Thermoreversible hydrogel scaffolds for articular cartilage engineering

Articular cartilage has limited potential for repair. Current clinical treatments for articular cartilage damage often result in fibrocartilage and are associated with joint pain and stiffness. To address these concerns, researchers have turned to the engineering of cartilage grafts. Tissue engineering, an emerging field for the functional restoration of articular cartilage and other tissues, is based on the utilization of morphogens, scaffolds, and responding progenitor/stem cells. Because articular cartilage is a water-laden tissue and contains within its matrix hydrophilic proteoglycans, an engineered cartilage graft may be based on synthetic hydrogels to mimic these properties. To this end, we have developed a polymer system based on the hydrophilic copolymer poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)]. Solutions of this polymer are liquid below 25 degrees C and gel above 35 degrees C, allowing an aqueous solution containing cells at room temperature to form a hydrogel with encapsulated cells at physiological body temperature. The objective of this work was to determine the effects of the hydrogel components on the phenotype of encapsulated chondrocytes. Bovine articular chondrocytes were used as an experimental model. Results demonstrated that the components required for hydrogel fabrication did not significantly reduce the proteoglycan synthesis of chondrocytes, a phenotypic marker of chondrocyte function. In addition, chondrocyte viability, proteoglycan synthesis, and type II collagen synthesis within P(PF-co-EG) hydrogels were investigated. The addition of bone morphogenetic protein-7 increased chondrocyte proliferation with the P(PF-co-EG) hydrogels, but did not increase proteoglycan synthesis by the chondrocytes. These results indicate that the temperature-responsive P(PF-co-EG) hydrogels are suitable for chondrocyte delivery for articular cartilage repair.     

Simultaneous anabolic and catabolic responses of human chondrocytes seeded in collagen hydrogels to long-term continuous dynamic compression

Cartilage repair strategies increasingly focus on the in vitro development of cartilaginous tissues that mimic the biological and mechanical properties of native articular cartilage. However, current approaches still face problems in the reproducible and standardized generation of cartilaginous tissues that are both biomechanically adequate for joint integration and biochemically rich in extracellular matrix constituents. In this regard, the present study investigated whether long-term continuous compressive loading would enhance the mechanical and biological properties of such tissues. Human chondrocytes were harvested from 8 knee joints (n=8) of patients having undergone total knee replacement and seeded into a collagen type I hydrogel at low density of 2×10(5)cells/ml gel. Cell-seeded hydrogels were cut to disks and subjected to mechanical stimulation for 28 days with 10% continuous cyclic compressive loading at a frequency of 0.3 Hz. Histological and histomorphometric evaluation revealed long-term mechanical stimulation to significantly increase collagen type II and proteoglycan staining homogenously throughout the samples as compared to unstimulated controls. Gene expression analyses revealed a significant increase in collagen type II, collagen type I and MMP-13 gene expression under stimulation conditions, while aggrecan gene expression was decreased and no significant changes were observed in the collagen type II/collagen type I mRNA ratio. Mechanical propertywise, the average value of elastic stiffness increased in the stimulated samples. In conclusion, long-term mechanical preconditioning of human chondrocytes seeded in collagen type I hydrogels considerably improves biological and biomechanical properties of the constructs, corroborating the clinical potential of mechanical stimulation in matrix-associated autologous chondrocyte transplantation (MACT) procedures.     

Hydrogel optimization for cultured elastic chondrocytes seeded onto a polyglycolic acid scaffold

The purpose of this study was to compare the effect of different hydrogels on the production of tissue-engineered cartilage based on polyglycolic acid (PGA). Chondrocytes were isolated from adult sheep auricles. Alginate, Type I collagen, methylcellulose, and pluronic F127 hydrogels were evaluated, as were controls prepared without hydrogels. Proliferated chondrocytes were mixed with each hydrogel at 20 x 10(6) cells/mL and seeded onto PGA (1 x 1 x 0.2 cm, n = 60). The constructs were cultured with serum-free medium containing 5 ng/mL TGF-beta(2) and 5 ng/mL des(1-3)IGF-I in rotational bioreactors for up to 6 weeks. The cellular morphology, histology, and biochemistry were analyzed. Type I collagen, methylcellulose, and pluronic F127 displayed improved cartilage matrix deposition in terms of histology and biochemistry compared to alginate. It was not concluded that the combined seeding of chondrocytes and hydrogels on a PGA scaffold had significantly better effects than cell seeding without hydrogels. However, the histology and other useful findings in this ECM analyses suggested that Type I collagen and MC hydrogels were the best candidates for cartilage regeneration, because of their stimulation for chondrocyte proliferation in a three-dimensional culture as well as cartilage regeneration.     

