner for the delivery of the hydrogel material. One example of a method of delivery is: in order to prevent heterotropic bone formation, decrease fibrosis, improve pain levels and increase joint function, hydrogel material can be administered to subjects receiving a TMJ joint prosthesis. After the prosthesis implantation (e.g. after the fossa and mandibular components have been stabilized), the articulating part of the prosthesis can be surrounded by hydrogel material. Alternatively, the hydrogel material can be administered at the same time that the prosthesis is implanted. The hydrogel material is administered through the endaural or preauricular incision made for the prosthesis implantation and the dead space around the articulating portion is filled. It is important to fill the entire dead space (See Wolford and Cassano in Shiffman (Ed) Autologous Fat Transfer (361-382) Springer- Verlag: Berlin; Wolford et al. Proc (Bayl Univ Med Cent) 2008 21 :248-254; Peterson and Adham Journal of Trauma-Injury Infection & Critical Care 2006 61 :392-5; Dimitroulis Int J Oral Maxillofac Surg 2004 33:755-760; which are incorporated by reference herein in their entirety).

Intractable skin ulcer

[00137] Intractable skin ulcers can form as a result of, for example, diabetes, ischemia, or collagen disease. Methods of delivering fat cells to an ulcer or wound, including via injection, incorporating into a wound dressing or direct application, are known in the art and can be adapted by the skilled practitioner for appropriate delivery of the hydrogel material to treat an intractable skin ulcer. For example: the hydrogel material is administered to a subject by injecting it underneath or into the ulcer or wound (Kim et al., J Derma Science 53(2009)96-102; PRS 2009 124(3) 765-774; which are incorporated by reference herein in their entirety). Alternatively, the hydrogel material can be directly placed in the ulcer or wound and the site then covered with a dressing, for example, Tegaderm (3-M Health Care, St. Paul, MN) or a silicon membrane, (see Kim et al. Journal of Dermatological Science. 2007 48: 15-24 and Mizuno and Nambu Methods Mol Biol. 2011 702:453-9, which are incorporated by reference herein in their entirety).

AESTHETIC APPLICATIONS

[00138] In one embodiment, the methods and compositions described herein are employed in aesthetic applications, e.g. to alter the subject's appearance for aesthetic reasons, not to improve their health. Such applications can include the augmentation of certain areas or the alteration of their appearance, e.g. firmer, smoother looking skin or reduced appearance of scarring (e.g. acne scarring). Breast Augmentation

[00139] Methods of delivering fat cells to breast tissue, including via injection, are well known in the art and can be adapted for delivery of the hydrogel material described herein. A non-limiting example for the purpose of breast augmentation and/or reconstruction is as follows: subjects receive general anesthesia or an epidural plus sedation and intercostal nerve blocks. The hydrogel material is grafted in 1 to 3 stages, with a total hydrogel volume of generally 200-300 cc per breast. The hydrogel material is delivered via blunt infiltration cannulas through 2-mm incisions using 5 mL syringes. Multiple incisions can be used, allowing placement from at least two directions into each area. Approximately 0.2 mL is delivered with each withdrawal of the cannula. Contouring can be achieved by layering the hydrogel material in different levels until the desired contour is achieved. In most cases, the largest portion of the hydrogel material is infiltrated into the pectoralis major muscle, followed by the retropectoral and prepectoral spaces. Shaping of the breast is accomplished with placement subcutaneously into the superficial breast planes. Placement into the parenchyma of the breast is limited and performed to further increase projection (Coleman and Saboeiro Plastic Reconstructive Surgery (2007) 119:775-784; Shu. (2010) in Shiffman (Ed) Autologous Fat Transfer (229-236) Springer-Verlag: Berlin; Takasu and Takasu (2010) in Shiffman (Ed) Autologous Fat Transfer (237-242) Springer-Verlag: Berlin which are incorporated by reference herein in their entirety). The outcome of a breast reconstruction or augmentation procedure can be determined and monitored by observation and/or measurement of breast size, breast shape, breast contour, longevity of shapes in breast shape, size, and/or contour, rate of liponecrotic cyst formation, and/or patient satisfaction.

Facial Contouring

[00140] Methods of administering fat cells to the temples to alter the contours of the face (e.g., reducing the appearance of wrinkles, depressions, sagging skin, etc.), including via injection, are known in the art and can be adapted for the administration of the hydrogel material described herein. A non-limiting example of a method is as follows: Using a blunt 18-gauge cannula attached to a 1 mL syringe, the hydrogel material is delivered to the desired area, depositing in the subcutaneous, submuscular, and intramuscular planes whenever possible. Each pass deposits approximately 0.1 mL of hydrogel material and each pass should leave a unique "tunnel" of hydrogel material in the tissue. Access to the forehead is preferably via incisions at the hairline, administering hydrogel composition subcutaneously and intramuscularly. Ridging should be smoothed out. An additional midforehead incision can be used to access the glabella and nasal root. Total hydrogel material delivered during a forehead contouring is typically 8-15 mL.

[00141] The cheek can be approached via incisions lateral to the nasolabial fold, administering about 5-7 mL threads of hydrogel composition around the zygomaticus muscle and through the subcutaneous fat. The cheek can also be approached via an incision lateral to the zygomatic arch to deposit hydrogel material in a cross-hatch or lattice pattern. In the buccal area, about 3-5 mL threads of hydrogel material should be administered, paying particular attention to blending. The area anterior to the ear can reviece about 4-5 mL of hydrogel material.

