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This patent application claims the benefit of priority from U.S. Provisional Application Serial No. 61/811,355, filed April 12, 2013, the content of which is herein incorporated by reference in its entirety.

Statement Regarding Federally Sponsored Research or

Development

This invention was made with government support under Grant Number 5R01AR55280 awarded by the National Institutes of Health - National Institute of Arthritis and

Musculoskeletal and Skin Diseases (NIH-NIAMS) . The

government has certain rights in the invention.

Field

The disclosed subject matter relates to biomimetic hydrogel scaffolds for promoting stable and integrative cartilage repair and methods for modulating chondrocyte biosynthesis and mineralization, enhancing matrix production by chrondrocytes or cells capable of chondrogenesis, and forming endochondral and/or osteochondral ossification mediated bone with these scaffolds.

Background

Osteoarthritis is a painful joint condition

characterized by cartilage degeneration. This disease currently affects over 27 million adults in the United

States and is a leading cause of disability among older Americans. Once damaged, cartilage has a limited capacity for self-repair due to its avascular nature. As a result, surgical intervention is often required. Current treatment strategies are limited, however, by fibrocartilage formation in the defect site and poor graft integration over time.

Osteochondral grafts have emerged as an alternative to surgery. While these grafts show promise for regenerating both cartilage and bone-like tissues, the clinical challenge which remains is the consistent formation of a stable osteochondral interface between these tissues.

Summary

An aspect of this application relates to scaffolds for promoting calcified cartilage and/or bone formation. The scaffolds of this application comprise a biomimetic hydrogel and a ceramic structure or mineral source selected to modulate biosynthesis and mineralization of chondrocytes.

Another aspect of this application relates to a method for modulating chondrocyte biosynthesis and mineralization in a hydrogel. The method comprises adding to a biomimetic hydrogel a ceramic structure or mineral source selected to modulate biosynthesis and mineralization of chondrocytes.

Another aspect of this application relates to a method for enhancing matrix production by chrondrocytes or cells capable of chondrogenesis . The method comprises culturing the cells on a scaffold comprising a biomimetic hydrogel and ceramic structure or mineral source selected to modulate biosynthesis and mineralization of chondrocytes.

Yet another aspect of this application relates to forming endochondral and/or osteochondral ossification mediated bone with these scaffolds.

Brief Description of the Figures

Figures 1A through 1C shows results of characterization of a CDA and TCP ceramic powder prior to scaffold

fabrication via scanning electron microscope (SEM; Figure 1A) , X-ray diffraction (XRD; Figure IB) and Fourier transfer infrared spectroscopy (FTIR; Figure 1C) .

Figures 2A and 2B show results of chondrocyte

proliferation and distribution analysis in CDA, TCP and CaP- free (ceramic free, hydrogel only) scaffolds. Figure 2A shows cell number at days 1, 7 and 14 while Figure 2B

provides results of hematoxylin and eosin staining at day 14.

Figures 3A and 3B show results of assessment of GAG content in the matrix deposition. Figure 3A is a bargraph comparing levels of GAG as a percentage of wet weight of the scaffold at days 1, 7 and 14 in CDA, TCP and CaP-free

scaffolds. Figure 3B compares Alcian Blue staining at day 14 in CDA, TCP and CaP-free scaffolds.

Figures 4A and 4B show results of assessment of

collagen content in the matrix deposition. Figure 4A is a bargraph comparing levels of collagen as a percentage of wet weight of the scaffold at days 1, 7 and 14 in CDA, TCP and CaP-free scaffolds. Figure 4B compares levels of collagen I-V, collagen I and collagen II at day 14 in CDA, TCP and CaP-free scaffolds.

Figures 5A and 5B shows results of the mineralization potential analyzed by measuring the ALP activity of

scaffolds further containing CDA or TCP as well as CaP-free scaffolds at days 1, 7 and 14 (see Figure 5A) and the

calcium content measured by Alizarin Red staining at days 1 and 14 (see Figure 5B) .

Figures 6A through 6C show the effects of scaffolds further containing CDA or TCP as well as CaP-free scaffolds on hypertrophic markers including collagen X at day 14

(Figure 6A) as well as collagen X, Indian hedgehog (Ihh) and matrix metalloproteinase 13 (MMP13) at days 1 (Figure 6B) and 14 (Figure 6C) . Figures 7A and 7B show the effects of scaffolds further containing CDA and TCP as well as CaP-free scaffolds on media ion concentrations of Ca ⁺² and P0 ₄ ^~3 . Detailed. Description

Definitions

In order to facilitate an understanding of the material which follows, one may refer to Freshney, R. Ian. Culture of Animal Cells - A Manual of Basic Technique (New York: Wiley- Liss, 2000) for certain frequently occurring methodologies and/or terms which are described therein.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. However, except as otherwise expressly provided herein, each of the following terms, as used in this application, shall have the meaning set forth below.