Incorporation of tissue-specific molecules alters chondrocyte metabolism and gene expression in photocrosslinked hydrogels

Hydrogels are highly swollen, insoluble networks which can entrap chondrocytes and provide a 3-D environment necessary for the re-growth of cartilaginous tissue. In this study, hydrogels were formulated with a synthetic poly(ethylene glycol) (PEG) component to provide control over the macroscopic gel properties and from a cartilage specific compound, chondroitin sulfate (ChSA), to capture features of the chondrocytes' native environment. PEG was chosen as the base hydrogel chemistry, because it forms a 3-D environment that maintains chondrocyte function. ChSA, a highly negatively charged main component of proteoglycans, was then selectively incorporated into the PEG gel. Macroscopic gel properties were manipulated to obtain high compressive moduli coupled with a high degree of swelling by formulating copolymer gels with these chemistries. The gel compressive modulus of cell-free PEG gels increased from 34 to 140 kPa with the incorporation of ChSA for similar degrees of swelling. When chondrocytes were encapsulated in pure ChSA gels, synthesis of collagen and glycosaminoglycans was inhibited. However, when PEG was introduced into the copolymer gels, both extracellular matrix components were stimulated. Total collagen content increased from non-detectable in the pure ChSA gels to 0.48+/-0.05 mg/g wet weight in the copolymer gels (40/60 ChSA/PEG). Gene expression for collagen type II was also enhanced by the incorporation of PEG into the gel, illustrating an important influence of gel chemistry on chondrocyte function; however, aggrecan gene expression was unaffected. This study demonstrates that the macroscopic properties of chondrocyte gel carriers can be controlled through the incorporation of charge into networks by ChSA, but the neutral, non-interactive base PEG chemistry facilitates extracellular matrix deposition.     

Surface elasticity and charge concentration-dependent endothelial cell attachment to copolymer polyelectrolyte hydrogel

The surface micromechanical properties of 2-hydroxyethyl methacrylate (HEMA) and 2-methacryloxyethyl trimethyl ammonium chloride (MAETAC) copolymer hydrogels are probed using atomic force microscopy. HEMA-MAETAC polyelectrolyte hydrogels with increasing positive charge concentrations ranging from 0 to 400mM in increments of 40mM, are fabricated using different proportions of HEMA and MAETAC monomers. Increasing proportions of positively charged MAETAC monomers produce hydrogels with increasingly swollen states and correspondingly decreasing measures of stiffness, or Young's modulus. Increasing the relative proportion of charged monomers also increases the hysteresis in the approaching and retracting components of the force spectroscopy curves. When these hydrogels are equilibrated in cell-culture media without fetal bovine serum and a pH-controlled CO(2) environment, precipitation reactions increase the variability of the Young's modulus estimates. Adding a buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, maintains physiological pH without the use of a CO(2) environment, and thus reduces salt precipitation reactions and the variability of the Young's modulus. The attachment of porcine pulmonary artery endothelial cells increases with increasing prepared hydrogel charge concentration and decreasing elasticity.     

