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1.  Comparison of polymer scaffolds in rat spinal cord: A step toward quantitative assessment of combinatorial approaches to spinal cord repair 
Biomaterials  2011;32(32):8077-8086.
The transected rat thoracic (T9/10) spinal cord model is a platform for quantitatively compa0ring biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ε-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF+ hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF+ polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies.
doi:10.1016/j.biomaterials.2011.07.029
PMCID: PMC3163757  PMID: 21803415
OPF; PLGA; PCLF; axon regeneration; spinal cord injury; Schwann cell
2.  TENSILE PROPERTIES OF ENGINEERED CARTILAGE FORMED FROM CHONDROCYTE- AND MSC-LADEN HYDROGELS 
Objective
The objective of this study was to determine the capacity of chondrocyte-and mesenchymal stem cell (MSC)-laden hydrogel constructs to achieve native tissue tensile properties when cultured in a chemically defined medium supplemented with transforming growth factor-beta3 (TGF-β3).
Design
Cell-laden agarose hydrogel constructs (seeded with bovine chondrocytes or MSCs) were formed as prismatic strips and cultured in a chemically defined serum-free medium in the presence or absence of TGF-β3. The effects of seeding density (10 versus 30 million cells/mL) and cell type (chondrocyte versus MSC) were evaluated over a 56 day period. Biochemical content, collagenous matrix deposition and localization, and tensile properties (ramp modulus, ultimate strain, and toughness) were assessed bi-weekly.
Results
Results show that the tensile properties of cell seeded agarose constructs increase with time in culture. However, tensile properties (modulus, ultimate strain, and toughness) achieved on day 56 were not dependent on either the initial seeding density or the cell type employed. When cultured in medium supplemented with TGF-β3, tensile modulus increased and plateaued at a level of 300–400 kPa for each cell type and starting cell concentration. Ultimate strain and toughness also increased relative to starting values. Collagen deposition increased in constructs seeded with both cell types and at both seeding densities, with exposure to TGF-β3 resulting in a clear shift towards type II collagen deposition as determined by immunohistochemical staining.
Conclusions
These findings demonstrate that the tensile properties, an important and often overlooked metric of cartilage development, increase with time in culture in engineered hydrogel-based cartilage constructs. Under the free-swelling conditions employed in the present study, tensile moduli and toughness did not match that of the native tissue, though significant time-dependent increases were observed with the inclusion of TGF-β3. Of note, MSC-seeded constructs achieved tensile properties that were comparable to chondrocyte-seeded constructs, confirming the utility of this alternative cell source in cartilage tissue engineering. Further work, including both modulation of the chemical and mechanical culture environment, is required to optimize the deposition of collagen and its remodeling to achieve tensile properties in engineered constructs matching the native tissue.
doi:10.1016/j.joca.2008.02.005
PMCID: PMC2601559  PMID: 18353693
Cartilage; Tissue Engineering; Tensile Testing; Chondrocytes; 3D Culture; Mesenchymal Stem Cells
3.  Osteochondral Tissue Regeneration using a Bilayered Composite Hydrogel with Modulating Dual Growth Factor Release Kinetics in a Rabbit Model 
Biodegradable oligo(poly(ethylene glycol) fumarate) (OPF) composite hydrogels have been investigated for the delivery of growth factors (GFs) with the aid of gelatin microparticles (GMPs) and stem cell populations for osteochondral tissue regeneration. In this study, a bilayered OPF composite hydrogel that mimics the distinctive hierarchical structure of native osteochondral tissue was utilized to investigate the effect of transforming growth factor-β3 (TGF-β3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) on osteochondral tissue regeneration in a rabbit full-thickness osteochondral defect model. The four groups investigated included (i) a blank control (no GFs), (ii) GMP-loaded IGF-1 alone, (iii) GMP-loaded IGF-1 and gel-loaded TGF-β3, and (iv) GMP-loaded IGF-1 and GMP-loaded TGF-β3 in OPF composite hydrogels. The results of an in vitro release study demonstrated that TGF-β3 release kinetics could be modulated by the GF incorporation method. At 12 weeks post-implantation, the quality of tissue repair in both chondral and subchondral layers was analyzed based on quantitative histological scoring. All groups incorporating GFs resulted in a significant improvement in cartilage morphology compared to the control. Single delivery of IGF-1 showed higher scores in subchondral bone morphology as well as chondrocyte and glycosaminoglycan amount in adjacent cartilage tissue when compared to a dual delivery of IGF-1 and TGF-β3, independent of the TGF-β3 release kinetics. The results suggest that although the dual delivery of TGF-β3 and IGF-1 may not synergistically enhance the quality of engineered tissue, the delivery of IGF-1 alone from bilayered composite hydrogels positively affects osteochondral tissue repair and holds promise for osteochondral tissue engineering applications.
doi:10.1016/j.jconrel.2013.03.013
PMCID: PMC3661728  PMID: 23541928
Hydrogel; osteochondral defect; transforming growth factor-β3; insulin-like growth factor-1
4.  MODULATION OF CHONDROCYTE BEHAVIOR THROUGH TAILORING FUNCTIONAL SYNTHETIC SACCHARIDE-PEPTIDE HYDROGELS 
Biomaterials  2012;33(26):6052-6060.
