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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Osteoarthritis Cartilage. Author manuscript; available in PMC 2010 October 1.
Published in final edited form as:
PMCID: PMC2753687
NIHMSID: NIHMS114512

Photo-crosslinked Alginate Hydrogels Support Enhanced Matrix Accumulation by Nucleus Pulposus Cells In Vivo

Alice I. Chou, B.S.E.,1 Sunday O. Akintoye, D.D.S, M.S.,2 and Steven B. Nicoll, Ph.D.1,3,*

Abstract

Objective

Intervertebral disc (IVD) degeneration is a major health concern in the United States. Replacement of the nucleus pulposus (NP) with injectable biomaterials represents a potential treatment strategy for IVD degeneration. The objective of this study was to characterize the extracellular matrix assembly and functional properties of NP cell-encapsulated, photo-crosslinked alginate hydrogels in comparison to ionically crosslinked alginate constructs.

Methods

Methacrylated alginate was synthesized by esterification of hydroxyl groups with methacrylic anhydride. Bovine nucleus pulposus cells were encapsulated in alginate hydrogels by ionic crosslinking using CaCl2 or through photo-crosslinking upon exposure to long-wave UV light in the presence of a photoinitiator. The hydrogels were evaluated in vitro by gross and histological analysis and in vivo using a murine subcutaneous pouch model. In vivo samples were analyzed for gene expression, extracellular matrix localization and accumulation, and equilibrium mechanical properties.

Results

Ionically crosslinked hydrogels exhibited inferior proteoglycan accumulation in vitro and were unable to maintain structural integrity in vivo. In further studies, photo-crosslinked alginate hydrogels were implanted for up to 8 weeks to examine NP tissue formation. Photo-crosslinked hydrogels displayed temporal increases in gene expression and assembly of type II collagen and proteoglycans. Additionally, hydrogels remained intact over the duration of the study and the equilibrium Young’s modulus increased from 1.24 ± 0.09 kPa to 4.31 ± 1.39 kPa, indicating the formation of functional matrix with properties comparable to those of the native NP.

Conclusions

These findings support the use of photo-crosslinked alginate hydrogels as biomaterial scaffolds for NP replacement.

Keywords: Nucleus pulposus, alginate, hydrogel, proteoglycan, type II collagen, Young’s modulus

Introduction

The intervertebral disc (IVD) is a fibro-cartilaginous tissue that confers flexibility to the spine by permitting limited bending and twisting movements between vertebral bodies. The disc is a heterogeneous structure divided into three anatomical regions: the outer and inner annulus fibrosus (AF) and the nucleus pulposus (NP). The outer AF is rich in type I collagen organized into concentric lamellae and allows the disc to resist tensile loads 13. The NP is composed primarily of negatively charged proteoglycans (i.e., aggrecan) and type II collagen that permit the disc to resist compressive loads by generation of osmotic swelling pressure 24. The inner AF is considered a transition zone between the highly organized collagenous structure of the outer annulus and the less organized and highly hydrated NP. The loss of proteoglycans from the NP that occurs with disc degeneration as a result of age or excessive trauma gives rise to a variety of health problems, ranging from lower back pain to paraplegia 57. Existing surgical interventions do not adequately restore disc function and often reduce joint mobility 8. Therefore, engineered constructs comprised of natural or synthetic materials have been explored as potential replacements for the disc.

Many naturally-derived materials have been examined for IVD tissue reconstruction, including but not limited to, collagen-derived scaffolds (i.e., atellocollagen, and types I and II collagen sponges), chitosan hydrogels, hyaluronic acid hydrogels, and alginate hydrogels 934. Most studies approximate the NP with a hydrogel-like scaffold that maintains the cells in a chondrocyte-like morphology. In particular, ionically crosslinked alginate hydrogels have been widely characterized and investigated for multiple tissue engineering applications. It is considered a routine method for in vitro culture of NP cells 1425. Alginate is a naturally derived polysaccharide extracted from brown algae, which possesses negatively charged carboxyl groups at physiologic pH. In the presence of divalent cations, the alginate is crosslinked to produce a three-dimensional construct. Previous studies have focused on characterizing gene expression and accumulation of extracellular matrix (ECM) macromolecules, such as type II collagen and proteoglycans. Few, however, have focused on the functional properties of accumulated matrix proteins. Baer et al. were first to study functional matrix production in cell-encapsulated ionically crosslinked alginate hydrogels 35. They found that cells were not capable of producing a functional matrix over 16 weeks in vitro. The authors hypothesized that diffusion and cellular uptake of the cationic crosslinkers were responsible for the adverse mechanical properties. Later, Mizuno et al. fabricated a composite IVD structure using a poly(glycolic acid)/poly(L-lactic acid) fiber mesh to approximate the AF and ionically crosslinked alginate to mimic the NP, and implanted the composite scaffold in the subcutaneous dorsa of athymic mice 27, 29. After 16 weeks post-implantation, the construct resembled native tissue and exhibited increased compressive properties similar to that of native tissue 27. These results suggest that further investigation of alginate hydrogels may be required to determine whether the biomaterial is appropriate for IVD tissue regeneration.