Modulation of chondrocyte functions and stiffness-dependent cartilage repair using an injectable enzymatically crosslinked hydrogel with tunable mechanical properties

We developed an injectable hydrogel system to evaluate the effect of hydrogel stiffness on chondrocyte cellular functions in a three-dimensional (3D) environment and its subsequent influence on ectopic cartilage formation and early-stage osteochondral defect repair in a rabbit model. The hydrogels, composed of gelatin-hydroxyphenylpropionic acid (Gtn-HPA) conjugate, were formed using oxidative coupling of HPA moieties catalyzed by hydrogen peroxide (H₂O₂) and horseradish peroxidase (HRP). The storage modulus (G′) of the hydrogels, which was tunable by changing the H₂O₂ and Gtn-HPA concentrations, ranged from 570 Pa to 2750 Pa. It was found that the cellular functions of chondrocytes encapsulated in hydrogels, including cell proliferation, biosynthesis of collagen and sulfated glycosaminoglycans (sGAG), as well as gene expression of type I (Col-I) and type II collagen (Col-II), were strongly affected by the stiffness of the hydrogels. Of note, chondrocytes cultured within the Gtn-HPA hydrogel of medium stiffness (G′ = 1000 Pa) produced highest level of sGAG production, as well as highest ratio of Col-II to Col-I gene expression among the Gtn-HPA hydrogels of different stiffness. Consistent with the results from in vitro and in vivo ectopic cartilage formation, osteochondral defect repair in a rabbit model showed stiffness-dependent tissue repair, with defects implanted with chondrocytes in hydrogels of medium stiffness having markedly more hyaline cartilage formation, smoother surface and better integration with adjacent cartilage, compared to defects treated with hydrogels of low or high stiffness. These results suggest that the tunable stiffness of Gtn-HPA hydrogels modulates chondrocyte cellular functions, and has a dramatic impact on cartilage tissue histogenesis and repair.     

Synthesis and in vitro evaluation of enzymatically cross-linked elastin-like polypeptide gels for cartilaginous tissue repair

Genetically engineered elastin-like polypeptide (ELP) hydrogels offer unique promise as scaffolds for cartilage tissue engineering because of the potential to promote chondrogenesis and to control mechanical properties. In this study, we designed and synthesized ELPs capable of undergoing enzyme-initiated gelation via tissue transglutaminase, with the ultimate goal of creating an injectable, in situ cross-linking scaffold to promote functional cartilage repair. Addition of the enzyme promoted ELP gel formation and chondrocyte encapsulation in a biocompatible process, which resulted in cartilage matrix synthesis in vitro and the potential to contribute to cartilage mechanical function in vivo. A significant increase in the accumulation of sulfated glycosaminoglycans was observed, and histological sections revealed the accumulation of a cartilaginous matrix rich in type II collagen and lacking in type I collagen, indicative of hyaline cartilage formation. These results provide evidence of chondrocytic phenotype maintenance for cells in the ELP hydrogels in vitro. In addition, the dynamic shear moduli of ELP hydrogels seeded with chondrocytes increased from 0.28 to 1.7 kPa during a 4-week culture period. This increase in the mechanical integrity of cross-linked ELP hydrogels suggests restructuring of the ELP matrix by deposition of functional cartilage extracellular matrix components.     

Cartilage tissue engineering using human auricular chondrocytes embedded in different hydrogel materials

To seek a suitable scaffold for cartilage tissue engineering, we compared various hydrogel materials originating from animals, plants, or synthetic peptides. Human auricular chondrocytes were embedded in atelopeptide collagen, alginate, or PuraMatrix, all of which are or will soon be clinically available. The chondrocytes in the atelopeptide collagen proliferated well, while the others showed no proliferation. A high-cell density culture within each hydrogel enhanced the expression of collagen type II mRNA, when compared with that without hydrogel. By stimulation with insulin and BMP-2, collagen type II and glycosaminoglycan were significantly accumulated within all hydrogels. Chondrocytes in the atelopeptide collagen showed high expression of beta1 integrin, seemingly promoting cell-matrix signaling. The N-cadherin expression was inhibited in the alginate, implying that decrease in cell-to-cell contacts may maintain chondrocyte activity. The matrix synthesis in PuraMatrix was less than that in others, while its Young's modulus was the lowest, suggesting a weakness in gelling ability and storage of cells and matrices. Considering biological effects and clinical availability, atelopeptide collagen may be accessible for clinical use. However, because synthetic peptides can control the risk of disease transmission and immunoreactivities, some improvement in gelling ability would provide a more useful hydrogel for ideal cartilage regeneration.     