As used herein, "ALP activity" shall mean alkaline phosphatase activity.

As used herein, a "biocompatible" material is a

synthetic or natural material used to replace part of a living system or to function in intimate contact with living tissue. Biocompatible materials are intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body. The biocompatible material has the ability to perform with an appropriate host response in a specific application and does not have toxic or injurious effects on biological systems. Nonlimiting examples of biocompatible materials include a biocompatible ceramic, a biocompatible mineral source, a biocompatible polymer or a biocompatible hydrogel.

As used herein, "biodegradable" means that the

material, once implanted into a host, will begin to degrade. As used herein, "biomimetic" shall mean a resemblance of a synthesized material to a substance that occurs naturally in a human body and which is not substantially rejected by (e.g., does not cause an unacceptable adverse reaction in) the human body. When used in connection with the tissue scaffolds, biomimetic means that the scaffold is substantially biologically inert (i.e., will not cause an unacceptable immune response/rejection) and is designed to resemble a structure (e.g., soft tissue anatomy) that occurs naturally in a mammalian, e.g., human, body and that

promotes healing when implanted into the body.

As used herein, "chondrocyte" shall mean a

differentiated cell responsible for secretion of

extracellular matrix of cartilage.

As used herein, "chondrogenesis" shall mean the

formation of cartilage tissue.

As used herein, "effective amount" shall mean a

concentration, combination or ratio of one or more

components added to the scaffold which promote growth and/or proliferation or cells and/or direct differentiation of stem cells to a selected cell type. Such components may include, but are not limited to, ceramic structures, mineral sources one or more extracellular matrix components, physical or mechanical stimulation and chemical stimulation such as media or growth factors which promote growth and/or

proliferation or cells and/or direct differentiation of stem cells to a selected cell type.

As used herein, "hydrogel" shall mean any colloid in which the particles are in the external or dispersion phase and water is in the internal or dispersed phase.

As used herein, "polymer" means a chemical compound or mixture of compounds formed by polymerization and including repeating structural units. Polymers may be constructed in multiple forms and compositions or combinations of

compositions .

By "osteochondral interface" it is meant a region composed of hypertrophic chondrocytes in a mineralized matrix. This interfacial zone is important because it serves to anchor the articular cartilage to the subchondral bone and allows for pressurization of articular cartilage during loading. This region also limits vascular invasion of the articular cartilage.

As used herein, "stem cell" means any unspecialized cell that has the potential to develop into many different cell types in the body, such as chondrocytes or chondrocyte progenitor cells. Nonlimiting examples of "stem cells" include mesenchymal stem cells, embryonic stem cells and induced pluripotent cells.

As used herein, "synthetic" shall mean that the

material is not of a human or animal origin.

As used herein, all numerical ranges provided are intended to expressly include at least the endpoxnts and all numbers that fall between the endpoxnts of ranges.

The following embodiments are provided to further illustrate the methods of tissue scaffold production of this application. These embodiments are illustrative only and are not intended to limit the scope of this application in any way.

Embodiments

The disclosed subject matter relates to scaffolds comprising a biomimetic hydrogel and a ceramic structure or mineral source selected to modulate biosynthesis and

mineralization of chondrocytes and methods for use of these scaffolds in modulating chondrocyte biosynthesis and

mineralization, enhancing matrix production by chrondrocytes or cells capable of chondrogenesis and/or forming endochondral and/or osteochondral ossification mediated bone. In one embodiment, the ceramic structure or mineral source is selected to modulate biosynthesis and

mineralization of deep zone chondrocytes and/or hypertrophic chondrocytes.

