Tissue-engineered Cartilage Constructs Research Articles

Engineering Lubrication in Articular Cartilage

Despite continuous progress toward tissue engineering of functional articular cartilage, significant challenges still remain. Advances in morphogens, stem cells, and scaffolds have resulted in enhancement of the bulk mechanical properties of engineered constructs, but little attention has been paid to the surface mechanical properties. In the near future, engineered tissues will be able to withstand and support the physiological compressive and tensile forces in weight-bearing synovial joints such as the knee. However, there is an increasing realization that these tissue-engineered cartilage constructs will fail without the optimal frictional and wear properties present in native articular cartilage. These characteristics are critical to smooth, pain-free joint articulation and a long-lasting, durable cartilage surface. To achieve optimal tribological properties, engineered cartilage therapies will need to incorporate approaches and methods for functional lubrication. Steady progress in cartilage lubrication in native tissues has pushed the pendulum and warranted a shift in the articular cartilage tissue-engineering paradigm. Engineered tissues should be designed and developed to possess both tribological and mechanical properties mirroring natural cartilage. In this article, an overview of the biology and engineering of articular cartilage structure and cartilage lubrication will be presented. Salient progress in lubrication treatments such as tribosupplementation, pharmacological, and cell-based therapies will be covered. Finally, frictional assays such as the pin-on-disk tribometer will be addressed. Knowledge related to the elements of cartilage lubrication has progressed and, thus, an opportune moment is provided to leverage these advances at a critical step in the development of mechanically and tribologically robust, biomimetic tissue-engineered cartilage. This article is intended to serve as the first stepping stone toward future studies in functional tissue engineering of articular cartilage that begins to explore and incorporate methods of lubrication.

Monitoring Tissue Engineering and Regeneration by Magnetic Resonance Imaging and Spectroscopy

In this article, based on the invited talk at the “Tissue Science 2012” meeting in Chicago on October 1-3, 2012, we describe some examples of characterization of engineered cartilage and bone tissue using magnetic resonance spectroscopy and imaging. Two different models of engineered cartilage and engineered bone tissue constructs were used for these studies: 1) chondrocyte based cartilage tissue engineering constructs: human and bovine chondrocytes seeded in alginate beads (Hydrogel scaffold model) or bovine chondrocytes grown as pellets (scaffold free model); 2) mesenchymal stem cell (MSC) based cartilage and bone tissue engineering constructs: human mesenchymal stem cell (HMSCs) seeded in cartilage biomimetic scaffolds (collagen/chitosan scaffold integrated with extracellular matrix of cartilage) or HMSCs seeded in collagen/chitosan scaffolds. Magnetic resonance spectroscopy and imaging experiments using 9.4 T (400 MHz proton frequency), 11.7 T (500 MHz proton frequency) or 14.1 T (600 MHz proton frequency) MR spectrometers/Imager were performed on these constructs over two to four weeks of tissue culture time. Specifically, water suppressed proton NMR spectroscopy; proton and sodium multi-quantum coherence spectroscopy and proton T1, T2 and ADC parametric MRI were used to study the chondrogenesis and osteogenesis of these tissues. We found that the change in MR relaxation and diffusion coefficient parameters correlate well with the growth of engineered tissues. We found that the MR parameters and the change in these parameters in growing tissue are strongly influenced by the choice of scaffolds. We also found as expected that the tissue-engineered cartilage lacked order or preference in collagen orientation. Further work is underway to elucidate these findings. We anticipate that in future, MRI will augment histological and immunohistochemical techniques by providing a complimentary and real time quantitative assessment of engineered tissue growth at all growth stages: (i) cell seeding to pre implantation; (ii) preclinical validation studies post implantation in small and large animal models; (iii) clinical studies of performance of engineered tissues.