Platelet-rich plasma loaded in situ-formed hydrogel enhances hyaline cartilage regeneration by CB1 upregulation

The efficacy of three-dimensional (3D) culture on the proliferation and maturation of chondrocytes seeded into a hydrogel scaffold was assessed. Three types of hydrogel were prepared for the 3D culture of primary isolated chondrocytes. Chondrocyte proliferation was assessed using a live/dead viability/cytotoxicity assay and semiquantitative RT-PCR after 3D culture in hydrogel. Cylindrical defects in the center of rat xyphoids were used for the implantation of platelet-rich plasma (PRP)/hydrogel composites. Rats were killed at day 7 postoperatively and evaluated histochemically and immunohistologically. Xyphoid chondrocytes proliferated well with time in hydrogels. In the PRP-containing hydrogels, xyphoid defects displayed early formation of chondroid matrix with massive peripheral infiltration of spindle cells. These results were consistent with Safranin-O staining for proteoglycans and immunohistochemistry for type II collagen. Gene expression analyses in vitro revealed aggrecan, type II collagen, and ChM-1 and CB1 upregulation by PRP/hydrogel. PRP/hydrogel provided a suitable environment for hyaline cartilaginous regeneration, leading to anti-inflammation by significant increase of CB1 and inhibiting vascular ingrowth via considerable upregulation of ChM-1. The results provide a valuable reference for the clinical application of hydrogel scaffolds for hyaline cartilage regeneration, as well as the use of autologous PRP to improve cellular proliferation and maturation of xyphoid repair. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3099-3107, 2012.     

A hydrogel-mineral composite scaffold for osteochondral interface tissue engineering

Osteoarthritis is the leading cause of physical disability among Americans, and tissue engineered cartilage grafts have emerged as a promising treatment option for this debilitating condition. Currently, the formation of a stable interface between the cartilage graft and subchondral bone remains a significant challenge. This study evaluates the potential of a hybrid scaffold of hydroxyapatite (HA) and alginate hydrogel for the regeneration of the osteochondral interface. Specifically, the effects of HA on the response of chondrocytes were determined, focusing on changes in matrix production and mineralization, as well as scaffold mechanical properties over time. Additionally, the optimal chondrocyte population for interface tissue engineering was evaluated. It was observed that the HA phase of the composite scaffold promoted the formation of a proteoglycan- and type II collagen-rich matrix when seeded with deep zone chondrocytes. More importantly, the elevated biosynthesis translated into significant increases in both compressive and shear moduli relative to the mineral-free control. Presence of HA also promoted chondrocyte hypertrophy and type X collagen deposition. These results demonstrate that the hydrogel-calcium phosphate composite supported the formation of a calcified cartilage-like matrix and is a promising scaffold design for osteochondral interface tissue engineering.     

Modulation of chondrocyte behavior through tailoring functional synthetic saccharide-peptide hydrogels

Tailoring three-dimensional (3D) biomaterial environments to provide specific cues in order to modulate function of encapsulated cells could potentially eliminate the need for addition of exogenous cues in cartilage tissue engineering. We recently developed saccharide-peptide copolymer hydrogels for cell culture and tissue engineering applications. In this study, we aim to tailor our saccharide-peptide hydrogel for encapsulating and culturing chondrocytes in 3D and examine the effects of changing single amino acid moieties differing in hydrophobicity/hydrophilicity (valine (V), cysteine (C), tyrosine (Y)) on modulation of chondrocyte function. Encapsulated chondrocytes remained viable over 21 days in vitro. Glycosaminoglycan and collagen content was significantly higher in Y-functionalized hydrogels compared to V-functionalized hydrogels. Extensive matrix accumulation and concomitant increase in mechanical properties was evident over time, particularly with the presence of Y amino acid. After 21 days in vitro, Y-functionalized hydrogels attained a modulus of 193 ± 46 kPa, compared to 44 ± 21 kPa for V-functionalized hydrogels. Remarkably, mechanical and biochemical properties of chondrocyte-laden hydrogels were modulated by change in a single amino acid moiety. This unique property, combined with the versatility and biocompatibility, makes our saccharide-peptide hydrogels promising candidates for further investigation of combinatorial effects of multiple functional groups on controlling chondrocyte and other cellular function and behavior.     