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.
doi:10.1016/j.biomaterials.2012.04.058
PMCID: PMC3387337  PMID: 22672831
tissue engineering; chondrocyte; cartilage; sacchride-peptide; amino acid; functional group
5.  Mechanically Strong Double Network Photocrosslinked Hydrogels from N, N-Dimethylacrylamide and Glycidyl Methacrylated Hyaluronan 
Biomaterials  2008;29(14):2153-2163.
Hyaluronan (HA) is a natural polysaccharide abundant in biological tissues and it can be modified to prepare biomaterials. In this work, HA modified with glycidyl methacrylate was photocrosslinked to form the first network (PHA), and then a series of highly porous PHA/N, N-dimethylacrylamide (DAAm) hydrogels (PHA/DAAm) with high mechanical strength were obtained by incorporating a second network of photocrosslinked DAAm into PHA network. Due to synergistic effect produced by double network (DN) structure, despite containing 90% of water, the resulting PHA/DAAm hydrogel showed a compressive modulus and a fracture stress over 0.5 MPa and 5.2 MPa, respectively. Compared to the photocrosslinked hyaluronan single network hydrogel, which is generally very brittle and fractures easily, the PHA/DAAm hydrogels are ductile. Mouse dermal fibroblast was used as a model cell line to validate in vitro non-cytotoxicity of the PHA/DAAm hydrogels. Cells deposited extracellular matrix on the surface of these hydrogels and this was confirmed by positive staining of Type I collagen by Sirius Red. The PHA/DAAm hydrogels were also resistant to biodegradation and largely retained their excellent mechanical properties even after two months of co-culturing with fibroblasts.
doi:10.1016/j.biomaterials.2008.01.012
PMCID: PMC2323023  PMID: 18272215
Hyaluronan; N, N-dimethylacrylamide; Hydrogels; Photocrosslinkable; Double network
6.  Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior 
Biomaterials  2012;33(30):7478-7488.
Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM.
doi:10.1016/j.biomaterials.2012.06.097
PMCID: PMC3600380  PMID: 22841922
Peptide; Hydrogel; Cell delivery; Self-assembly; Chondrocyte; Tissue engineering
7.  On the role of hydrogel structure and degradation in controlling the transport of cell-secreted matrix molecules for engineered cartilage 
Damage to cartilage caused by injury or disease can lead to pain and loss of mobility, diminishing one’s quality of life. Because cartilage has a limited capacity for self-repair, tissue engineering strategies, such as cells encapsulated in synthetic hydrogels, are being investigated as a means to restore the damaged cartilage. However, strategies to date are suboptimal in part because designing degradable hydrogels is complicated by structural and temporal complexities of the gel and evolving tissue along multiple length scales. To address this problem, this study proposes a multi-scale mechanical model using a triphasic formulation (solid, fluid, unbound matrix molecules) based on a single chondrocyte releasing extracellular matrix molecules within a degrading hydrogel. This model describes the key players (cells, proteoglycans, collagen) of the biological system within the hydrogel encompassing different length scales. Two mechanisms are included: temporal changes of bulk properties due to hydrogel degradation, and matrix transport. Numerical results demonstrate that the temporal change of bulk properties is a decisive factor in the diffusion of unbound matrix molecules through the hydrogel. Transport of matrix molecules in the hydrogel contributes both to the development of the pericellular matrix and the extracellular matrix and is dependent on the relative size of matrix molecules and the hydrogel mesh. The numerical results also demonstrate that osmotic pressure, which leads to changes in mesh size, is a key parameter for achieving a larger diffusivity for matrix molecules in the hydrogel. The numerical model is confirmed with experimental results of matrix synthesis by chondrocytes in biodegradable poly(ethylene glycol)-based hydrogels. This model may ultimately be used to predict key hydrogel design parameters towards achieving optimal cartilage growth.
doi:10.1016/j.jmbbm.2012.10.016
PMCID: PMC3606675  PMID: 23276516
8.  Degradation Improves Tissue Formation in (Un)Loaded Chondrocyte-laden Hydrogels 
Background
Photopolymerizable poly(ethylene glycol) (PEG) hydrogels offer a platform to deliver cells in vivo and support three-dimensional cell culture but should be designed to degrade in sync with neotissue development and endure the physiologic environment.