[00142] The mandible and chin can be approached via multiple incisions perpendicular to the mandible, fanning laterally and inferiorly to within 1 cm below the bony border on the superior portion of the neck. As much as 20 mL of hydrogel composition can be administered to this area (See Donofrio. Aesthetic Surgery Journal 2008 28:681-7; Glashofer and Lawrence Dermatologic Therapy 2006 19: 169-176; Kranendonk and Obagi 2007 33:572-578; Lambros Aesthetic Surgery Journal 2011 31:89-94; Shiffman and Kaminski (2010) in Shiffman (Ed) Autologous Fat Transfer (113-122) Springer-Verlag: Berlin; Erian and Hafeez (2010) in Shiffman (Ed) Autologous Fat Transfer (135-146) Springer-Verlag: Berlin; Giebler (2010) in Shiffman (Ed) Autologous Fat Transfer (147-152) Springer-Verlag: Berlin; which are incorporated by reference herein in their entirety). Sunken Upper Eyelid

[00143] Methods of administering fat cells to the temples to decrease the appearance of a sunken, darkened, or wrinkled upper eyelids including via injection, are well known to those of ordinary skill in the art and can be adapted to the delivery of the hydrogel material described herein. A non-limiting example of a method for the purpose of decreasing the appearance of sunken upper eyelids is as follows: Using a blunt 18-gauge cannula attached to a 1 mL syringe, the hydrogel material is delivered to the desired area, depositing in the subcutaneous, submuscular, and intramuscular planes whenever possible. Each pass deposits approximately 0.1 mL as a "droplet" in the lid crease, pulling the tail of the "droplet" superiorly. Each deposit of hydrogel material should leave a unique "tunnel" of hydrogel composition in the tissue. Access to upper eyelid is preferably via incisions at the eyebrow. Total hydrogel material delivered during a forehead contouring is typically about 1 mL per eyelid (See Donofrio Aesthetic Surgery Journal 2008 28:681-7; Glashofer and Lawrence Dermatologic Therapy 2006 19: 169-176; Kranendonk and Obagi 2007 33:572-578; Lambros Aesthetic Surgery Journal 2011 31:89-94; Park (2010) in Shiffman (Ed) Autologous Fat Transfer (155-164) Springer- Verlag: Berlin; which are incorporated by reference herein in their entirety).

Dark Circle in Lower Eyelid

[00144] Methods of administering fat cells as described herein to the temples to decrease the appearance of dark circles or lines in the area of the lower eyelid, including via injection, are known in the art and can be adapted to the delivery of the hydrogel material described herein. One example of a method of administering a hydrogel composition for the purpose of decreasing dark under-eye circles is as follows: Using a blunt 18-gauge cannula attached to a 1 mL syringe, the hydrogel composition is delivered to the desired area, depositing in the subcutaneous, submuscular, and intramuscular planes whenever possible. Each pass deposits approximately 0.1 mL. Each deposit of hydrogel composition should leave a unique "tunnel" of hydrogel composition in the tissue. Access to the lower eyelid is via incisions in the central midcheek. The hydrogel composition is injected inferior to the arcus marginalis and the orbital septum is never violated. 0.1 mL deposits of hydrogel composition are placed as substantially round deposits along the orbital rim, moving medial to lateral, starting in a place deep to the zygomaticus muscles and ascending through the muscle into the subcutaneous fat. Hydrogel compositions can also be placed deep to the orbicularis oculi muscle superior to the orbital rim. Total hydrogel composition delivered during a forehead contouring is typically about 1 mL superior to the orbital rim and about 1 to 4 mL overall (See Donofrio Aesthetic Surgery Journal 2008 28:681-7; Glashofer and Lawrence Dermatologic Therapy 2006 19: 169-176; Kranendonk and Obagi 2007 33:572-578; Lambros Aesthetic Surgery Journal 2011 31 :89-94).

Deepened Nasolabial Fold

[00145] Methods of administering fat cells to the temples to decrease the extent of the nasolabial fold, including via injection, are known in the art and can be adapted to the delivery of the hydrogel material described herein. One example of decreasing the appearance of a deep nasolabial fold is as follows: using an incision site lateral to the crease, the hydrogel material is administered across the crease, blending into the cutaneous portion of the upper lip. Typically, about 1-2 mL of hydrogel material can be administered to each crease (See Donofrio. Aesthetic Surgery Journal 2008 28:681-7; Glashofer and Lawrence Dermatologic Therapy 2006 19: 169-176; Kranendonk and Obagi 2007 33:572-578; Lambros Aesthetic Surgery Journal 2011 31:89-94; Dryden and Heringer (2010) in Shiffman (Ed) Autologous Fat Transfer (189-196) Springer- Verlag: Berlin; which are incorporated by reference herein in their entirety).

Temple Depression

[00146] Methods of administering fat cells to the temples to decrease the extent of depressions or concavity at the temples, including via injection, are known in the art and can be adapted to the delivery of the hydrogel material described herein. A non-limiting example of decreasing temple depressions is as follows: using a blunt 18-gauge cannula attached to a 1 mL syringe, the hydrogel material is delivered to the desired area, depositing in the subcutaneous, submuscular, and

intramuscular planes whenever possible. Each pass deposits approximately 0.1 mL of hydrogel composition and each pass should leave a unique "tunnel" of hydrogel material in the tissue. Access to the temple is via a hairline incision superior to the temporal fossa or from a site superior to the zygoma. A curved Amar no. 7 cannula can be useful to deliver hydrogel material to the temporalis muscle. Total hydrogel material delivered during a forehead contouring is typically 2-4 mL (See Donofrio Aesthetic Surgery Journal 2008 28:681-7; Glashofer and Lawrence Dermatologic Therapy 2006 19: 169-176; Kranendonk and Obagi 2007 33:572-578; Lambros Aesthetic Surgery Journal 2011 31:89-94; which are incorporated by reference herein in their entirety).

Lip Augmentation

[00147] Methods of administering fat cells to the lips, including via injection, are known in the art and can be adapted to the delivery of the hydrogel material described herein. A non-limiting example of lip augmentation is as follows: the hydrogel material is administered to the lip

subcutaneously using a small needle or grafting cannula to deposit the hydrogel material in horizontal tunnels within the lip tissue. The amount and location of the depositions are varied to achieve the size and contour desired (See Donofrio Aesthetic Surgery Journal 2008 28:681-7; Tobin and Karas.

Journal of Oral and Maxillofacial Surgery 1998 56:722-7; Gatti Annals of Plastic Surgery 1999 42:376-380; Castor et al. Aesthetic Plastic Surgery 1999 23:218-223; Hopping et al (2010) in

Shiffman (Ed) Autologous Fat Transfer (197-202) Springer- Verlag: Berlin;which are incorporated by reference herein in their entirety).

Hand Rejuvenation

[00148] Methods of delivering fat cells to the hands, including via injection, are known in the art and can be adapted to the delivery of the hydrogel material described herein.. Such treatments can, for example, reduce the appearance of wrinkles, depressions, and the extent to which the underlying bone, tendon, and muscle structures are visible. A non-limiting example of a method of hand rejuvenation and/or reconstruction is as follows: the hydrogel material as described herein is injected subcutaneously on the dorsum of the hands (back of the hand). Each hand can receive, for example, about 15-20 mL of hydrogel composition (See Coleman Plastic and Reconstructive Surgery vol 110 7 2002 1731-44; Bidic et al.. Plastic & Reconstructive Surgery. 2010 126: 163-168; Edelson, J Cosmetic Dermatology 2009 8:44-51 ; Giunta et al. Handchir Mikrochir Plast Chir 2010 42:143-7; Fournier (2010) in Shiffman (Ed) Autologous Fat Transfer (273-280) Springer- Verlag: Berlin; which are incorporated by reference herein in their entirety).