Evaluation of the implant type tissue-engineered cartilage by scanning acoustic microscopy

The tissue-engineered cartilages after implantation were nonuniform tissues which were mingling with biodegradable polymers, regeneration cartilage and others. It is a hard task to evaluate the biodegradation of polymers or the maturation of regenerated tissues in the transplants by the conventional examination. Otherwise, scanning acoustic microscopy (SAM) system specially developed to measure the tissue acoustic properties at a microscopic level. In this study, we examined acoustic properties of the tissue-engineered cartilage using SAM, and discuss the usefulness of this devise in the field of tissue engineering. We administered chondrocytes/atelocollagen mixture into the scaffolds of various polymers, and transplanted the constructs in the subcutaneous areas of nude mice for 2 months. We harvested them and examined the sound speed and the attenuation in the section of each construct by the SAM. As the results, images mapping the sound speed exhibited homogenous patterns mainly colored in blue, in all the tissue-engineered cartilage constructs. Contrarily, the images of the attenuation by SAM showed the variation of color ranged between blue and red. The low attenuation area colored in red, which meant hard materials, were corresponding to the polymer remnant in the toluidine blue images. The localizations of blue were almost similar with the metachromatic areas in the histology. In conclusion, the SAM is regarded as a useful tool to provide the information on acoustic properties and their localizations in the transplants that consist of heterogeneous tissues with various components.

Comutational Study of Oxygen and Glucose Transport in Engineered Cartilage Constructs

ABSTRACTThis paper characterizes the mass transfer and replenishment of glucose and oxygen in tissue engineered cartilage constructs by a numerical approach. Cell population growth modulated by glucose and oxygen is incorporated in the mathematic model. The distribution of synthesized type II collagen and its influence on mediating the chondrocyte growth over scaffold are also investigated. Results from simulation are compared with the experiments in literature to verify the formulation and predictions. It is found that, under static culture, the oftentimes observed phenomenon that the overall cell number densities in thick scaffolds are smaller than in thin scaffolds is mainly due to depletion of glucose rather than oxygen. Cell growth is found to be more sensitive to the change in glucose concentration for thick scaffolds, whereas to be more sensitive to the change in oxygen concentration for thin scaffolds. Results also demonstrate the modulation of chondrocyte growth by type II collagen, presenting the biphasic impact of type II collagen which promotes chondrocyte growth in the initial phase of cultivation, while inhibits cell growth in the long term. The numerical model provides a useful reference for developing cartilaginous constructs in tissue engineering.

Finite element study of a tissue-engineered cartilage transplant in human tibiofemoral joint

Most tissue-engineered cartilage constructs are more compliant than native articular cartilage (AC) and are poorly integrated to the surrounding tissue. To investigate the effect of an implanted tissue-engineered construct (TEC) with these inferior properties on the mechanical environment of both the engineered and adjacent native tissues, a finite element study was conducted. Biphasic swelling was used to model tibial cartilage and an implanted TEC with the material properties of either native tissue or a decreased elastic modulus and fixed charged density. Creep loading was applied with a rigid impermeable indenter that represented the femur. In comparison with an intact joint, compressive strains in the transplant, surface contact stress in the adjacent native AC and load partitioning between different phases of cartilage were affected by inferior properties of TEC. Results of this study may lead to a better understanding of the complex mechanical environment of an implanted TEC.

Feasibility and limitations of the round robin test for assessment of in vitro chondrogenesis evaluation protocol in a tissue-engineered medical product

Tissue-engineered medical products (TEMPs) should be evaluated before implantation. Therefore, it is indispensable to establish evaluation protocols in regenerative medicine. Whether or not such evaluation protocols are reasonable is generally verified through a 'round robin' test. However, the round robin test for TEMPs intrinsically includes a deficiency, because 'identical' specimens can not be prepared for TEMPs. The aim of the study was to assess the feasibility and limitations of the round robin test for TEMPs by using a prepared evaluation protocol. We adopted tissue-engineered cartilage constructs as delivered specimens and a protocol of measuring sGAG content as an evaluation protocol proposed to ISO TC150/SC7, which is an invasive, but usually applied, method, although non-invasive methods are keenly required in evaluating TEMPs. The results showed that: (a) the coefficient of variation (CV) of the measured sGAG contents in intralaboratory tests was ~5% at most; (b) the CV of sGAG content in the scheme where each participating laboratory measured different constructs was comparable with that in the scheme where each participating laboratory measured one half of a construct along with the organizing laboratory; (c) the CV caused by factors other than the specimen was ~15%, comparable to that in reproducible experiments in biomedical fields. Based on these results, the study concludes that a round robin test for a TEMP could be valuable, under the condition that the delivered TEMPs are sufficiently reproducible so that the CV of the measured values is < 5% in the organizing laboratory.