Questions/purposes
We asked whether (1) incorporation of degradation into PEG hydrogels facilitates tissue development comprised of essential cartilage macromolecules; (2) with early loading before pericellular matrix formation, the duration of load affects matrix production; and (3) dynamic loading in general influences macroscopic tissue development.
Methods
Primary bovine chondrocytes were encapsulated in hydrogels (n = 3 for each condition). The independent variables were hydrogel degradation (nondegrading PEG and degrading oligo(lactic acid)-b-PEG-b-oligo(lactic acid) [PEG-LA]), culture condition (free swelling, unconfined dynamic compressive loading applied intermittently for 1 or 4 weeks), and time (up to 28 days). The dependent variables were neotissue deposition through biochemical contents, immunohistochemistry, and compressive modulus.
Results
Degradation led to 2.3- and 2.9-fold greater glycosaminoglycan and collagen contents, respectively; macroscopic cartilage-like tissue formation comprised of aggrecan, collagen II and VI, link protein, and decorin; but decreased moduli. Loading, applied early or throughout culture, did not affect neotissue content in either hydrogel but affected neotissue spatial distribution in degrading hydrogels where 4 weeks of loading appeared to enhance hydrogel degradation resulting in tissue defects.
Conclusions
PEG-LA hydrogels led to macroscopic tissue development comprised of key cartilage macromolecules under loading, but hydrogel degradation requires further tuning.
Clinical Relevance
PEG-LA hydrogels have potential for delivering chondrocytes in vivo to replace damaged cartilage with a tissue-engineered native equivalent, overcoming many limitations associated with current clinical treatments.
doi:10.1007/s11999-011-1823-0
PMCID: PMC3171547  PMID: 21347817
9.  Cartilage-like mechanical properties of poly (ethylene glycol)-diacrylate hydrogels 
Biomaterials  2012;33(28):6682-6690.
Hydrogels prepared from poly-(ethylene glycol) (PEG) have been used in a variety of studies of cartilage tissue engineering. Such hydrogels may also be useful as a tunable mechanical material for cartilage repair. Previous studies have characterized the chemical and mechanical properties of PEG-based hydrogels, as modulated by precursor molecular weight and concentration. Cartilage mechanical properties vary substantially, with maturation, with depth from the articular surface, in health and disease, and in compression and tension. We hypothesized that PEG hydrogels could mimic a broad range of the compressive and tensile mechanical properties of articular cartilage. The objective of this study was to characterize the mechanical properties of PEG hydrogels over a broad range and with reference to articular cartilage. In particular, we assessed the effects of PEG precursor molecular weight (508 Da, 3.4 kDa, 6 kDa, and 10 kDa) and concentration (10–40%) on swelling property, equilibrium confined compressive modulus (HA0), compressive dynamic stiffness, and hydraulic permeability (kp0) of PEG hydrogels in static/dynamic confined compression tests, and equilibrium tensile modulus (Eten) in tension tests. As molecular weight of PEG decreased and concentration increased, hydrogels exhibited a decrease in swelling ratio (31.5–2.2), an increase in HA0 (0.01–2.46 MPa) and Eten (0.02–3.5 MPa), an increase in dynamic compressive stiffness (0.055–42.9 MPa), and a decrease in kp0 (1.2 × 10−15 to 8.5 × 10−15 m2/(Pa s)). The frequency-dependence of dynamic compressive stiffness amplitude and phase, as well as the strain-dependence of permeability, were typical of the time- and strain-dependent mechanical behavior of articular cartilage. HA0 and Eten were positively correlated with the final PEG concentration, accounting for swelling. These results indicate that PEG hydrogels can be prepared to mimic many of the static and dynamic mechanical properties of articular cartilage.
doi:10.1016/j.biomaterials.2012.06.005
PMCID: PMC3572364  PMID: 22749448
PEG; Biomechanics; Crosslink; Compression; Tension; Modulus
10.  Biochemical and Structural Characterization of Neocartilage Formed by Mesenchymal Stem Cells in Alginate Hydrogels 
PLoS ONE  2014;9(3):e91662.