Buttock Augmentation

[00149] Methods of delivering fat cells to buttocks tissue, including via injection, are well known to those of ordinary skill in the art and can be adapted to the delivery of the hydrogel material described herein. A non-limiting example of a method of buttocks augmentation and/or

reconstruction is as follows: subjects receive general anesthesia and administration is conducted using grafting cannula 2 mm in diameter by 15 cm in length with a blunt tip and a single side hole. Three to five small incisions are made in each lateral buttock. The hydrogel material is deposited beginning medially and deep into the muscle and fat just above the bone. No more than 0.3cc is deposited in each pass, requiring some 3000 tunnels per side for an the average augmentation which totals roughly 600-1000 cc to each buttock. Administration is preferably begun inferomedially, then proceeds to the mid-medial buttock, then the superior-medial buttock, then the lateral buttock and thighs, each to the extent desired. Suction is optionally used to sculpt the desired shape. Drains are inserted on each side of the patient and a compressive bandage is placed over the sacrum to prevent fluid accumulation (Roberts et al Clinics in Plastic Surgery 2006 33: 371-394; Aiache (2010) in Shiffman (Ed)

Autologous Fat Transfer (297-300) Springer- Verlag: Berlin; which are incorporated by reference herein in their entirety).

Penile Augmentation

[00150] Methods of delivering fat cells to penile tissue, including via injection, are well known to those of ordinary skill in the art and can be applied to the delivery of the hydrogel material described herein. Non-limiting examples of methods of penile augmentation and/or reconstruction are as follows: the subject is locally anesthetized and either the penis or the prepubic area is partially circumcised and the hydrogel material is inserted into the penile tissue, in the location and amount necessary to achieve the desired contouring, size and shape. Alternatively, a penile augmentation can be performed as follows: a penile skin segment directly behind the glans is transversely incised and the hydrogel material is administered between the subglans margin and tunica albuginea.

Alternatively, a distal part of the penis is minimally incised and the hydrogel composition is applied to the Buck's fascia layer and optionally, the subglans space. Alternatively, the penis is incised at a part immediately below the glans in a transverse direction and hydrogel material is administered to the space between Buck's fascia and the dartos fascia, the glans zone, a distal zone, and/or a proximal zone (See U.S. Patent No. 7,806,821 and U.S. Patent Publication No. 2006/0096603, and Hernandez- Perez et al (2010) in Shiffman (Ed) Autologous Fat Transfer (217-222) Springer-Verlag: Berlin; which are incorporated by reference herein in their entirety).

Depressed Scars

[00151] Methods of delivering fat cells to the site of a scar, for example, for the purpose of decreasing the appearance of the scar, reducing a depression associated with a scar or improving the characteristics of the affected skin, including via injection, are well known to those of ordinary skill in the art and can be applied to the delivery of the hydrogel material described herein. A non-limiting example of a method of treating a scar is as follows: the hydrogel material is administered by small 0.1-0.2 mm cannulas at the dermal-hypodermal junction in the scar areas (Klinger et al. (Aesth Plast Surg (2008) 32:465-469; Raskin (2010) in Shiffman (Ed) Autologous Fat Transfer (69-78) Springer- Verlag: Berlin; which are incorporated herein by reference in their entirety).

Anti-Wrinkle effect, Skin Tone

[00152] In one embodiment, the compositions and methods described herein are employed in minimizing the appearance of wrinkles and/or for minimizing the effects of aging on skin. In one embodiment, the wrinkles or aesthetically displeasing skin appearance is present on the face, on the neck, on the hands, or any other part of the subject's body. By way of non-limiting example, the hydrogel material described herein can be administered to a subject by injecting the hydrogel material underneath the wrinkle, line, loose skin, depression, scar, or other undesired feature (Kim et al., J Derma Science 53(2009)96-102; PRS 2009 124(3) 765-774; which are incorporated by reference herein in their entirety).

Kits

[00153] Another aspect of the present invention relates to kits comprising components for performing the methods described herein. In one embodiment, the kit comprises the high molecular weight and low molecular weight alginate combined to produce the pre-matrix composition described herein. The kit may further comprise a cross-linking agent or precursor thereof, described herein. The HMW and LMW alginate may be in the kit stored in combination (precombined) or alternatively present separately in amounts convenient for combination as to the intended final hydrogel product. In one embodiment, the HMW and LMW alginate are present in combination at a ratio of from about 1 :2 to about 1:4. In one embodiment, the HMW and LMW alginate are present in a convenient amount that is ready for dilution with cells in media, addition of cross-linker, and then delivery into a subject. In one embodiment, the HMW and LMW alginate are present in a 2X, 3X or 4X concentration, for dilution with cells/media (e.g., 1 : 1 dilution for a 2X concentration). In one embodiment, the HMW and LMW alginate are present at a concentration of greater than or equal to 1 %, or to 2%, or to 3% weight/volume. In one embodiment, one or both of the HMW and LMW alginate is coupled to an adhesion peptide. In one embodiment, the kit further comprises one or more growth factors (e.g., adipogenic growth factor and/or angiogenic factor. The HWM and/or LMW alginate and any additional components of the kit can be present in the kit in any convenient form described herein (e.g, solution, solid, semi-solid, powder, etc.) In one embodiment, one or both the HMW and LMW alginate are in solution. In one embodiment, one or both the HMW and LMW alginate are in lyophilized form. [00154] The kit of the present invention may further comprise directions for the appropriate preparation and/or use of the components therein. The kit may further comprise useful tools for preparation and/or delivery of the hydrogel materials to a subject, as described herein.

[00155] The description of embodiments of the invention is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[00156] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[00157] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00158] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. 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 is defined solely by the claims.

[00159] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used to described the present invention, in connection with percentages means +1%.

[00160] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the invention ("consisting essentially of). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments, the inventions, compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method ("consisting of).

[00161] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[00162] All patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[00163] The invention is further illustrated by the following examples, which should not be construed as further limiting.