It is expected that any polymer chain hydrogel useful as a tissue scaffold can be used. Examples include, but are not limited to, agarose, carrageenan, polyethylene oxide, polyethylene glycol, tetraethylene glycol, triethylene glycol, trimethylolpropane ethoxylate, pentaerythritol ethoxylate, hyaluronic acid, thiosulfonate polymer

derivatives, polyvinylpyrrolidone-polyethylene glycol-agar, collagen, dextran, heparin, hydroxyalkyl cellulose,

chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, dextran sulfate, pentosan polysulfate, chitosan, alginates, pectins, agars, glucomannans ,

galactomannans, maltodextrin, amylose, polyalditol,

alginate-based gels cross-linked with calcium, polymeric chains of methoxypoly (ethylene glycol ) monomethacrylate, chitin, poly (hydroxyalkyl methacrylate), poly (electrolyte complexes), poly (vinylacetate) cross-linked with

hydrolysable bonds, water-swellable N-vinyl lactams,

carbomer resins, starch graft copolymers, acrylate polymers, polyacrylamides , polyacrylic acid, ester cross-linked polyglucans, and derivatives and combinations thereof.

In one embodiment, the hydrogel used in the scaffolds of this application is agarose. Agarose offers a controlled inert matrix which supports a rounded chondrocyte phenotype. Furthermore, agarose allows for accumulation and retention of matrix products.

Scaffolds of this application further comprise a ceramic structure or alternative mineral source selected to modulate biosynthesis and mineralization of chondrocytes. Nonlimiting examples of ceramic structures or mineral sources which can be used include, bioactive glasses,

hydroxyapatite, calcium deficient apatite, biphasic calcium phosphate, and combinations of these. In one embodiment the ceramic structure is a calcium phosphate. A nonlimiting example of a calcium phosphate ceramic is beta-tricalcium phosphate (TCP) . As will be understood by the skill artisan upon reading this disclosure, however, alternative ceramic structures and mineral sources can be selected. Further, as will also be understood by the skilled artisan upon reading this disclosure, parameters including, but not limited to, chemistry, crystallinity and/or particle size of the ceramic structure or mineral source can be selected to modulate and/or direct chondrocyte response. In one embodiment, the selected ceramic structure is calcium-deficient apatite

(CDA) .

In one embodiment, a ceramic structure is added at a concentration ranging from about 0.5% to 4.5% of the ceramic.

In one embodiment, the concentration of ceramic

structure or mineral source added is selected to mimic physiologic levels or superphysiologic levels of the

selected cell type and selected ceramic.

Without being bound to any particular theory, it is believed that the ceramic structure and/or mineral source will serve as part of any bone or calcified cartilage formed on the scaffold.

The tissue engineered scaffolds may further comprise chondrogenic cells or chondrocytes. By "chondrogenic cells" is it meant to include any cell capable of chrondrogenic differentiation. In one embodiment, the tissue engineered scaffold is seeded with mesenchymal stem cells. In one embodiment, the mesenchymal stem cells are human mesenchymal stem cells. Examples of alternative cells for seeding

include, but are not limited to, adipose derived stem cells, synovium derived stem cells, induced pluripotent stem cells, embryonic stem cells, and fibrochondrocytes .

Scaffolds of this application comprising the biomimetic hydrogel agarose and the ceramic structures TCP and CDA, selected to modulate biosynthesis and mineralization of chondrocytes were fabricated. CaP-free agarose scaffolds were also fabricated as a control.

Before scaffold fabrication, the ceramic powders were characterized via SEM, XRD (see Figure IB), FTIR (Figure 1C) , and ICP (see Table 1) . From the SEM imaging depicted in

Figure 1A, it was clear that the TCP powder had a clearly defined, rhombic particle shape while the CDA powder did not. XRD spectra depicted in Figure IB showed broad peaks for the CDA powder, indicative of small crystallite size and poor crystallinity, whereas TCP resulted in sharp peaks matching the spectra for TCP powder. FTIR depicted in Figure 1C showed the presence of carbonate in the CDA powder whereas the TCP powder was carbonate free and marked by split

phosphate bending curves. ICP analysis, results of which are depicted in Table 1, showed no difference in calcium

phosphate ratio between the two powders.

Table 1 : Results of ICP Analysis

After acellular characterization was completed, cell viability and distribution of cells seeded on hydrogel

scaffolds further containing CDA or TCP was assessed. See Figures 2A and 2B. Cell number was determined at days 1, 7 and 14. Cell number increased on scaffolds further

containing CDA or TCP as well as CaP-free scaffolds.

However, significantly higher number of cells were observed on days 7 and 14 for the scaffold further containing CDA (see Figure 2A) .

Once it was determined that the cells were viable in the scaffolds, matrix deposition was assessed by looking at both collagen and GAG content.

Results for GAG content are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, GAG content increased in scaffolds further containing CDA or TCP as well as CaP-free scaffolds and no differences in GAG deposition were observed between the CaP-free and TCP containing scaffolds. The highest GAG deposition observed was in the CDA containing scaffold on day 14.