Administration of the insulin into the scaffold atelocollagen for tissue‐engineered cartilage

Three-dimensional culture of the tissue-engineered cartilage constructs may increase the matrix production, but central necrosis must occur if the construct becomes large. To increase the cell viability in the middle part of constructs and to enhance the in vivo cartilage regeneration, we attempted to administer the insulin into the scaffold. Insulin is known to strongly enhance the matrix production in the chondrocytes. The pellets of human auricular chondrocytes with atelocollagen hydrogel were 3D-cultured in the medium. The comparison among three groups (insulin mixed in the atelocollagen, insulin added to the medium, and control group, i.e.; insulin in neither atelocollagen nor medium) revealed that both insulin mixed in the atelocollagen and that in the medium could effectively promoted the cell viability and matrix synthesis of the chondrocytes. The daily assay also showed the gradual release of insulin from the atelocollagen hydrogel, suggesting that this material may work as a control release of insulin. We actually transplanted the poly-L-lactide porous scaffolds carrying the chondrocytes and the atelocollagen mixed with or without insulin, into the nude mice, showing that glycosaminoglycan accumulation was evident in the group with insulin although less without insulin. We thus showed the possibility to enhance the in vivo cartilage regeneration, when administered insulin into the atelocollagen hydrogel.

Neovaskularisation und freier mikrochirurgischer Transfer von in vitro gezüchteten Knorpelkonstrukten

Clinical imperatives for new cartilage to replace or restore the function of traumatized or missing tissue as a consequence of trauma, inherent malformations or disease has led to the need for therapies or procedures to generate cartilage for clinical applications. To ensure shape, function, and survival, in vitro cartilage-engineered constructs need to be revascularized. This study presents a viable method for neovascularization and free microsurgical transfer of these in vitro constructs. Twelve female Chinchilla Bastard rabbits were operated. Cartilage-engineered constructs were created by isolating chondrocytes from auricular biopsies, amplifying in monolayer culture, and then seeding them onto polycaprolactone scaffolds. In each prefabricated skin flap, three in vitro cartilage-engineered constructs measuring 2×2×0.5cm and one construct without cells, which served as the control, were implanted beneath an 8×15-cm random-pattern skin flap, neovascularized by implantation of an arteriovenous vascular pedicle with maximal blood flow. After 6weeks, the neovascularized flaps with embedded cartilage-engineered constructs were completely removed based on the newly implanted vascular pedicle, and then freely retransferred into position using microsurgery. Macroscopic observation, histology, selective microangiography, and immunohistochemistry were performed to determine the construct vitality, neovascularization, and new cartilage formation. All neovascularized skin flaps with embedded tissue-engineered cartilage constructs were effectively free-transferred as free flaps. The implanted constructs were protected and well integrated within the flap. All constructs were well neovascularized and showed histologically stability in both form and size. Immunohistology showed the existence of cartilage-like tissue with extracellular matrix neosynthesis. Our experimental study revealed the reliable ability of neovascularization and free microsurgical transplantation of cartilage-engineered constructs using prefabricated flaps. With respect to effective clinical application, engineered cartilage composed of a patient's own cells can become a feasible option for the reconstruction of large cartilage defects or auricular reconstruction using this method. The procedure also represents a promising alternative for clinical practice due to minimal donor site morbidity and favorable aesthetic outcomes.

Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly(urethane urea) scaffolds

The chemical and physical properties of scaffolds affect cellular behaviour, which ultimately determines the performance and outcome of tissue-engineered cartilage constructs. The objective of this study was to assess whether a degradable porous poly(urethane urea) scaffold could be a suitable material for cartilage tissue engineering. We also investigated whether the post-expansion redifferentiation and cartilage tissue formation of in vitro expanded adult human chondrocytes could be regulated by controlled modifications of the scaffold architecture. Scaffolds with different pore sizes, < 150 µm, 150-300 µm and 300-500 µm, were seeded with chondrocytes and subjected to chondrogenic and osteogenic induction in vitro. The poly(urethane urea) scaffold with the smaller pore size enhanced the hyaline-like extracellular matrix and thus neocartilage formation. Conversely, the chondrocytes differentiated to a greater extent into the osteogenic pathway in the scaffold with the larger pore size. In conclusion, our results demonstrate that poly(urethane urea) may be useful as a scaffold material in cartilage tissue engineering. Furthermore, the chondrogenic and the osteogenic differentiation capacity of in vitro expanded human articular chondrocytes can be influenced by the scaffold architecture. By tailoring the pore sizes, the performance of the tissue-engineered cartilage constructs might be influenced and thus also the clinical outcome in the long run.