[00164] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting.

[00165] Some embodiments of the invention described herein can be defined according to any of the following numbered paragraphs:

1. A cell implantation pre-matrix composition comprising, a solution of oxidized high molecular weight (HMW) alginate and oxidized low molecular weight (LMW) alginate, wherein:

a) the oxidized HMW alginate and oxidized LMW alginate are present at a ratio of from about 1 :3 to about 1 :4;

b) the oxidized HMW alginate and oxidized LMW alginate are present at a weight/volume concentration of from about 1% to about 4%;

c) the oxidized HMW alginate and oxidized LMW alginate are oxidized at about 2% of their sugar residues;.

d) one or both of the high molecular weight alginate and the low molecular weight alginate are coupled to an adhesion peptide at a yield of about 2 peptides per alginate polymer chain.

2. The composition of paragraph 1, further comprising adipogenic growth factors.

3. The composition of paragraph 1, further comprising one or more angiogenic factors. 4. The composition of paragraph 3, wherein the angiogenic factor is vascular endothelial growth factor.

5. The composition of paragraph 4, wherein the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration of about 4%.

6. The composition of paragraph 1 , wherein the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration from about 1 % to about 3 %.

7. The composition of paragraph 6, wherein the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration of about 2%.

8. The composition of any one of paragraphs 1 - 7, further comprising cells.

9. The composition of paragraph 8, wherein the cells are selected from the group consisting of preadipocytes, adult mesenchymal stem cells, human adipose stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

10. The composition of any one of paragraphs 8-9, wherein the cells are primary cells.

11. The composition of any one of paragraphs 8-10, wherein the cells are predifferentiated cells.

12. The composition of paragraph 11, wherein the predifferentiated cells are adipocytes.

13. The composition of paragraph 1, wherein the adhesion peptide comprises the amino acid sequence G4RGDASSKY (SEQ ID NO:2).

14. The composition of any one of paragraphs 1-13, further comprising a cross-linking agent or precursor thereof.

15. The composition of any one of paragraphs 1-14, that is cross-linked to produce a hydrogel.

16. A kit comprising:

a) the cell implantation pre -matrix composition of any one of paragraphs 1-15;

b) directions for use.

17. The kit of paragraph 16, further comprising one or more of an adipogenic growth factor and an angiogenesis factor.

18. A method of generating adipose tissue at a specific site in a subject, the method comprising: administering to the specific site cells encapsulated in a hydrogel, under conditions suitable for generating new adipose tissue, wherein the hydrogel is generated from crosslinking the cell implantation matrix of any one of paragraphs 1-14.

19. The method of paragraph 18, wherein administering is performed by a method selected from the group consisting of subcutaneous injection, submuscular injection, intramuscular injection, and subfascial injection.

20. The method of any one of paragraphs 18-19, wherein the cells are primary cells.

21. The method of any one of paragraphs 18-20, wherein the cells are autologous.

22. The method of any one of paragraphs 18 -21, wherein the cells are selected from the group consisting of adipocytes, preadipocytes, adult mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof. 23. The method of any one of paragraphs 18-22, wherein the specific site is located at a region selected from the group consisting of the face, the breast, the buttocks, the hand, and the penis.

24 The method of paragraph 23, wherein the specific site is located at the region of the face and is selected from the group consisting of lip, nasolabial fold, temple, lower eye lid, and upper eye lid.

25. The method of any one of paragraphs 18-22, wherein the specific site is a scar of the subject.

26. The method of paragraph 25, wherein the scar is a depression scar.

27. The method of paragraph 25, wherein the scar is a post-burn contracture.

28. The method of paragraph 25, wherein the scar is from post-irradiation soft tissue atrophy.

29. The method of paragraph 25, wherein the scar is a post-traumatic soft tissue deficiency.

30. The method of any one of paragraphs 18-22, wherein the specific site is in a region of congenital anomaly.

31. The method of paragraph 30, wherein the congenital anomaly is hemifacial microsomia or facial asymmetry.

EXAMPLES

Example 1: Adipose Tissue Engineering using injectable, oxidized alginate hydrogels

[00166] Described herein are experiments demonstrating the feasibility of a degradable alginate hydrogel system with pre-conditioned, cryopreserved human adipose stem cells (hADSCs) to engineer adipose tissue. hADSCs were differentiated into adipogenic cells, and encapsulated in alginate hydrogels made susceptible to hydrolysis by partial periodate oxidation of the polymer chains. Cell-laden gels were injected subcutaneously into the chest wall of male nude mice, and a cell suspension without alginate served as control. After 10 weeks, specimens were harvested and analyzed morphologically, histologically and with immunoblotting of tissue extractions. Newly generated tissues were semitransparent and soft in all experimental mice, grossly resembling adipose tissue. Analysis using confocal live imaging, immunohistochemisty and Western blot analysis revealed that the newly generated tissue was adipose tissue.

Materials and Methods

[00167] Human adipogenic stem cells(hADSCs): preparation and adipogenic

differentiation in vitro. Passage 1 hADSCs were purchased from Lonza (Basel, Switzerland).

hADSCs were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL) supplemented with 10%(v/v) fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Invitrogen), and used between passages 5-6. Cells were maintained at lower than 80% confluency in culture, and trypsinized and replated on T225 flasks (Falcon) at a density of 4x 10⁶ cells per flask for predifferentiation. Cells were incubated in standard medium overnight to allow adherence to the bottom of flasks, then the medium was replaced with adipogenic differentiation medium containing DMEM with 10%FBS, ΙμΜ dexamethasone (Sigma), ΙΟμΜ human recombinant insulin (Sigma), 200μΜ indomethacin (Sigma), and 0.5μΜ 3-isobutyl-l-methylxanthine (IB MX; Sigma) for 2weeks. The adipogenic medium was exchanged every 3 days. The adipogenic differentiation of hADSCs was assessed by the presence of intracellular lipid accumulation without staining, Nile Red staining, and indirect Immunostaining.

[00168] Nile Red staining. Nile Red staining was used to detect the presence of intracellular lipid-filled droplets. Briefly, cells were fixed in 4% paraformaldehyde (PFA) for 30 min, washed with PBS, and stained with 1 μg/mL Nile Red (Sigma) in PBS for 20 min at room temperature. The nuclei of cells were counter-stained with 4' ,6-diamidino-2-phenylindole (DAPI;Invitrogen), and stained cells were treated with PROLONG GOLD™ antifade reagent (Invitrogen). After two intensive rinses with deionized water, photomicrographs were taken using a fluorescence microscope (NIKON ECLIPSE E800™).