A popular approach to make neocartilage in vitro is to immobilize cells with chondrogenic potential in hydrogels. However, functional cartilage cannot be obtained by control of cells only, as function of cartilage is largely dictated by architecture of extracellular matrix (ECM). Therefore, characterization of the cells, coupled with structural and biochemical characterization of ECM, is essential in understanding neocartilage assembly to create functional implants in vitro. We focused on mesenchymal stem cells (MSC) immobilized in alginate hydrogels, and used immunohistochemistry (IHC) and gene expression analysis combined with advanced microscopy techniques to describe properties of cells and distribution and organization of the forming ECM. In particular, we used second harmonic generation (SHG) microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) to study distribution and assembly of collagen. Samples with low cell seeding density (1e7 MSC/ml) showed type II collagen molecules distributed evenly through the hydrogel. However, SHG microscopy clearly indicated only pericellular localization of assembled fibrils. Their distribution was improved in hydrogels seeded with 5e7 MSC/ml. In those samples, FIB/SEM with nm resolution was used to visualize distribution of collagen fibrils in a three dimensional network extending from the pericellular region into the ECM. In addition, distribution of enzymes involved in procollagen processing were investigated in the alginate hydrogel by IHC. It was discovered that, at high cell seeding density, procollagen processing and fibril assembly was also occurring far away from the cell surface, indicating sufficient transport of procollagen and enzymes in the intercellular space. At lower cell seeding density, the concentration of enzymes involved in procollagen processing was presumably too low. FIB/SEM and SHG microscopy combined with IHC localization of specific proteins were shown to provide meaningful insight into ECM assembly of neocartilage, which will lead to better understanding of cartilage formation and development of new tissue engineering strategies.
doi:10.1371/journal.pone.0091662
PMCID: PMC3953515  PMID: 24626259
11.  NELL-1 Promotes Cartilage Regeneration in an In Vivo Rabbit Model 
Tissue Engineering. Part A  2011;18(3-4):252-261.
Repair of cartilage due to joint trauma remains challenging due to the poor healing capacity of cartilage and adverse effects related to current growth factor-based strategies. NELL-1 (Nel-like molecule-1; Nel [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]), a protein first characterized in the context of premature cranial suture fusion, is believed to accelerate differentiation along the osteochondral lineage. We previously demonstrated the ability of NELL-1 protein to maintain the cartilaginous phenotype of explanted rabbit chondrocytes in vitro. Our objective in the current study is to determine whether NELL-1 can affect endogenous chondrocytes in an in vivo cartilage defect model. To generate the implant, NELL-1 was incorporated into chitosan nanoparticles and embedded into alginate hydrogels. These implants were press fit into 3-mm circular osteochondral defects created in the femoral condylar cartilage of 3-month-old New Zealand White rabbits (n=10). Controls included unfilled defects (n=8) and defects filled with phosphate-buffered saline-loaded chitosan nanoparticles embedded in alginate hydrogels (n=8). Rabbits were sacrificed 3 months postimplantation for histological analysis. Defects filled with alginate containing NELL-1 demonstrated significantly improved cartilage regeneration. Remarkably, histology of NELL-1-treated defects closely resembled that of native cartilage, including stronger Alcian blue and Safranin-O staining and increased deposition of type II collagen and absence of the bone markers type I collagen and Runt-related transcription factor 2 (Runx2) as demonstrated by immunohistochemistry. Our results suggest that NELL-1 may produce functional cartilage with properties similar to native cartilage, and is an exciting candidate for tissue engineering-based approaches for treating diverse pathologies of cartilage defects and degeneration.
doi:10.1089/ten.tea.2011.0142
PMCID: PMC3267973  PMID: 21902605
12.  Incorporation of phosphate group modulates bone cell attachment and differentiation on oligo(polyethylene glycol) fumarate hydrogel 
Acta biomaterialia  2012;8(4):1430-1439.
In this work, we have investigated the development of a synthetic hydrogel that contains a negatively charged phosphate group for use as a substrate for bone cell attachment and differentiation in culture. The photoreactive, phosphate-containing molecule, bis(2-(methacryloyloxy)ethyl)phosphate (BP), was incorporated into oligo(polyethylene glycol) fumarate hydrogel and the mechanical, rheological and thermal properties of the resulting hydrogels were characterized. Our results showed changes in hydrogel compression and storage moduli with incorporation of BP. The modification also resulted in decreased crystallinity as recorded by differential scanning calorimetry. Our data revealed that incorporation of BP improved attachment and differentiation of human fetal osteoblast (hFOB) cells in a dose-dependent manner. A change in surface chemistry and mineralization of the phosphate-containing surfaces verified by scanning electron microscopy and energy dispersive X-ray analysis was found to be important for hFOB cell attachment and differentiation. We also demonstrated that phosphate-containing hydrogels support attachment and differentiation of primary bone marrow stromal cells. These findings suggest that BP-modified hydrogels are capable of sustaining attachment and differentiation of both bone marrow stromal cells and osteoblasts that are critical for bone regeneration.
doi:10.1016/j.actbio.2011.12.031
PMCID: PMC3970912  PMID: 22277774
Hydrogel; Bone regeneration; Osteoblast; Rabbit marrow stromal cells
13.  Cartilage Matrix Formation by Bovine Mesenchymal Stem Cells in Three-dimensional Culture Is Age-dependent 
Background
Cartilage degeneration is common in the aged, and aged chondrocytes are inferior to juvenile chondrocytes in producing cartilage-specific extracellular matrix. Mesenchymal stem cells (MSCs) are an alternative cell type that can differentiate toward the chondrocyte phenotype. Aging may influence MSC chondrogenesis but remains less well studied, particularly in the bovine system.