Coculture of human mesenchymal stem cells and articular chondrocytes reduces hypertrophy and enhances functional properties of engineered cartilage

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair; however, it still remains a challenge to recapitulate the functional properties of native articular cartilage using only MSCs. Additionally, MSCs may exhibit a hypertrophic phenotype under chondrogenic induction, resulting in calcification after ectopic transplantation. With this in mind, the objective of this study was to assess whether the addition of chondrocytes to MSC cultures influences the properties of tissue-engineered cartilage and MSC hypertrophy when cultured in hyaluronic acid hydrogels. Mixed cell populations (human MSCs and human chondrocytes at a ratio of 4:1) were encapsulated in the hydrogels and exhibited significantly higher Young's moduli, dynamic moduli, glycosaminoglycan levels, and collagen content than did constructs seeded with only MSCs or chondrocytes. Furthermore, the deposition of collagen X, a marker of MSC hypertrophy, was significantly lower in the coculture constructs than in the constructs seeded with MSCs alone. When MSCs and chondrocytes were cultured in distinct gels, but in the same wells, there was no improvement in biomechanical and biochemical properties of the engineered tissue, implying that a close proximity is essential. This approach can be used to improve the properties and prevent calcification of engineered cartilage formed from MSC-seeded hydrogels with the addition of lower fractions of chondrocytes, leading to improved clinical outcomes.     

Delivery of TGF-beta1 and chondrocytes via injectable, biodegradable hydrogels for cartilage tissue engineering applications

In this work, novel hydrogel composites, based on the biodegradable polymer, oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles (MPs) were utilized as injectable cell and growth factor carriers for cartilage tissue engineering applications. Specifically, bovine chondrocytes were embedded in composite hydrogels co-encapsulating gelatin MPs loaded with transforming growth factor-beta1 (TGF-beta1). Hydrogels with embedded cells co-encapsulating unloaded MPs and those with no MPs served as controls in order to assess the effects of MPs and TGF-beta1 on chondrocyte function. Samples were cultured up to 28 days in vitro. By 14 days, cell attachment to embedded gelatin MPs within the constructs was observed via light microscopy. Bioassay results showed that, over the 21 day period, there was a statistically significant increase in cellular proliferation for samples containing gelatin MPs, but no increase was exhibited in samples without MPs over the culture period. The release of TGF-beta1 further increased cell construct cellularity. Over the same time period, glycosaminoglycan content per cell remained constant for all formulations, suggesting that the dramatic increase in cell number for samples with TGF-beta1-loaded MPs was accompanied by maintenance of the cell phenotype. Overall, these data indicate the potential of OPF hydrogel composites containing embedded chondrocytes and TGF-beta1-loaded gelatin MPs as a novel strategy for cartilage tissue engineering.     

Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage

Transplantation of cultured chondrocytes can regenerate cartilage tissue in cartilage defects. This method requires serial cell passages to expand chondrocytes to a large number of cells for transplantation. However, as chondrocytes are expanded in number in monolayer culture, the cells gradually lose their differentiated phenotype and may not form cartilage tissue. This study investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype in three-dimensional scaffolds and form cartilage tissue in vitro and in vivo. The growth rate, viability, synthesis of collagen type I and II, and apoptotic activity of chondrocytes with passage number of 1, 2 and 5 were compared during in vitro culture. As the passage number increased, the cell growth rate and viability decreased and apoptotic cell increased. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a low expression of collagen type II and a high expression of collagen type I, indicating chondrocyte dedifferentiation. To examine the ability of chondrocytes to regenerate cartilage tissues in vitro and in vivo, chondrocytes were expanded in vitro to passage number of 1 or 5, seeded onto biodegradable polymer scaffolds, and maintained in vitro or implanted into subcutaneous spaces of athymic mice for 1 month. Histological and immunohistochemical analyses of cartilage tissues engineered in vitro and in vivo with passage 1 chondrocytes showed mature and well-formed cartilage and the presence of highly sulfated glycosaminoglycans and type II collagen, a collagen type produced by differentiated chondrocytes. In contrast, tissues engineered in vitro and in vivo with passage 5 chondrocytes did not have chondrocyte morphology or cartilage-specific extracellular matrices (i.e., glycosaminoglycans and type II collagen). The results of this study show that chondrocyte passage number is an important factor affecting the quality of cartilage tissue-engineered with the chondrocytes, and that chondrocytes.     