[00169] Immunostaining for peroxisome proliferating antigen receptor gamma(PPAR-y).

After 10 days of adipogenic culture in Lab-Tek chamber slides (Thermo scientific), cells were rinsed in PBS and fixed in 4% PFA. The samples were blocked in a solution containg 10% normal goat serum, 0.5% BSA (bovine serum albumin), and 0.1% Triton in PBS for 30min, incubated overnight in primary PPAR-γ rabbit antibody (Cell Signaling) diluted at 1 : 1000 in 1% BSA-PBS solution, and then incubated for 2 hr in Daylight 549-conjugated anti-rabbit antibody (Jackson Immunolabs) as secondary antibody diluted 1 :500 in 1% BSA-PBS solution. After samples were counterstained with DAPI, and treated with PROLONG GOLD™ antifade reagent, photomicrographs were taken using the camera system described above.

[00170] Alginate modification/Hydrogel Processing. Ultrapure alginates were purchased from ProNova Biomedical (Norway). MVG alginate (M/G:40/60) was used as the high molecular weight component to prepare gels. Low molecular weight (LMW) alginate was obtained by γ- irradiating high molecular weight(HMW) alginate with a cobalt-60 source for 4h at a γ-dose of 5.0 Mrad (Phoenix Lab, University of Michigan, Ann Arbor, MI,USA)¹⁴.

[00171] Both alginate polymers were diluted to 1% w/v in double-distilled H₂0, and 2% of the sugar residues in the polymer chains were oxidized by treatment with sodium periodate (Sigma). Oxidation was performed in the dark for 17 hours at room temperature ¹⁵. An equimolar amount of ethylene glycol (Fisher) was added to stop the reaction and the solution was subsequently dialyzed (MWCO 1000, Spectra/Por®) over 3 days.

[00172] Following oxidation, the adhesion peptide sequence G4RGDASSKY-OH (SEQ ID NO: 3) (Peptides International) was coupled to both HMW and LMW alginate using carbodiimide chemistry¹⁸. The concentration of peptides and polymer was adjusted to yield 2 peptides per polymer chain. Following peptide modification, alginate was dialyzed, treated with activated charcoal, filter sterilized (0.22μπι), freeze-dried and stored at -20 °C. To prepare gels, each molecular weight alginate was reconstituted at 4% w/v in media without serum or phenol red. Gels were formed using a combination mixture of the two different molecular weight polymers at a ratio of 3: 1 (low:high).

[00173] Injection of differentiated hADSCs-alginate hydrogel mixture in vivo and harvest.

To form injectable hydrogels containing cells, 0.2 mL of a suspension containing predifferentiated cells (2xl0⁶/mL) was mixed with 0.2mL of the 4% (w/v) alginate solution using two syringes coupled by a connector. 0.4mL of the resulting 2% (w/v) alginate solution was gelled by combining with an aqueous slurry of calcium sulfate (0.21g CaSCVrnL distilled H20) at a ratio of 25: 1 (40 μί_^ of CaSCVlmL of 2% w/v alginate solution) in the syringe subsequently used to inject the mixture.

[00174] Under inhalation general anesthesia using methoxyfluorane, hydrogels were injected subcutaneously into two locations on either the chest wall or abdomen of each male nude mice (Jackson Laboratory) using a 23 gauge needle. The cell-encapsulating hydrogels (Experimental Group) were on one side, a cell suspension without alginate hydrogel (Control Group) was on the other side. Mice (n=5) were euthanized 10 week after injection in a C0₂ gas chamber. Newly formed tissue specimens were harvested for gross and histological evaluation.

[00175] Histology, Immuostaining, Western blot analysis, Scanning Electron

Microscopic(SEM) examination. Specimens were sequentially sectioned at 7-10 μπι thickness and stained with hematoxylin and eosin, oil red O(ORO), or subject immunostaining. For immunostaining, PPARy (Cell Signaling Technology) was used as the primary antibody for tissue analysis. For western blot analysis, lOOmg samples of the newly generated tissue were homogenized and lysed in Radio Immunoprecipitation Assay (RIP A) buffer (Sigma) with Minitab Protease Inhibitors (Roche) on ice for 20 min. The lysate was collected, and centrifuged at 12,000 rpm for 10 min at 4°C.

Aliquots of the supernatants containing 20μg of protein (determined using the Pierce BCA protein assay kit) were subjected to protein gel electrophoresis using 4-20% Tris-Glycine gel (Invitrogen), transferred onto a nitrocellulose membrane (Amersham), and treated with 20% methanol in Tris- glycine buffer. After being blocked with PBS containing 3% BSA for lh at room temperature, the membranes were incubated with 1 : 1000 diluted primary monoclonal PPARy (Cell Signaling

Technology), Adiponectin (Cell Signaling Technology), C/EBP alpha/beta (Cell Signaling

Technology) and β-actin (Sigma) antibodies at 4°C overnight and then with 1 : 10,000 diluted HRP- conjugated monoclonal secondary antibody (Jackson Immunolabs) for lh at room temperature.

Signals were detected by bioluminescence using X-ray films (Kodak MR,Sigma-Aldrich) and the Enhanced Chemiluminescence substrate system(Thermo Scientific). For scanning electron

miscroscopic (SEM) examination, newly generated tissues were fixed in 2.5% glutaraldehyde (Sigma) at 4°C for lh, postfixed for 2h in 1 % osmium tetraoxide (Polyscience), and dehydrated in a series of ethanol solutions(30,50,70,90,100%: Sigma). After completion of critical point drying, samples were coated with platinum by sputtering at an accelerating voltage of 15kV, and then examined by

SEM(FESEM Supra55VP, Zeiss) to evaluate the morphology of the newly generated tissue. [00176] Whole-mount staining of living tissue. Visualization of newly generated living tissue was performed as described²⁰. Briefly, freshly harvested tissue was cut into 2-3 mm pieces within 2h after sacrifice, and incubated for lh with three different colored immunofluorescent reagents: 5μΜ BODIPY 493/503(Invitrogen) to stain adipocytes, 2mM Alexorfluor 568-congugated isolectin GS- IB₄(Invitrogen) to stain endothelial cells, and 40μΜ hoechst 33342(Molecular Probes) to stain all nuclei. Samples were then washed and observed directly with a confocal microscope system

(Olympus 1X81) equipped with a Coolsnap HQ2 camera(Prior Scientific, Rockland, MA) and a Carv II Niplow-type Spinning Disc Confocal Attachment (BD bioscences, San Jose, CA).