Questions/purposes
The objectives of this study were (1) to confirm age-related changes in bovine articular cartilage, establish how age affects chondrogenesis in cultured pellets for (2) chondrocytes and (3) MSCs, and (4) determine age-related changes in the biochemical and biomechanical development of clinically relevant MSC-seeded hydrogels.
Methods
Native bovine articular cartilage from fetal (n = 3 donors), juvenile (n = 3 donors), and adult (n = 3 donors) animals was analyzed for mechanical and biochemical properties (n = 3–5 per donor). Chondrocyte and MSC pellets (n = 3 donors per age) were cultured for 6 weeks before analysis of biochemical content (n = 3 per donor). Bone marrow-derived MSCs of each age were also cultured within hyaluronic acid hydrogels for 3 weeks and analyzed for matrix deposition and mechanical properties (n = 4 per age).
Results
Articular cartilage mechanical properties and collagen content increased with age. We observed robust matrix accumulation in three-dimensional pellet culture by fetal chondrocytes with diminished collagen-forming capacity in adult chondrocytes. Chondrogenic induction of MSCs was greater in fetal and juvenile cell pellets. Likewise, fetal and juvenile MSCs in hydrogels imparted greater matrix and mechanical properties.
Conclusions
Donor age and biochemical microenvironment were major determinants of both bovine chondrocyte and MSC functional capacity.
Clinical Relevance
In vitro model systems should be evaluated in the context of age-related changes and should be benchmarked against human MSC data.
doi:10.1007/s11999-011-1869-z
PMCID: PMC3171558  PMID: 21424832
14.  Synthesis and Characterization of Hybrid Hyaluronic Acid-Gelatin Hydrogels 
Biomacromolecules  2013;14(4):1085-1092.
Biomimetic hybrid hydrogels have generated broad interest in tissue engineering and regenerative medicine. Hyaluronic acid (HA) and gelatin (hydrolyzed collagen) are naturally derived polymers and biodegradable under physiological conditions. Moreover, collagen and HA are major components of the extracellular matrix (ECM) in most of the tissues (e.g. cardiovascular, cartilage, neural). When used as a hybrid material, HA-gelatin hydrogels may enable mimicking the ECM of native tissues. Although HA-gelatin hybrid hydrogels are promising biomimetic substrates, their material properties have not been thoroughly characterized in the literature. Herein, we generated hybrid hydrogels with tunable physical and biological properties by using different concentrations of HA and gelatin. The physical properties of the fabricated hydrogels including swelling ratio, degradation, and mechanical properties were investigated. In addition, in vitro cellular responses in both two and three dimensional (2D and 3D) culture conditions were assessed. It was found that the addition of gelatin methacrylate (GelMA) into HA methacrylate (HAMA) promoted cell spreading in the hybrid hydogels. Moreover, the hybrid hydrogels showed significantly improved mechanical properties compared to their single component analogs. The HAMA-GelMA hydrogels exhibited remarkable tunability behavior and may be useful for cardiovascular tissue engineering applications.
doi:10.1021/bm3019856
PMCID: PMC3643200  PMID: 23419055
hyaluronic acid; gelatin; hydrogel; extracellular matrix; tissue engineering
15.  Coculture of Human Mesenchymal Stem Cells and Articular Chondrocytes Reduces Hypertrophy and Enhances Functional Properties of Engineered Cartilage 
Tissue Engineering. Part A  2011;17(7-8):1137-1145.
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.
doi:10.1089/ten.tea.2010.0531
PMCID: PMC3063700  PMID: 21142648
16.  Effects of Auricular Chondrocyte Expansion on Neocartilage Formation in Photocrosslinked Hyaluronic Acid Networks 
Tissue engineering  2006;12(9):2665-2673.
The overall objective of this study was to examine the effects of in vitro expansion on neocartilage formation by auricular chondrocytes photoencapsulated in a hyaluronic acid (HA) hydrogel as a next step towards the clinical application of tissue engineering therapies for treatment of damaged cartilage. Swine auricular chondrocytes were encapsulated either directly after isolation (p = 0), or after further in vitro expansion (p = 1 and p = 2) in a 2 wt%, 50 kDa HA hydrogel and implanted subcutaneously in the dorsum of nude mice. After 12 weeks, constructs were explanted for mechanical testing and biochemical and immunohistochemical analysis and compared to controls of HA gels alone and native cartilage. The compressive equilibrium moduli of the p = 0 and p = 1 constructs (51.2 ± 8.0 and 72.5 ± 35.2 kPa, respectively) were greater than the p = 2 constructs (26.8 ± 14.9 kPa) and the control HA gel alone (12.3 ± 1.3 kPa) and comparable to auricular cartilage (35.1 ± 12.2 kPa). Biochemical analysis showed a general decrease in glycosaminoglycan (GAG), collagen, and elastin content with chondrocyte passage, though no significant differences were found between the p = 0 and p = 1 constructs for any of the analyses. Histological staining showed intense and uniform staining for aggrecan, as well as greater type II collagen versus type I collagen staining in all constructs. Overall, this study illustrates that constructs with the p = 0 and p = 1 auricular chondrocytes produced neocartilage tissue that resembled native auricular cartilage after 12 weeks in vivo. However, these results indicate that further expansion of the chondrocytes (p = 2) can lead to compromised tissue properties.