Recent work in the synthesis of in situ photo-crosslinkable polymers may provide an alternative method for producing alginate hydrogels for NP replacement 3642. In this system, polymers are modified with functional groups (i.e., methacrylates) that undergo free radical polymerization in the presence of a photo-initiator and upon exposure to UV light. This polymerization reaction induces a fluid-solid phase transformation under physiologic conditions and is ideal for encapsulation of cells in situ 4345. Elisseeff et al. used this methodology to successfully encapsulate chondrocytes in poly(ethylene oxide)-based hydrogels for potential use in cartilage repair applications 42. The technique was modified by Bryant et al. to incorporate hydrolytically degradable lactic acid units into photo-crosslinked poly(ethylene glycol)-based hydrogels to enhance the spatial distribution of ECM components in these otherwise inert polymers36. Methacrylated alginate and hyaluronic acid, which are more analogous to the negatively charged mucopolysaccharides in cartilaginous tissues (in comparison to the synthetic ethylene glycol derivatives), have also been studied as platforms for tissue engineering replacement strategies 43, 44, 46, 47. However, photo-crosslinked alginate has not been used previously for cell encapsulation or for NP repair. Therefore, the objective of this study was to characterize photo-crosslinked alginate hydrogels for NP tissue formation in vivo, specifically focusing on the ability of these materials to support functional ECM assembly by encapsulated NP cells. In this study, modified alginate hydrogels were ionically and photo-crosslinked, and cultured in vitro (4 weeks) as well as evaluated in vivo using a murine subcutaneous pouch model (over 8 weeks). Hydrogels were analyzed for gene expression, ECM accumulation and distribution, and mechanical properties. We hypothesized that photo-crosslinked alginate hydrogels would exhibit increased structural integrity, ECM accumulation, and mechanical properties compared to ionically crosslinked alginate constructs.

Methods

Primary Cell Isolation

Adult bovine tails were obtained from a local abattoir and caudal disc tissue excised from levels C2-C4 in a sterile environment. The NP was separated through gross visual inspection. All tissue was maintained in Dulbecco’s Modified Essential Medium (DMEM) with 20% Fetal Bovine Serum (FBS, Hyclone, Logan, UT), 2.5 μg/mL fungizone reagent, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.075% NaHCO3 (primary media) for 1 day at 37°C in a humidified atmosphere with 5% CO2 to confirm that no contamination occurred during the harvesting process. Tissue was diced and cells were released through collagenase digestion as previously described 48. Released cells were designated passage 0 and serially passaged twice according to previous protocols 15, 49.

Macromer Synthesis

Methacrylated alginate (MA-LVALG) was synthesized through esterification of hydroxyl groups based on protocols previously described 43, 44. Briefly, a 1% solution of low viscosity alginate (LVALG, Sigma, St. Louis, MO) was prepared in deionized water and adjusted to pH 8 using 5N NaOH. Methacrylic anhydride (Sigma, St. Louis, MO) at 20-fold excess was added to the alginate solution slowly at 4°C and the pH was periodically adjusted to 8 using 5N NaOH (approximately 15 times over the course of the 24-hour reaction). The solution was allowed to react for 24 hours at 4°C. The modified alginate was purified via dialysis against sterile water (Spectra/Por 1, MW 5–8kDa, Rancho Dominguez, CA) for 48 hours to remove excess methacrylic anhydride and the final product was recovered by lyophilization. The degree of substitution was confirmed using 1H-NMR analysis (DMX 360) with D2O as the solvent. The relative integrations of the methacrylate proton peaks (methylene, δ= 6.0 and 5.6 ppm and the methyl peak, δ= 1.8 ppm) to carbohydrate protons were used to determine molar percent of methacrylation. A 3.5% modified MA-LVALG was synthesized and used for all subsequent studies.