Results

[00177] In vitro culture of preadipocytes.

[00178] The original number of cryopreserved hADSCs was 1.2xl0⁶, and after primary culture in the control medium, hADSCs demonstrated the typical elongated fibroblast-like morphology. hADSCs incubated in the adipogenic medium for 10 days exhibited accumulation of small lipid vacuoles in the cytoplasm (Figure 1). hADSCs were stained with Nile red and DAPI, allowing the lipid droplets to be visualized with red staining and the nucleus with blue staining. The intercellular lipid vacuoles stained positively with Nile red. Immunostaining for PPAR-γ protein and BODIPY lipid staining revealed a nuclear localization of PPAR-γ protein in differentiated cells.

[00179] Analysis of newly generated tissue.

[00180] After injection of cell-encapsulating hydrogels, the volume of the grafts gradually decreased through the subsequent 3-4 weeks to a volume -1/2 the initial. The volume then remained stable until sacrifice. The gel constructs did not migrate away from the injection location, or invade the surrounding tissue. There was no gross inflammation, swelling, or redness through the experimental period. The injection sites of the experimental group could be identified distinctively at tissue harvest, and the newly formed tissues obtained from cell-alginate constructs were harvested at 10 weeks after injection. Gel volumes ranging from 0.1-0.42 mL were injected into mice, and the average volume of engineered fat tissue was 50+12 % of the original injection volume at the time of tissue harvest. The explants were pale yellowish, semi-transparent, soft on palpation, and exhibited evidence of neovascularization (Figure 2). In contrast, injection of cells without gel carrier (control) resulted in no tissue formation at sacrifice, no new tissue could be identified

[00181] Histological and immunohistochemical analysis was performed on the newly formed tissue. Sections of tissues from the experimental group and native inguinal fat tissue were stained with H&E (Figure 3A) and ORO (Figure 3B) and demonstrated well-organized adipose tissue with small fragments of alginate hydrogel interspersed in the new tissue, but without any signs of inflammatory cell infiltration or evidence of tissue necrosis, cystic spaces or fibrosis. Lipid staining revealed that the cells comprising the new tissue were largely adipocytes. Immunofluorescent staining of PPARy (visualized as red) and DAPI (visualized as blue) was performed on newly formed tissue at 10 weeks. The newly formed tissue was seen to have characteristics typical of adipose tissue with respect to PPARy and DAPI localization and expression. Immunostaining for adiponectin revealed a concentration at the periphery of the cytoplasm of adipocytes in the newly formed tissue, confirming that adipose tissue was successfully regenerated from the adipogenically differentiated hADSC-alginate construct. Western blot analysis of lysates from newly formed adipose tissue and native inguinal fat tissue demonstrated expression of PPARy, adiponectin and C/EBP alpha/beta in the engineered tissues (Figure 3C). SEM analysis of the tissue formed from cell-encapsulating hydrogels demonstrated intact adipocytes surrounded by extracellular matrix fibers (Figure 4).

[00182] Whole-mount staining of living tissue. The overall structure of the engineered fat tissue was next analyzed with whole-mount staining. Photomicrograhs depicted living tissue revealing the presence of capillaries running alongside adipocytes to form a well-organized network in newly generated tissue, compared with native inguinal fat tissue. A cell was regarded as an adipocyte when the nucleus was localized within the BODIPY-positive lipid area. When the nucleus was within or attached to capillaries, they were regarded as a vascular associated cells (e.g., adipose-derived stromal cells, endothelial cells, pericytes), and when localization of the nucleus did not meet either of these two conditions, the cells were classified as other cell types (eg. as blood cells or fibroblasts). The overall morphology was very similar to native adipose tissue. Spherical adipocytes dominated the volume of the new tissue, supporting the histology analysis, and the majority of the cells in the tissue were adipocytes. An extensive series of capillaries was evident, running alongside adipocytes to form a well-organized network. Small capillaries could be appreciated growing out from larger vessels, suggesting an active remodeling of the vessel network.

Discussion

[00183] Demonstrated herein is the ability of a degradable alginate hydrogel system with preconditioned cryopreserved hADSCs to generate living adipose tissue via minimally invasive injections, and these results highlight the potential clinical application of this approach for contour improvement in applications ranging from trauma to congenital abnormality to cosmetic improvement. Adipose tissue engineering has been an area of relatively intense research, because soft tissue replacement using autologous tissue flaps, autologous fat injections, and artificial fillers all have significant pitfalls for both surgeons and patients. The limitations of current therapies include volume loss and donor site morbidity over time (1).

[00184] Potential cell sources for adipose tissue engineering include terminally differentiated adipocytes, preadipocytes, adult mesenchymal stem cells, embryonic stem cell (ESCs), and induced pluripotent stem cells (iPS cells). Lineage -committed precursor cell populations, ESCs and iPS cells have been limited in use because of issues with availability, danger of tumor formation, or ethical issues. Adult mesenchymal stem cells, having a multipotent differentiation potential, are currently the best candidate for use as donor cell. Adult mesenchymal stem cell populations can be harvested from mature adipose tissue (hADSCs), bone marrow (hBMSCs), trabecular bone, periosteum, articular cartilage, synovium, synovial fluid, muscles, tendons, blood, blood vessels, skin, spleen, and thymus²¹'²². A comparison of hBMSCs to hADSCs has shown that both types of cells can be differentiated toward multiple lineages, and both exhibit similar cytokine secretory profiles²³. Since adipose tissue is available in large quantities, is easy to obtain and causes minimal patient discomfort and donor site morbidity when harvested, it provides an abundant reservoir of stem cells for clinical use. hADSCs were reported to maintain their ability to undergo adipogenesis over 160 population doublings²⁴, providing a significant advantage to their use in cell therapies. Preadipocytes, found in the stromal vascular fraction of adipose tissue, are able to differentiate to mature adipocytes, these cells, however, lose their capacity to differentiate following elevated numbers of passages⁷.