Results for collagen content are shown in Figures 4A and 4B. As shown in Figures 4A and 4B, collagen content increased in scaffolds further containing CDA or TCP as well as CaP-free scaffolds and no differences in collagen

deposition were observed between the CaP-free and TCP

containing scaffolds. The highest collagen deposition observed was in the CDA containing scaffold on day 14 which tested positive for collagen II.

Mineralization potential was analyzed by measuring the ALP activity of scaffolds further containing CDA or TCP as well as CaP-free scaffolds at days 1, 7 and 14 (see Figure 5A) and the calcium content at days 1 and 14 (see Figure 5B) . ALP activity decreased in all scaffolds (see Figure 5A) while only scaffolds containing a ceramic structure were positive for calcium via Alizarin staining (see Figure 5B) .

The effect of scaffolds further containing CDA or TCP on hypertrophic markers collagen X, Ihh and MMP13 was also examined. Results are shown in Figures 6A through 6C. As shown in Figure 6B, hypertrophic markers were downregulated at day 1 in all the scaffolds tested. Further, collagen X was lowest in scaffolds further containing CDA at day 14 (see Figures 6A and 6C) . However, as shown in Figure 6C, MMP13 was upregulated in scaffolds containing TCP at day 14.

In addition to cell biosynthesis and mineralization, the media was analyzed to determine relevant changes in media ion concentrations of Ca ²⁺ and P0 ₄ ^3~ . Results are shown in Figures 7A and 7B. On day 1, Ca ²⁺ media concentrations were decreased in scaffolds further containing CDA (see Figure 7A) while P0 ₄ ^3_ concentrations were increased in scaffolds further containing TCP (see Figure 7B) . After day 1, however, there were no differences in ion concentration in media between the scaffolds.

These results show that ceramic type modulated

chondrocyte response. While not being bound to any

particular theory, it is believed that the change in cell response may be due to parameters which were altered during sintering. Table 2 shows ceramic characteristics of CDA and TCP following sintering.

Table 2 : CDA vs . TCP Ceramics

For instance, the particle shape was changed from irregular to rhombic during the sintering and the crystallite size increased from small to large which was detected by

decreasing peak width in the XRD spectra. Finally the carbonate which was present in the CDA was removed during sintering. Both ceramic types had the same Ca/P molar ratio.

Also shown by these experiments is that CDA promoted cell proliferation and enhanced matrix deposition while TCP did not. Further, while ALP activity decreased over time in all the scaffolds, only CDA downregulated hypertrophic markers. Similar experiments were conducted comparing scaffolds of this application stimulated by addition of

triiodothyronine or thyroid hormone, also referred to as T3. This hormone stimulates the hypertrophic phenotype in deep zone chondrocytes. T3 (25nM) was added in the day 1 and day 3 feeding with no subsequent treatment after that.

The CDA containing scaffold in the presence and absence of T3 stimulation measured the highest cell number at day 14. Hematoxylin and eosin staining revealed uniform cell

distribution throughout all the T3 stimulated as well as unstimulated scaffolds at day 14. The CDA scaffolds also measured the highest collagen deposition on day 14 in the presence and absence of T3 stimulation. Picrosirius red staining showed positive collagen staining throughout all the scaffolds with the most positive staining observed for CDA containing scaffolds on day 14 in the presence and absence of T3 stimulation. The CDA scaffolds also measured the highest proteoglycan deposition on day 14 in the

presence and absence of T3 stimulation. Alcian blue

staining showed positive GAG staining throughout all the scaffolds with the most positive staining observed for CDA containing scaffolds on day 14 in the presence and absence of T3 stimulation. The CDA scaffolds also measured the highest ALP activity on day 1 with low ALP activity

thereafter in the presence and absence of T3. Finally, TCP and CDA containing scaffolds exhibited downregulated Col X and Ihh on day 1. The CDA containing scaffolds also

exhibited the lowest ALP expression and the highest PTHrP. On day 14, the untreated CDA containing scaffold exhibited the lowest Col X, Ihh, and ALP, although there were no significant differences between scaffolds for these genes. MMP13 was upregulated at day 14 for the unstimulated TCP containing scaffold. A T3-stimulated HA containing scaffold exhibited upregulated Ihh on day 14. All stimulated groups exhibited decreased MMP13 expression with respect to the stimulated control group.