doi:10.1089/ten.2006.12.2665
PMCID: PMC2678567  PMID: 16995800
cartilage; auricular chondrocyte; hyaluronic acid; hydrogel; passage number
17.  The role of hydrogels with tethered acetylcholine functionality on the adhesion and viability of hippocampal neurons and glial cells 
Biomaterials  2011;33(8):2473-2481.
In neural tissue engineering, designing materials with the right chemical cues is crucial in providing a permissive microenvironment to encourage and guide neuronal cell attachment and differentiation. Modifying synthetic hydrogels with biologically active molecules has become an increasingly important route in this field to provide a successful biomaterial and cell interaction. This study presents a strategy of using the monomer 2-methacryloxyethyl trimethylammonium chloride (MAETAC) to provide tethered neurotransmitter acetylcholine-like functionality with a complete 2-acetoxy-N,N,N-trimethylethanaminium segment, thereby modifying the properties of commonly used, non-adhesive PEG-based hydrogels. The effect of the functional monomer concentration on the physical properties of the hydrogels was systematically studied, and the resulting hydrogels were also evaluated for mice hippocampal neural cell attachment and growth. Results from this study showed that MAETAC in the hydrogels promotes neuronal cell attachment and differentiation in a concentration-dependent manner, different proportions of MAETAC monomer in the reaction mixture produce hydrogels with different porous structures, swollen states, and mechanical strengths. Growth of mice hippocampal cells cultured on the hydrogels showed differences in number, length of processes and exhibited different survival rates. Our results indicate that chemical composition of the biomaterials is a key factor in neural cell attachment and growth, and integration of the appropriate amount of tethered neurotransmitter functionalities can be a simple and effective way to optimize existing biomaterials for neuronal tissue engineering applications.
doi:10.1016/j.biomaterials.2011.12.005
PMCID: PMC3262018  PMID: 22196899
PEG-based hydrogels; Neurotransmitters; Acetylcholine functionality; Concentration-dependent manner
18.  High-Fidelity Tissue Engineering of Patient-Specific Auricles for Reconstruction of Pediatric Microtia and Other Auricular Deformities 
PLoS ONE  2013;8(2):e56506.
Introduction
Autologous techniques for the reconstruction of pediatric microtia often result in suboptimal aesthetic outcomes and morbidity at the costal cartilage donor site. We therefore sought to combine digital photogrammetry with CAD/CAM techniques to develop collagen type I hydrogel scaffolds and their respective molds that would precisely mimic the normal anatomy of the patient-specific external ear as well as recapitulate the complex biomechanical properties of native auricular elastic cartilage while avoiding the morbidity of traditional autologous reconstructions.
Methods
Three-dimensional structures of normal pediatric ears were digitized and converted to virtual solids for mold design. Image-based synthetic reconstructions of these ears were fabricated from collagen type I hydrogels. Half were seeded with bovine auricular chondrocytes. Cellular and acellular constructs were implanted subcutaneously in the dorsa of nude rats and harvested after 1 and 3 months.
Results
Gross inspection revealed that acellular implants had significantly decreased in size by 1 month. Cellular constructs retained their contour/projection from the animals' dorsa, even after 3 months. Post-harvest weight of cellular constructs was significantly greater than that of acellular constructs after 1 and 3 months. Safranin O-staining revealed that cellular constructs demonstrated evidence of a self-assembled perichondrial layer and copious neocartilage deposition. Verhoeff staining of 1 month cellular constructs revealed de novo elastic cartilage deposition, which was even more extensive and robust after 3 months. The equilibrium modulus and hydraulic permeability of cellular constructs were not significantly different from native bovine auricular cartilage after 3 months.
Conclusions
We have developed high-fidelity, biocompatible, patient-specific tissue-engineered constructs for auricular reconstruction which largely mimic the native auricle both biomechanically and histologically, even after an extended period of implantation. This strategy holds immense potential for durable patient-specific tissue-engineered anatomically proper auricular reconstructions in the future.
doi:10.1371/journal.pone.0056506
PMCID: PMC3577892  PMID: 23437148
19.  An Interplay between Electrostatic and Polar Interactions in Peptide Hydrogels 
Biopolymers  2013;100(2):10.1002/bip.22194.