Scaffold-dependent differentiation of human articular chondrocytes

Matrix-associated autologous chondrocyte transplantation (MACT) is a tissue-engineered approach for the treatment of cartilage defects and combines autologous chondrocytes seeded on biomaterials. The objective of the study is the analysis of growth and differentiation behaviour of human articular chondrocytes grown on three different matrices used for MACT. Human articular chondrocytes were kept in monolayer culture for 42 days and then seeded on matrices consisting of either collagen type I/III, hyaluronan, or gelatine. During the culture time of 4 weeks the constructs were analyzed weekly. Morphological criteria were studied by scanning and transmission electron microscopy. The expression of the main type collagens was analyzed by real-time PCR. The collagen type I/III matrix supported a differentiation that closely resembled the tissue organisation of native cartilage, but cell number and type II collagen synthesis were low and differentiation occurred rather late in the cultivation period. The hyaluronan matrix and the gelatine-based matrix supported a rather rapid differentiation, with a high number of cells and a relatively high amount of type II collagen, but there was no spatial assembly that mimicked native cartilage. These facts indicate that the nature of the matrix is of great influence in the differentiation behaviour of dedifferentiated chondrocytes.     

Alginate/lactose-modified chitosan hydrogels: a bioactive biomaterial for chondrocyte encapsulation

A new bioactive scaffold was prepared from a binary polysaccharide mixture composed of a polyanion (alginate) and a polycation (a lactose-modified chitosan, chitlac). Its potential use for articular chondrocytes encapsulation and cartilage reconstructive surgery applications has been studied. The hydrogel combines the ability of alginate to act as a 3D supporting structure with the capability of the second component (chitlac) to provide interactions with porcine articular chondrocytes. Physico-chemical characterization of the scaffold was accomplished by gel kinetics and compression measurements and demonstrated that alginate-chitlac mixture (AC-mixture) hydrogels exhibit better mechanical properties when compared with sole alginate hydrogels. Furthermore, biochemical and biological studies showed that these 3D scaffolds are able to maintain chondrocyte phenotype and particularly to significantly stimulate and promote chondrocyte growth and proliferation. In conclusion, the present study can be considered as a first step towards an engineered, biologically active scaffold for chondrocyte in vitro cultivation, expansion, and cell delivery.     

Manipulations in hydrogel chemistry control photoencapsulated chondrocyte behavior and their extracellular matrix production

In engineering a cell-carrier to support cartilage growth, hydrogels provide a unique, largely aqueous environment for 3-dimensional chondrocyte culture that facilitates nutrient transport yet provides an elastic framework dictating tissue shape and supporting external loads. Although the gel environment is often >90% water, we demonstrate that slight variations in hydrogel chemistry control gel degradation, evolving macroscopic properties, and ultimately the secretion and distribution of extracellular matrix molecules. Specifically, biodegradable poly(ethylene glycol)-co-poly(lactic acid) hydrogels were fabricated via photopolymerization. When chondrocytes were photoencapsulated in these gels, changes in the poly(ethylene glycol)-co-poly(lactic acid) repeat unit ratio from 19 to 7 increased total collagen synthesis 2.5-fold after 6 weeks in vitro. Furthermore, the ratio of collagen to glycosaminoglycans varied from glycosaminoglycan-rich, 0.33 +/- 0.13, to collagen-rich, 4.58 +/- 1.21, depending on gel chemistry and in vitro versus in vivo culture environment. By tuning scaffold chemistry, and subsequently, gel structure and degradation behavior, we can better guide tissue evolution and development.