Alginate Hydrogel Preparations

All ionically and photo-crosslinked cell-encapsulated alginate constructs were prepared with 3.5% modified MA-LVALG at 2 w/v% based on our previous studies 50. Prior to dissolution, lyophilized MA-LVALG was sterilized by exposure to germicidal UV light for 30 minutes. MA-LVALG was dissolved in a 0.05% filter-sterilized solution of the photoinitiator 2-methyl-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959, I2959, Ciba Specialty Chemicals, Basel, Switzerland) in calcium and magnesium-free Dulbecco’s Phosphate Buffered Saline (DPBS) at 2.2 w/v%. An NP cell suspension in DPBS (10×106 cells/mL final MA-LVALG) was added and homogenously mixed with MA-LVALG and cast at a final concentration of 2 w/v%. For photo-crosslinked MA-LVALG hydrogels, a cell-polymer solution was cast in a custom-made glass casting device and exposed to long-wave UV light (EIKO, Shawnee, KS, peak 368 nm, 1.2 W) for 10 minutes to produce 2-mm thick, 8-mm diameter hydrogels. For ionically crosslinked MA-LVALG hydrogels, a cell-polymer solution was cast into a custom-designed gel casting device between two sheets of filter paper soaked in 102 mM CaCl2. After 10 minutes, the top of the cast was removed and submerged in a 102 mM CaCl2 bath for an additional 10 minutes to produce 2-mm thick, 8-mm diameter hydrogels. Acellular hydrogel controls were also prepared for all groups.

Preliminary In Vitro and In Vivo Crosslinking Study

Using MA-LVALG, ionically and photo-crosslinked alginate hydrogels were prepared as described above. Ionically and photo-crosslinked samples were incubated in vitro with 3 mL of growth medium (DMEM with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.075% NaHCO3) and supplemented with vitamin C (50 μg/mL) at day 1 and replaced every 2–3 days. All in vitro hydrogels were maintained at 37°C in a humidified atmosphere with 5% CO2. Ionically and photo-crosslinked hydrogels were also implanted subcutaneously in a murine pouch model as described below. Samples were isolated at 4 weeks for gross inspection and immunohistochemical analysis.

In Vivo Murine Subcutaneous Pouch Model

Cell-seeded and acellular hydrogel constructs were prepared as described above and implanted subcutaneously in immunocompromised 6–8 week-old female beige mice (Strain: NIH-III-nu, Charles River Laboratories, Wilmington, MA) in accordance with University of Pennsylvania guidelines for the use of vertebrate animals for research (animal protocol # 800209). Surgery was performed aseptically under anesthesia induced by injection of 140 mg/kg body weight of ketamine (Fort Dodge, Animal Health, Fort Dodge IA), 7 mg/kg body weight xylazine (Phoenix Pharmaceuticals, St Joseph MO) and 1 mg/kg body weight acepromazine (Burns Veterinary Supply, Farmers Branch, TX). A mid-longitudinal sagittal skin incision on the dorsum of each mouse was expanded by blunt dissection to create separate subcutaneous pockets for a set of 4 individual hydrogel implants. Each mouse received the 4 implants to reduce variability among samples. The skin incision was closed with absorbable 3-0 Vicryl® sutures (Ethicon Inc., Somerville, NJ). Implants were harvested at 4 (ionically and photo-crosslinked hydrogels) and 8 (photo-crosslinked hydrogels only) weeks. The animals were euthanized by CO2 asphyxiation in accordance with the guidelines established by the American Veterinary Medical Association Panel on Euthanasia.