Nevertheless, preadipocytes and adipocytes have advantages for clinical application, as they can allow one to avoid the culture and pre-differentiation processes. The source and purity of the stem cells are important variables, and those used herein were specified by the vendor (29 yr old, female, manufactured September 14, 2007; experiments described herein spanned 2010-2011, virus testing- negative, CD13,CD29,CD44,CD73,CD90 >90%positive, CD14,CD31, <5% positive).

[00185] It is demonstrated herein that cryopreserved hADSCs (>3 year) could be used to engineer tissue successfully. Freshly prepared cell populations are preferred, but from a clinical application point of view, the ready availability of cryopreserved hADSCs or preadipocytes has a significant advantage for the surgeon and patients, because liposuction procedures would not have to be performed each time cells are needed. Further, for optimal contour improvement, serial touch up processes over a scheduled time period will likely be necessary.

[00186] Various synthetic materials such as poly(lactide) (PLA), poly(glycolide) (PGA), poly(lactide-co-glycolide) (PLG), polyethylene glycol(PEG), and natural biomaterials such as adipose-derived ECM, collagen, gelatin, and hyaluronic acid have been explored for adipose tissue engineering. These materials demonstrate varying levels of biocompatibility, and different mechanical and chemical properties, and degradability. The ability to consistently control material properties of synthetics typically provides a considerable advantage over natural materials, but natural materials have been shown to provide advantages with respect to compatibility, extent of adipose tissue formation and degradation properties²⁵. Previously researched materials may not be suitable for clinical application since many are not injectable. Alginate is commonly used in many tissue engineering applications because of its biocompatibility, safety, relatively low cost and mild gelation behavior with divalent cations²⁶.

[00187] A previous study performed with collagen sponges and gelatin microspheres containing basic fibroblast growth factors (bFGF) also demonstrated a robust induction of adipose tissue²⁷. As that study did not use any transplanted cells, the induction of new tissue generation was solely dependent on the viability and potency of cells in the surrounding host tissue as well as cells that home to the implant site²⁸. Transplantation of exogenous cells, preferably autogenous, can overcome this host dependency and may significantly add to procedure consistency and provide an efficacious and predictable surgical outcome. Another study using cultured ADSCs encapculated in non- modified alginate gels demonstrated the potential of alginate gel as a filler material²⁹. Alginate gels with widely varying chemical, physical and degradative properties have been developed over the past decade³⁰, and these materials can be designed to tightly regulate the gene expression of encapsulated cells via manipulation of their cell binding capability and mechanical properties³¹.

[00188] Since cell adhesion is a strict requirement for survival, the alginate gels described herein contained covalently coupled RGD peptides (SEQ ID NO: l) in order to provide an adhesive cue from the gel. Previous studies, using a variety of cell types including MSCs, have demonstrated this modification enhances cell adhesion, survival, proliferation, and allows differentiation of MSCs to adipocytes 18 ' 32 ' 33. The current experiment demonstrates that degradable alginate hydrogels can be useful injectable scaffolds for adipose engineering, following the ideal dictum of the artificial scaffold material being replaced over time by the newly regenerating tissue that it induces. Although the degradability of materials can be exactly measured in vitro, the in vivo degradation is less precisely predicted due to multiple tissue and subject variations such as the local tissue enzymes and proteases, physical forces, degree of immune response, among others.

[00189] A key issue in adipose tissue engineering is the vascularization of the new tissue, as this is crucial to maintain the viability of the transplanted cells and prevent large-scale shrinkage of the tissue over time. The use of confocal microscopy allows one to determine that degradable alginate gels allowed significant ingrowth of capillaries that grew alongside the adipocytes in the newly generated adipose tissue by 10 weeks as seen by histological staining with BODIPY and DAPI . The engineered adipose tissue demonstrated typical characteristics of adipose tissue, grossly,

microscopically and at the molecular level.

Example 2: Hydrogel Compositions

[00190] The hydrogels described in Example 1 were composed of 2% gels with a ratio of 1 :3 high molecular weight (HMW): low molecular weight (LMW) alginate. The performance of hydrogels of varying compositions was investigated and is described herein. In addition to the 2% (1 :3) hydrogel, a 2% (1:4) hydrogel and a 1% (1 :3) hydrogel were examined (Table 1).

Table 1 : Hydrogel Compositions

[00191] The hydrogels were injected into nude mice via subcutaneous injection with a 23 gauge needle as described in detail above. The performance of the hydrogel was examined 10-12 weeks after injection.

[00192] The performance of different cell types was also examined. The change in volume of compositions including pre-differentiated adipose cells or human adipose-derived stem cells

(hADSC's) in a 2% (1:3) gel were compared 10 weeks after injection (Figures 5A-5B, Table 2). Pre- differentiated cells retained a slightly greater, but statistically significant percentage of the originally injected volume.

Table 2: Comparative Performance by Cell Type

Pre-differentiated Cells hADSCs

2%, 1 :3 (HMW:LMW) 2%, 1 :3 (HMW:LMW)

Initial Final % Initial Final %

Remaining Remaining

Average ± 315.71 ± 164.29 ± 50.949 ± 278.33 + 130.00 + 47.312 +

SD 111.931 70.912 0.100 91.742 41.473 0.056

There was statistical significance between control and experimental group, (paired t-test, /?<0.001)

[00193] When the ratio of HMW:LMW alginate and their final, collective concentration within the hydrogel was varied, the 2% (1 :4) hydrogel retained more of the original injection volume than the 1% (1 :3) hydrogel 10 weeks after implantation (Figures 6A-6B; Table 3).

Table 3: Comparative performance of Alginate Hydrogels

2% 1%

1 :4 (HMW:LMW) 1 :3 (HMW:LMW)

Initial Final % Initial Final %

Average + 287.778 + 145.556 + 46.864 + 304.444 + 63.333 + 19.789 +

SD 122.350 72.992 0.120 98.121 33.166 0.073

[00194] As shown in Figures 7A-7B; Figures 8A-8B; Figures 9A-9B; Figures 10A-10B and Table 4, dermal thickness, which is indicative of increased collagen deposition was observed following implantation of a alginate hydrogel comprising hADSCs. The results were analyzed by SPSS 12.0 (IBM, NY, USA). Epiermal and dermal thickness comparisons between control and experimental group were performed using paired t-test. The normality was not violated by

Kolmogorov-Smirnov and Shapiro-Wilk test. The difference was considered if p was <0.05.