Inherent chemical programmability available in peptide-based hydrogels has allowed diversity in the development of these materials for use in biomedical applications. Within the 20 natural amino acids, a range of chemical moieties are present. Here we used a mixing-induced self-assembly of two oppositely charged peptide modules to form a peptide-based hydrogel. To investigate electrostatic and polar interactions on the hydrogel, we replace amino acids from the negatively charged acidic glutamic acid (E) to the uncharged polar glutamine (Q) on a negatively charged peptide module, while leaving the positively charged module unchanged. Using dynamic rheology, the mechanical properties of each hydrogel were investigated. It was found that the number, but not the location, of electrostatic interactions (E residues) dictate the elastic modulus (G′) of the hydrogel, compared to polar interactions (Q residues). Increased electrostatic interactions also promote faster peptide assembly into the hydrogel matrix, and result in the decrease of T2 relaxation times of H2O and TFA. Small-angle X-ray scattering (SAXS) showed that changing from electrostatic → polar interactions affects the ability to form fibrous networks: from the formation of elongated fibers to no fiber assembly. This study reveals the systematic effects that the incorporation of electrostatic and polar interactions have when programmed into peptide-based hydrogel systems. These effects could be used to design peptide-based biomaterials with predetermined properties.
doi:10.1002/bip.22194
PMCID: PMC3869455  PMID: 23616100
hydrogels; dynamic rheometry; viscoelasticity; transport properties; small-angle X-ray scattering (SAXS); nuclear magnetic resonance (NMR)
20.  A Hydrogel-Mineral Composite Scaffold for Osteochondral Interface Tissue Engineering 
Tissue Engineering. Part A  2011;18(5-6):533-545.
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.
doi:10.1089/ten.tea.2011.0279
PMCID: PMC3286822  PMID: 21919797
21.  Hydrogels for the Repair of Articular Cartilage Defects 
The repair of articular cartilage defects remains a significant challenge in orthopedic medicine. Hydrogels, three-dimensional polymer networks swollen in water, offer a unique opportunity to generate a functional cartilage substitute. Hydrogels can exhibit similar mechanical, swelling, and lubricating behavior to articular cartilage, and promote the chondrogenic phenotype by encapsulated cells. Hydrogels have been prepared from naturally derived and synthetic polymers, as cell-free implants and as tissue engineering scaffolds, and with controlled degradation profiles and release of stimulatory growth factors. Using hydrogels, cartilage tissue has been engineered in vitro that has similar mechanical properties to native cartilage. This review summarizes the advancements that have been made in determining the potential of hydrogels to replace damaged cartilage or support new tissue formation as a function of specific design parameters, such as the type of polymer, degradation profile, mechanical properties and loading regimen, source of cells, cell-seeding density, controlled release of growth factors, and strategies to cause integration with surrounding tissue. Some key challenges for clinical translation remain, including limited information on the mechanical properties of hydrogel implants or engineered tissue that are necessary to restore joint function, and the lack of emphasis on the ability of an implant to integrate in a stable way with the surrounding tissue. Future studies should address the factors that affect these issues, while using clinically relevant cell sources and rigorous models of repair.
doi:10.1089/ten.teb.2011.0077
PMCID: PMC3171151  PMID: 21510824
22.  Synthesis and Characterization of Tunable Poly(Ethylene Glycol): Gelatin Methacrylate Composite Hydrogels 
Tissue Engineering. Part A  2011;17(13-14):1713-1723.
Poly(ethylene glycol) (PEG) hydrogels are popular for cell culture and tissue-engineering applications because they are nontoxic and exhibit favorable hydration and nutrient transport properties. However, cells cannot adhere to, remodel, proliferate within, or degrade PEG hydrogels. Methacrylated gelatin (GelMA), derived from denatured collagen, yields an enzymatically degradable, photocrosslinkable hydrogel that cells can degrade, adhere to and spread within. To combine the desirable features of each of these materials we synthesized PEG-GelMA composite hydrogels, hypothesizing that copolymerization would enable adjustable cell binding, mechanical, and degradation properties. The addition of GelMA to PEG resulted in a composite hydrogel that exhibited tunable mechanical and biological profiles. Adding GelMA (5%–15% w/v) to PEG (5% and 10% w/v) proportionally increased fibroblast surface binding and spreading as compared to PEG hydrogels (p<0.05). Encapsulated fibroblasts were also able to form 3D cellular networks 7 days after photoencapsulation only within composite hydrogels as compared to PEG alone. Additionally, PEG-GelMA hydrogels displayed tunable enzymatic degradation and stiffness profiles. PEG-GelMA composite hydrogels show great promise as tunable, cell-responsive hydrogels for 3D cell culture and regenerative medicine applications.
doi:10.1089/ten.tea.2010.0666
PMCID: PMC3118706  PMID: 21306293
23.  Crosslinking and degradation of step-growth hydrogels formed by thiol-ene photo-click chemistry 
Biomacromolecules  2012;13(7):2003-2012.