Gene Expression

At 4 and 8 weeks post-implantation, RNA was isolated from freshly harvested in vivo constructs to evaluate gene expression 48. Cells were released from the photo-crosslinked alginate hydrogels by addition of 20 mg/mL of alginate lyase (Sigma, St. Louis, MO) in DPBS (1 mL per hydrogel), and incubated on an orbital shaker at 37°C until the alginate was fully dissolved (approximately 30 minutes). Cells were pelleted by centrifugation (2000 rcf, 5 minutes) and RNA was extracted and purified using the RNeasy kit (Qaigen, Valencia, CA). Purity and concentration of all RNA were assessed spectrophotometrically. Reverse transcription (RT) was performed using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) and real-time PCR was conducted using the SYBR Green Master Mix Kit (Applied Biosystems, Foster City, CA) and an Applied Biosystems 7300 real-time PCR system. Primers for collagen type I, collagen type II, aggrecan, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as published 51. Types I and II collagen and aggrecan gene expression were normalized to GAPDH expression and fold differences were calculated using the ΔΔCt method.

Collagen Quantification

Protein expression of type II collagen was quantified on freshly isolated samples using an indirect enzyme-linked immunosorbant assay (ELISA) as previously described 48, 52. Briefly, cells encapsulated in alginate hydrogels were released as for RNA isolation and stored in 100–200 μL of 0.05 N acetic acid at −20°C. To extract proteins, all samples were removed from acetic acid and treated with 3 M guanidine HCl (GuHCl) and 2 mg/ml pepsin. Digests were plated in a 96-well Nunc Maxisorp plate for collagen protein quantification. Antibodies to type II collagen (1:4,000) (Rabbit anti-bovine type II collagen polyclonal antibody, Chemicon, Temecula, CA) were used along with a peroxidase-based detection system employing a biotinylated secondary antibody (goat anti-rabbit IgG (H+L), Vector Labs, Burlingame, CA) and a streptavidin-horseradish peroxidase enzyme conjugate (R&D Systems, Minneapolis, MN). Total reacted substrate was spectrophotometrically analyzed at 450 nm using a Bio-Tek Synergy-HT microplate reader (Winooski, Vermont) and total collagen was determined from standard curves of bovine type II collagen (isolated from bovine nasal cartilage, Rockland, Gilbertsville, PA).

Sulfated Glycosaminoglycan Quantification

Production of sulfated glycosaminoglycans (GAGs) was quantified on freshly harvested samples using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay 53. Protein extracts (Pepsin and GuHCl digests) were allowed to react with DMMB at pH 1.5 (Sigma-Aldrich, St. Louis, MO) and spectrophotometrically analyzed at 595 nm using a Bio-Tek Synergy-HT microplate reader (Winooski, Vermont) 54. Total GAG was determined from standard curves of chondroitin-6-sulfate C isolated from shark cartilage (Sigma, St. Louis, MO).

Mechanical Testing

Unconfined compression testing was conducted on excised in vivo hydrogels at 4 and 8 weeks post-implantation to evaluate the equilibrium Young’s modulus. Prior to testing, stereomicrographs were captured using a Zeiss Stemi 2000-C stereomicroscope and analyzed for hydrogel surface area using Scion image software. The mechanical testing device is based on a similar set-up described by Soltz and Ateshian 55. Samples were compressed between two impermeable glass platens in a DPBS bath. A protease inhibitor cocktail (10 mM N-ethylmaleimide, 1 mM phenyl-methyl-sulfonyl fluoride, 5 mM benzamadine) was added to the DPBS bath to prevent matrix degradation over the course of the test 55. The unconfined compression testing protocol consisted of a creep test followed by a multi-ramp stress-relaxation test 56. For the creep test, a 1-g tare load was applied at a 10 μm/sec ramp velocity for 2100 seconds, which corresponded to the time when equilibrium was reached (equilibrium criteria: <10 μm change in 10 minutes). The multi-ramp stress-relaxation test consisted of three 5% strain ramps, each followed by a 2000 second relaxation period (equilibrium criteria for stress-relaxation: <0.5 g change in 10 minutes). Equilibrium stress was calculated at each ramp using surface area data and plotted against the applied strain. From this equilibrium stress versus strain curve, an equilibrium Young’s modulus was determined and reported for each sample. After mechanical testing, hydrogels were fixed in acid formalin for 30 minutes at 4°C for immunohistochemical analysis.