Accordingly, as shown by these experiments, crystal structure, ceramic chemistry and particle size are critical parameters for calcified cartilage scaffold design. As further shown, selection of the ceramic structure or mineral source to be added to a biomimetic hydrogel can modulate biosynthesis and mineralization of chondrocytes and enhance matrix production by chrondrocytes or cells capable of chondrogenesis . As also shown by these experiments, a preferred embodiment of the present invention is a

biomimetic hydrogel scaffold further containing CDA.

In one embodiment, a stimulant . such as T3 is added to the scaffold.

The tissue engineered scaffolds of this application are useful in studying chrondrogenesis , promoting proliferation and chondrogenesis of chondrogenic cells and producing functional cartilage.

These scaffolds can be used in combination with a cartilage graft to promote functional integration at the cartilage-bone interface. This can be done by layering this scaffold with a mineral free cartilage scaffold or an allo- and autograft. Cartilage scaffolds can be made from a variety of hydrogels, including, but not limited to alginate PEG, chitosan and hyaluronic acid.

Biomimetic hydrogel and selected ceramic or mineral interface scaffolds of this application are useful in regenerating calcified cartilage, promoting stable and integrative cartilage repair and for osteochondral

ossification mediated and/or endochondral bone formation.

The disclosed subject matter is further illustrated by the following nonlimiting example.

The following disclosure should not be construed as limiting the invention in any way. One of skill in the art will appreciate that numerous modifications, combinations, rearrangements, etc. are possible without exceeding the scope of the invention. While this invention has been

described with an emphasis upon various embodiments, it will be understood by those of ordinary skill in the art that variations of the disclosed embodiments can be used, and that it is intended that the invention can be practiced otherwise than as specifically described herein.

EXAMPLES

Example 1 : Scaffold, Production

The chondrocytes were encapsulated at a density of 10 million cells/ml in sterile 2% low gelling agarose (Agarose Type VII, Sigma, St. Louis, MO) and a biopsy punch (Sklar Instruments, West Chester, PA) was used to core cylindrical scaffolds (0 = 5 mm, height = 2.4 mm). Acellular and cellular agarose scaffolds with 1.5 w/v% ceramic (Sigma) and corresponding samples without ceramic were fabricated. All samples were cultured under humidified conditions at 37 C and 5% C0 ₂ , and maintained in ITS culture medium composed of DMEM supplemented with 1% ITS+ Premix (BD Biosciences, San Jose, CA) , 1% penicillin-streptomycin, 0.1% gentamicin sulfate, 0.1% antifungal, and 40 pg/ml L-proline (Sigma). The medium was changed every other day and freshly supplemented with 50 pg/mL ascorbic acid (Sigma) . The responses of deep zone chondrocytes were compared in CDA-1, CDA-2, and ceramic-free scaffolds over a two-week culture period.

Example 2 : Methods used to characterize ceramic powder

Ceramic particle shape was assessed using scanning electron microscopy (SEM, Hitachi 4700 FE-SEM, 5kV, lOOOx) . Particles were sputter-coated with gold for 20 seconds before SEM imaging (Cressington 108 Auto, Watford, UK) .

Ceramic calcium and phosphorus content was determined using inductively coupled plasma analysis (ICP, Thermo Jarrell Ash, Trace Scan Advantage) . Briefly, 10 mg of ceramic was

dissolved in several drops of 17% HC1 and brought to 100 ml with double distilled water. The resulting solutions were pumped through argon plasma excited by a 2 k /27.12 MHz radiofrequency generator. The concentrations of each

element were determined using their characteristic

wavelengths (Ca, 317.9 A ; P, 213.6 A). The crystal

structure of the ceramics was evaluated with X-ray

diffraction (XRD, X-ray Diffractometer, Inel, Artenay,

France). The samples were evaluated over a range of 0-120°, with a step size of 0.029°. Ceramic chemistry was examined using Fourier transform infrared spectroscopy (FTIR, FTS 3000MX Excalibur Series, Digilab, Randolph, MA), wherein the samples were dehydrated, mixed with potassium bromide and FTIR spectra were collected in absorbance mode (400 scans, 4 cm ^-1 resolution) .