Thiol-ene photo-click hydrogels have been used for a variety of tissue engineering and controlled release applications. In this step-growth photopolymerization scheme, multi-arm poly(ethylene glycol) norbornene (PEG4NB) was crosslinked with di-thiol containing crosslinkers to form chemically crosslinked hydrogels. While the mechanism of thiol-ene gelation was well described in the literature, its network ideality and degradation behaviors are not well-characterized. Here, we compared the network crosslinking of thiol-ene hydrogels to Michael-type addition hydrogels and found thiol-ene hydrogels formed with faster gel points and higher degree of crosslinking. However, thiol-ene hydrogels still contained significant network non-ideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEG-norbornene macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, we found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network crosslinking. In an attempt to manipulate network crosslinking and degradation of thiol-ene hydrogels, we incorporated peptide crosslinkers with different sequences and characterized the hydrolytic degradation of these PEG-peptide hydrogels. In addition, we incorporated a chymotrypsin-sensitive peptide as part of the crosslinkers to tune the mode of gel degradation from bulk degradation to surface erosion.
doi:10.1021/bm300752j
PMCID: PMC4016800  PMID: 22708824
Thiol-ene chemistry; hydrogel; photopolymerization; degradation
24.  Biomimetic Poly(ethylene glycol)-Based Hydrogels as Scaffolds for Inducing Endothelial Adhesion and Capillary-like Network Formation 
Biomacromolecules  2012;13(3):706-713.
The extracellular matrix (ECM) is an attractive model for designing synthetic scaffolds with a desirable environment for tissue engineering. Here, we report on the synthesis of ECM-mimetic poly(ethylene glycol) (PEG) hydrogels for inducing endothelial cell (EC) adhesion and capillary-like network formation. A collagen type I-derived peptide, GPQGIAGQ (GIA)-containing PEGDA (GIA-PEGDA) was synthesized with the collagenase-sensitive GIA sequence attached in the middle of the PEGDA chain, which was then copolymerized with RGD capped-PEG monoacrylate (RGD-PEGMA) to form biomimetic hydrogels. The hydrogels degraded in vitro with the rate dependent on the concentration of collagenase, and also supported the adhesion of human umbilical vein ECs (HUVECs). Biomimetic RGD/GIA-PEGDA hydrogels with incorporation of 1% RGD-PEGDA into GIA-PEGDA hydrogels induced capillary-like organization when HUVECs were seeded on the hydrogel surface, while RGD/PEGDA and GIA-PEGDA hydrogels did not. These results indicate that both cell adhesion and biodegradability of scaffolds play important roles in the formation of capillary-like networks.
doi:10.1021/bm201596w
PMCID: PMC3310151  PMID: 22296572
biomimetic hydrogel; poly(ethylene glycol) (PEG); enzyme-sensitive peptide; capillary-like network; tissue engineering; human vein umbilical endothelial cell (HUVEC)
25.  Synergistic Effect of TGF-β1 And BMP–7 on Chondrogenesis and Extracellular Matrix Synthesis: An In Vitro Study 
Introduction:
The purpose of the present study seeks to determine the signal timing of BMP–7 and TGF-β1 from a novel chitosan based hydrogel system that may affect chondrocyte proliferation resulting in the presence of a synergism seen conspicuously in consecutive controlled delivery.
Methods:
Four groups of cultured chondrocytes were seeded on a novel designed chitosan based hydrogel. The hydrogel was left empty (control) in one group and loaded with BMP–7, TGF-β1 and their combination in the other groups, respectively. Hydrogel structure was analyzed with scanning electron microscope. The release kinetics of Growth Factors (GFs) was determined with ELISA. Chondrocyte viability and toxicity after being tested with MTS and collagen type II synthesis, were quantified with western blotting. Canonical regression analysis was used for measuring statistical evaluation.
Results:
Chitosan based hydrogel allowed controlled release of GFs in different time intervals for BMP–7 and TGF-β1. Double peak concentration gradient was found to be present in the group loaded with both GFs. In this group, substantially higher chondrocyte growth and collagen synthesis were also detected.
Conclusions:
We concluded that, chitosan based hydrogel systems may be adjusted to release GFs consecutively during biodegradation at the layers of surface, which may increase the cell number and enhance collagen type II synthesis.
doi:10.2174/1874325001206010406
PMCID: PMC3447182  PMID: 23002411
Chondrocyte culture; hydrogel; chitosan; controlled release; BMP-7; TGF-β1; cartilage.

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