Immunohistochemistry

Fixed samples were paraffin-embedded, sectioned using a Lieca microtome (Model 2030, Nussloch, Germany), and processed for immunohistochemistry to visualize ECM deposition. For collagen immunohistochemistry, samples were pre-treated with hyaluronidase for 30 minutes at 37°C and 0.5 N acetic acid for 2 hours at 4°C. Samples for chondroitin sulfate proteoglycan immunohistochemistry were only treated with 0.5 N acetic acid for 2 hours at 4°C. Antibodies to type II collagen (1:40) (Polyclonal Rabbit anti-bovine type II collagen polyclonal antibody, Chemicon, Temecula, CA) and chondroitin sulfate proteoglycan (1:100) (Sigma, St. Louis, MO) were applied. A peroxidase-based detection system (Vectastain Elite ABC, Vector Labs, Burlingame, CA) and 3,3′ diaminobenzidine as the substrate chromagen were used to detect protein localization. Non-immune controls were included without primary antibody. Stained samples were viewed with a Zeiss Axioskop 40 optical microscope and images were captured using AxioVision software.

Statistical Analysis

Data are presented as the mean ± standard deviation (n=4). A one-way ANOVA was used to determine the effect of time on gene expression, ECM accumulation (normalized type II collagen and GAG content) and the equilibrium Young’s modulus. Pairwise comparisons were made using a Tukey post-hoc test and significance was set at p<0.05. Statistical analyses were performed using JMP software (SAS Institute, Cary, NC).

Results

Preliminary Analysis of Ionically and Photo-crosslinked Alginate Hydrogels In Vitro and In Vivo

Nucleus pulposus cells were encapsulated in ionically and photo-crosslinked methacrylated alginate and cultured in vitro as well as evaluated in vivo using a murine subcutaneous pouch model. At 4 weeks in vitro, NP cells remained viable in all cultures, with photo-crosslinked hydrogels exhibiting greater viability than ionic alginate hydrogels (data not shown). After 4 weeks in vivo, all hydrogels were encapsulated in a thin fibrous capsule consistent with a mild foreign body response. Nevertheless, ionically crosslinked alginate hydrogels had undergone severe dissolution by that time and were unable to be processed for immunohistochemistry (Figure 1A). Photo-crosslinked hydrogels remained intact and displayed no apparent changes in structural integrity and shape (Figure 1B). Nucleus pulposus cells maintained a rounded morphology in all intact hydrogels (Figure 2), however, immunohistochemistry revealed more extensive ECM accumulation in photo-crosslinked hydrogels in comparison to ionic alginate hydrogels (Figure 2A versus 2B and 2C). Furthermore, photo-crosslinked hydrogel constructs produced greater amounts of type II collagen (data not shown) and chondroitin sulfate proteoglycan (Figure 2B versus 2C) in vivo compared to those cultured in vitro. Based on these initial results, photo-crosslinked alginate hydrogels were evaluated in the murine subcutaneous pouch model in all subsequent studies.

Figure 1
Methacrylated alginate hydrogels after 4 weeks in vivo. Ionically (A) and photo-crosslinked (B) hydrogels. Arrows indicate location of the hydrogel.
Figure 2
Chondroitin sulfate proteoglycan immunohistochemistry of ionically and photo-crosslinked alginate hydrogels after 4 weeks of in vitro and in vivo culture. (A) ionically crosslinked alginate hydrogel cultured in vitro, (B) photo-crosslinked alginate hydrogel ...

Gene Expression

Gene expression at 4 and 8 weeks revealed that photo-crosslinked alginate hydrogels support expression of characteristic ECM macromolecules by encapsulated NP cells. Both type II collagen and aggrecan expression increased while type I collagen expression remained unchanged over time (Figure 3). However, only aggrecan displayed a significant increase in gene expression (Figure 3). The RNA yields from acellular controls were not sufficient for quantitative analysis.

Figure 3
Quantitative gene expression of types I (COL I) and II (COL II) collagen and aggrecan (AGG) normalized to GAPDH expression at 4 and 8 weeks post-implantation. * denotes a significant difference (p<0.05) between 4 and 8 weeks.