Example 3 : Cell proliferation and distribution analysis

Cell proliferation (n=5) was determined using the

Quanti-iT™ PICOGREEN dsDNA assay kit (Molecular Probes,

Eugene, OR) following sample digestion. Briefly, the

samples were exposed to a freeze-thaw cycle in 500.. μΐ of 0.1% Triton-X solution (Sigma) in order to lyse the cells. The samples were desiccated for 12 hours (CentriVap

Concentrator, Labconco Co., Kansas City, MO) and digested with papain (8.3 activity units/ml) in 0.1M sodium acetate (Sigma) , 10 mM cysteine HC1 (Sigma) , and 50 mM

ethylenediaminetetraacetate (Sigma) at 65 °C for 18 hours. A 25 μΐ aliquot of the sample was mixed with 175 μΐ of the PICOGREEN working solution and fluorescence was measured with a microplate reader (Tecan, Research Triangle Park, NC) , at the excitation and emission wavelengths of 485 and 535 nm, respectively. The conversion factor of 7.7 pg DNA/cell was used to determine cell number. Example 4: Assessment of GAG content

Sample glycosaminogylcan content (GAG, n=5) was

determined with a modified 1 , 9-dimethylmethylene blue (DMMB) binding assay, with chondrotin-6-sulfate (Sigma-Aldrich, St. Louis, MO) as the standard. Briefly, a 10 μΐ aliquot of sample was diluted (1:4) with dH ₂ 0 and mixed with 250 μΐ of DMMB dye. The absorbance difference between 540nm and 595nm was used to improve the sensitivity in signal detection.

Alician Blue histology staining was used to qualitatively assess proteoglycan distribution (n=2) .

Example 5: Assessment of collagen content

Total collagen content (n=5) was quantified using a modified hydroxyproline assay with a bovine collagen type I solution (Biocolor, UK) as a standard. All matrix values were normalized by sample wet weight in order to account for any differences in sample size. Additionally, collagen distribution (n=2) was ^' evaluated via histology. Briefly, dehydrated samples were embedded in paraffin (Paraplast X- tra Tissue Embedding Medium, Fisher Scientific) and 7 μπι sections were obtained from the center of the scaffold.

Total collagen distribution was visualized using Picrosirius Red staining (n ^~ 2) while collagen types I and II were assessed using immunohistochemistry. Specifically,

monoclonal antibodies for collagen type I (1:00 dilution) and collagen type II (1:100 dilution) were purchased from Abeam (Cambridge, MA) . After fixation, samples were treated with 1% hyaluronidase for 30 minutes at 37 °C and incubated with primary antibody overnight. All samples were

counterstained with DAPI (Sigma) . A FITC-conjugated

secondary antibody (1:200 dilution, LSAB2 Abeam) was used and samples were imaged under confocal microscopy (Olympus Fluoview 1X70) at excitation and emission wavelengths of 488nm and 568nm, respectively.

Example 6 : Mineralization Potential Analysis

Alkaline phosphatase (ALP) activity (n=5) was measured using an colorimetric assay based on the hydrolysis of p- nitrophenyl phosphate (pNP-P0 ₄ ) to p-nitrophenol (pNP) .

Briefly, the samples were lysed in 0.1% TRITON X solution, exposed to a freeze-thaw cycle, and crushed with a mortar. A 25 μΐ aliquot was added to pNP-P0 ₄ solution (Sigma) and incubated for 10 min at 37 °C. Absorbance was measured at 405 nm using a microplate reader (Tecan) . In addition, calcium distribution (n=2) was evaluated using Alizarin Red staining as an indicator of overall mineral distribution.

Example 7 : Measurement of Hypertrophic Markers

The expression (n=3) of collagen type X, matrix

metalloproteinase-13 (MMP-13) , Indian Hedgehog (Ihh) , Runt- related transcription factor 2 (Runx 2), and parathyroid hormone-related protein (PTHrP) were measured at day 7 using reverse transcription followed by polymerase chain reaction (RT-PCR) . Briefly, total RNA was isolated via TRIzol

(Invitrogen, Carlsbad, CA) extraction, and then was reverse- transcribed into cDNA using the Superscript III First-Strand Synthesis System (Invitrogen) . The cDNA product was

amplified with recombinant Platinum Taq DNA polymerase

(Invitrogen) . Expression band intensities of relevant genes were analyzed semi-quantitatively and normalized to the housekeeping gene glyceraldehydes 3-phosphate dehydrogenase (GAPDH) .

Example 8 : Measurement of media ion concentrations

Media calcium concentrations (n=5) were quantified using the Arsenazo III dye (Pointe Scientific, Lincoln Park, MI) , with absorbance measured at 620 nm using a microplate reader. Media aliquots were collected at each feeding and the BioVision Phosphate Assay Kit was used to analyze media phosphate levels. Briefly, media was diluted with water in a 1:10 ratio and allowed to react with 30ul of dye for 30 minutes. Absorbance was measured at 650 nm using a microplate reader (Tecan) .