Masson's trichrome staining revealed increased collagen in the dermis when hydrogel with hADSCs was implanted (Figures 9A-9B). New collagen deposition was observed with hematoxylin and eosin (H&E) staining (Figures 8A-8B). [00195] Picosirius red staining of the tissue also revealed increased collagen in subjects receiving hydrogels comprising hADSCs (Figure 10B). In areas In areas without grafts, the host dermis, as visualized by picosirus red staining under polarizing light conditions, exhibited orange-red staining, indicative of thick, existing collagen deposits. Host dermis in areas with grafts as described herein, exhibited the staining indicative of new, thinner collagen deposition. The areas occupied by the hydrogel implant exhibited green yellow staining (staining not shown) indicative of the presence of new, thinner collagen deposition.

[00196] Collagen deposition is an important predictive parameter for clinical results such as improvement in skin tone and wrinkle appearance (Mojallal et al., Plast Reconstr Surg. 2009

Sep;124(3):765-74).

Table 4: Epidermal and Dermal Thickness

Epidermis Dermis

Control Experimental Control Experimental

Average+SD 13.917+1.880 15.422+3.667 226.198+20.155 298.144+34.870

[00197] The rate of hydrogel degradation increased as the gel percentage was lowered or the amount of LMW was increased. For the purposes of adipose tissue engineering, a 2% gel with (1 :3) or (1:4) HMW:LMW is preferred to a 1% gel. This is in contrast to the use of hydrogels as filler material, where a higher hydrogel concentration and increased amounts of HMW alginates is desirable.

Example 3: Clinical Applications

[00198] Once prepared as described above, alginate hydrogel compositions as described herein can be administered to subjects using the same methods and techniques as conventional fat injections. The alginate hydrogel compositions can be administered for reconstructive applications, e.g.

congenital abnormalities (e.g., hemifacial microsomia, facial asymmetry); following cancer ablation (e.g., breast reconstruction, facial reconstruction, depressed scars following large skin cancer excision such as for melanoma); post-irradiation soft tissue atrophy; post-burn scar contracture; post-traumatic soft tissue deficiency; soft tissude padding around a joint area; intractable skin ulcers from, e.g., diabetes, ischemia, or collagen diseases. The hydrogel compositions are administered for aesthetic applications, e.g. breast augmentation; facial contouring; sunken upper eyelid; dark circle in lower eyelid; deepened nasolabial fold; temple depression; lip augmentation; hand rejuvenation; buttock augmentation; penile augmentation; depressed scars; anti-wrinkle treatments; anti-aging treatments; or improvement of skin tone and texture. Tables 5 and 6 list a number of applications of the hydrogel compositions described herein and citations further describing the clinical applications of traditional fat injections. Table 5: Reconstructive Applications

Table 6: Aesthetic Applications

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Claims

What is claimed herein:

1. A cell implantation pre-matrix composition comprising, a solution of oxidized high molecular weight (HMW) alginate and oxidized low molecular weight (LMW) alginate, wherein:

a) the oxidized HMW alginate and oxidized LMW alginate are present at a ratio of from about 1 :3 to about 1 :4;

b) the oxidized HMW alginate and oxidized LMW alginate are present at a

weight/volume concentration of from about 1% to about 4%;

c) the oxidized HMW alginate and oxidized LMW alginate are oxidized at about 2% of their sugar residues;.

d) one or both of the high molecular weight alginate and the low molecular weight alginate are coupled to an adhesion peptide at a yield of about 2 peptides per alginate polymer chain.

2. The composition of claim 1, further comprising adipogenic growth factors.

3. The composition of claim 1, further comprising one or more angiogenic factors.

4. The composition of claim 3, wherein the angiogenic factor is vascular endothelial growth factor.

5. The composition of claim 4, wherein the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration of about 4%.

6. The composition of claim 1 , wherein the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration from about 1 % to about 3 %.

7. The composition of claim 6, wherein the oxidized HMW and oxidized LMW alginate are present at a weight/volume concentration of about 2%.

8. The composition of any one of claims 1 - 7, further comprising cells.

9. The composition of claim 8, wherein the cells are selected from the group consisting of

preadipocytes, adult mesenchymal stem cells, human adipose stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

10. The composition of any one of claims 8 - 9, wherein the cells are primary cells.

11. The composition of any one of claims 8 - 10, wherein the cells are predifferentiated cells.

12. The composition of claim 11, wherein the predifferentiated cells are adipocytes.

13. The composition of claim 1, wherein the adhesion peptide comprises the amino acid sequence G4RGDASSKY (SEQ ID NO:2).

14. The composition of any one of claims 1 - 13, further comprising a cross-linking agent or precursor thereof.

15. The composition of any one of claims 1 - 14, that is cross-linked to produce a hydrogel.

16. A kit comprising:

a) the cell implantation pre -matrix composition of any one of claims 1-15;

b) directions for use.

17. The kit of claim 16, further comprising one or more of an adipogenic growth factor and an angiogenesis factor.

18. A method of generating adipose tissue at a specific site in a subject, the method comprising: administering to the specific site cells encapsulated in a hydrogel, under conditions suitable for generating new adipose tissue, wherein the hydrogel is generated from crosslinking the cell implantation matrix of any one of claims 1-14.

19. The method of claim 18, wherein administering is performed by a method selected from the group consisting of subcutaneous injection, submuscular injection, intramuscular injection, and subfascial injection.

20. The method of any one of claims 18 - 19, wherein the cells are primary cells.

21. The method of any one of claims 18 - 20, wherein the cells are autologous.

22. The method of any one of claims 18 - 21, wherein the cells are selected from the group

consisting of adipocytes, preadipocytes, adult mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, and combinations thereof.

23. The method of any one of claims 18 - 22, wherein the specific site is located at a region selected from the group consisting of the face, the breast, the buttocks, the hand, and the penis.

24 The method of claim 23, wherein the specific site is located at the region of the face and is selected from the group consisting of lip, nasolabial fold, temple, lower eye lid, and upper eye lid.

25. The method of any one of claims 18 - 22, wherein the specific site is a scar of the subject.

26. The method of claim 25, wherein the scar is a depression scar.

27. The method of claim 25, wherein the scar is a post-burn contracture.

28. The method of claim 25, wherein the scar is from post-irradiation soft tissue atrophy.

29. The method of claim 25, wherein the scar is a post-traumatic soft tissue deficiency.

30. The method of any one of claims 18 - 22, wherein the specific site is in a region of congenital anomaly.

31. The method of claim 30, wherein the congenital anomaly is hemifacial microsomia or facial asymmetry.