Biochemistry

Production of type II collagen and sulfated glycosaminoglycans was quantified for photo-crosslinked hydrogels over the 8-week in vivo period. At 8 weeks, cell-seeded constructs contained 0.15 ± 0.03×10−3 μg COL II/mg wet weight and 6.3 ± 2.5×10−3 μg GAG/mg wet weight. Similar to the gene expression results, greater amounts of both type II collagen and sulfated glycosaminoglycans were detected at 8 weeks compared to the 4-week time point; however, only sulfated glycosaminoglycans exhibited a significant increase in accumulation (Figures 4A, 4B). Acellular controls did not produce measurable amounts of sulfated glycosaminoglycans or type II collagen.

Figure 4
Total type II collagen (A) and sulfated glycosaminoglycan (B) accumulation normalized to the wet weight of the hydrogels at 4 and 8 weeks post-implantation. * denotes a significant difference between 4 and 8 weeks.

Immunohistochemistry

Extracellular matrix macromolecules secreted by encapsulated NP cells were distributed pericellularly as shown by the immunohistochemical staining of type II collagen and chondroitin sulfate proteoglycan. At 4 weeks, there was relatively little expression of type II collagen in comparison to chondroitin sulfate proteoglycan (Figures 5A, 5B). However, by 8 weeks post-implantation, there was an increase in the number of cells producing these characteristic ECM components (Figures 5C, 5D). Acellular controls were transparent with no cellular infiltration or detectable matrix accumulation, although remnants of the thin fibrous capsule that enveloped the samples were present on the periphery of the gels (not shown).

Figure 5
Immunohistochemistry of type II collagen (A, C) and chondroitin sulfate proteoglycan (B, D) extracellular matrix elaboration of photo-crosslinked alginate hydrogels at 4 (A, B) and 8 (C, D) weeks post-implantation. Scale bar = 100 μm.

Mechanical Properties

The equilibrium Young’s modulus of cell-seeded photo-crosslinked constructs reached 4.31 ± 1.39 kPa by 8 weeks in vivo. At both 4 and 8 weeks post-implantation, the mechanical properties of the photo-crosslinked hydrogels were significantly increased compared to pre-implanted hydrogels (Figure 6A), but there was no significant difference between 4-week and 8-week constructs. Stereomicrographs confirmed an increase in opacity of hydrogels over time (Figure 6B, 6C, 6D). The equilibrium Young’s modulus of acellular controls (2.34 ± 0.33 kPa) was significantly lower than cell-seeded hydrogels at 4 and 8 weeks.

Figure 6
Equilibrium Young’s Modulus of photo-crosslinked alginate hydrogels cultured in vivo (A). Representative stereomicrographs of NP cell-encapsulated, photo-crosslinked alginate hydrogels pre-implantation (B), at 4 weeks post-implantation (C), and ...

Discussion

In this study, NP cells were encapsulated in ionically and photo-crosslinked alginate hydrogels and evaluated in vitro and in vivo. Ionically crosslinked alginate hydrogels were unable to maintain their structural integrity and displayed decreased cell numbers and characteristic ECM accumulation compared to photo-crosslinked hydrogels. In further studies, NP cell-encapsulated, photo-crosslinked alginate hydrogels exhibited a significant increase in aggrecan gene expression and total sulfated proteoglycan accumulation over time. Furthermore, the equilibrium Young’s modulus demonstrated significant increases, indicating the production of a mechanically functional ECM. These findings support the use of photo-crosslinked alginate hydrogels for functional matrix accumulation by encapsulated NP cells.

Ionically crosslinked hydrogels experienced significant dissolution in vivo compared to corresponding covalently crosslinked hydrogels. These results underscore the importance of crosslinking method in maintaining hydrogel structural integrity. Although previous reports support this observation, there are other formulations of alginate that may give rise to better structural properties in vivo 57. Alginate is composed of (1–4)-linked β-D-manuronic acid (M) and α-L-guluronic acid (G) monomers. The relative amounts of M and G monomers vary between species of algae. During ionic crosslinking, carboxyl functional groups within the G subunits of adjacent alginate chains are ionically linked to form a semi-solid structure. As ionic crosslinking is dependent on the available G monomers, alginate polymers composed of a higher percentage of G monomers produce stiffer hydrogels and are more stable in vivo 57. For the purpose of this study, modified alginate was used for all hydrogels to eliminate the effect of modification on the behavior of encapsulated cells. Even though the alginate used here may not have been optimal, analyses revealed increased cellular viability and protein deposition in photo-crosslinked alginate. Moreover, the pericellular localization of ECM macromolecules in the photo-crosslinked alginate constructs was more analogous to the native tissue structure than the microenvironment observed in the ionically crosslinked alginate.

Significant increases in gene expression and ECM accumulation were measured in photo-crosslinked alginate hydrogels at 8 weeks in vivo. Increased expression of type II collagen and aggrecan combined with low expression of type I collagen indicate that encapsulated cells maintained their differentiated phenotype. A significant increase in proteoglycan expression was detected between 4 and 8 weeks, while type II collagen exhibited an increasing trend that was not statistically different. Of particular interest is the increase in proteoglycan content, which has been shown to be important for functional compressive properties 27, 58. However, compared to the glycosaminoglycan content of native sheep NP (20.88 ± 4.82 μg/mg tissue), these cell-seeded constructs only reached 3% of native levels 27. It is important to consider that a low cell seeding density of 10×106 cells/mL was used here to specifically examine the effect of the biomaterial on cellular behavior. Typically, cell-seeded hydrogel constructs for tissue regeneration use between 20 and 60×106 cells/mL. As such, the low initial seeding density may have contributed to the low levels of ECM production 59. It should also be noted that the modification of alginate for covalent crosslinking alters the in vitro and in vivo degradation profile of the polymer. In general, the degradation of the biomaterial scaffold may allow for increased ECM accumulation in tissue-engineered constructs 57, 60, 61. Therefore, it is possible that the improved stability of the photo-crosslinked alginate hydrogels afforded by the covalent interchain crosslinks also prevented the uniform distribution and assembly of ECM molecules from reaching native tissue levels. Although most of the materials currently under development for intradiscal replacement are non-degradable synthetic polymers, degradable subunits may be incorporated into the alginate polymer backbone to study the effect of degradation on new matrix assembly. This approach has been used successfully for both poly(ethylene glycol) and hyaluronic acid-based photo-crosslinked hydrogels 36, 62.

In addition to increased accumulation of characteristic ECM proteins, mechanical properties of cell-encapsulated, photo-crosslinked hydrogels exhibited significant increases over time, indicating the production of a mechanically functional matrix. At 4 weeks in vivo, acellular controls appeared transparent and had an equilibrium Young’s modulus that was significantly lower than cell-laden hydrogels. This implies that the opacity of the constructs was due to the activity of implanted cells rather than to host cells migrating into the constructs. This also demonstrates that the increased mechanical properties were a result of cellular activity as opposed to a change in hydrogel structure. Interestingly, despite no apparent ECM accumulation, the modulus of acellular photo-crosslinked hydrogels was significantly greater than pre-implantation cell-laden constructs. This was likely due to the presence of fragments from the thin fibrous capsule that enveloped the hydrogels in vivo, which may have contributed to modest contraction and stiffening of the samples. After 8 weeks in vivo, NP cell-encapsulated, photo-crosslinked alginate hydrogels had an equilibrium Young’s modulus of 4.31 ± 1.39 kPa, which is close to the native NP equilibrium modulus reported in previous studies that employed similar unconfined compression testing protocols to determine the material properties of native NP tissue (5–6.7 kPa) 32, 63, 64.

In conclusion, the results of this study support the use of photo-crosslinked alginate constructs for NP repair, as the hydrogel maintains the differentiated phenotype and allows for the assembly of functional matrix. The purpose of this investigation was to analyze the interaction between photo-crosslinked alginate and cellular behavior, particularly focusing on characteristic ECM production and function. Future studies will focus on optimizing culture conditions for NP tissue regeneration. The effect of cell seeding density, mechanical stimulation, and additional polymer chemistries may be investigated to increase the accumulation of ECM macromolecules. Furthermore, various cell sources may be used to explore the inductive potential of the photo-crosslinked alginate environment. Refinement of these parameters may lead to an injectable therapy for disc degeneration, and possibly for the repair of other cartilaginous tissues, such as hyaline articular cartilage.

Acknowledgments

National Institutes of Health and the Wallace H. Coulter Foundation

This work was supported by grants from the NIH (EB002425) and the Wallace H. Coulter Foundation. The authors would like to thank Dr. Jason Burdick and his laboratory for assistance with experimental methods and helpful discussions and Monika Damek-Poprawa for her assistance in conducting animal studies.

Footnotes

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