Magnetic Resonance Studies of Macromolecular Content in Engineered Cartilage Treated with Pulsed Low-Intensity Ultrasound

Noninvasive monitoring of matrix development in tissue-engineered cartilage constructs would permit ongoing assessment with the ability to modify culture conditions during development to optimize tissue characteristics. In this study, chondrocytes seeded in a collagen hydrogel were exposed for 20 min/day to pulsed low-intensity ultrasound (PLIUS) at 30 mWcm(-2) and cultured for up to 5 weeks. Biochemical assays, histology, immunohistochemistry, Fourier transform infrared spectroscopy, and magnetic resonance imaging (MRI) were performed at weeks 3 and 5 after initiation of growth. The noninvasive MRI measurements were correlated with those from the invasive studies. In particular, MRI transverse relaxation time (T2) and magnetization transfer rate (k(m)) correlated with macromolecular content, which was increased by application of PLIUS. This indicates the sensitivity of MR techniques to PLIUS-induced changes in matrix development, and highlights the potential for noninvasive assessment of the efficacy of anabolic interventions for engineered tissue.

Macromol. Biosci. 11/2010

Back Cover: Tissue-engineered cartilage constructs were fabricated using 3D woven poly(ε-caprolactone) (PCL) scaffolds infiltrated with a slurry of homogenized cartilage-derived matrix (CDM) and seeded with human adipose-derived stem cells. Fiber-reinforced CDM scaffolds and 3D woven PCL scaffolds maintained their cartilage biomimetic mechanical properties throughout the culture period, while supporting the accumulation of a cartilaginous extracellular matrix. Images show different cross-sections of the scaffolds showing viable cells fluorescently labeled (green) and PCL fibers (all other colors). Further details can be found in the full paper by F. T. Moutos, B. T. Estes, and F. Guilak* on page 1355.

Computational fluid dynamics modeling of momentum transport in rotating wall perfused bioreactor for cartilage tissue engineering

In this study, computational fluid dynamics (CFD) analysis of a rotating-wall perfused-vessel (RWPV) bioreactor is performed to characterize the complex hydrodynamic environment for the simulation of cartilage development in RWPV bioreactor in the presence of tissue-engineered cartilage constructs, i.e., cell-chitosan scaffolds. Shear stress exerted on chitosan scaffolds in bioreactor was calculated for different rotational velocities in the range of 33–38 rpm. According to the calculations, the lateral and lower surfaces were exposed to 0.07926–0.11069 dyne/cm 2 and 0.05974–0.08345 dyne/cm 2, respectively, while upper surfaces of constructs were exposed to 0.09196–0.12847 dyne/cm 2. Results validate adequate hydrodynamic environment for scaffolds in RWPV bioreactor for cartilage tissue development which concludes the suitability of operational conditions of RWPV bioreactor.

Culturing functional cartilage tissue under a novel bionic mechanical condition

Bioreactor, which is used for in vitro construction of tissue-engineered cartilage, has been extensively studied by researchers. The growth and development of articular cartilage tissue are affected by biomechanical and biochemical factors, especially mechanical condition. Kinds of mechanical conditions including compressive and shear force, fluid flow, hydrostatic pressure, and tissue deformation, were developed in the past years. However, most mechanical conditions of improved bioreactor involve only one or two external force, which is merely partial for engineering cartilage tissue. No bioreactor which can simulate a normal articular cartilage in terms of structure and function has been reported. Consequently, simulation of bionic mechanical environment of a normal articular cartilage is considered to be the optimal environment for culturing the functional articular cartilage in vitro. Based upon this purpose, we designed a rolling-compression loading bioreactor. It could provide cultures with multi-mechanical stimulations and sufficiently mimic the complex mechanical environment of a normal articular cartilage. We propose that this comprehensive rolling-compression loading bioreactor can enhance the cultivation of functional cartilage constructs in vitro.

Strategies for Articular Cartilage Lesion Repair and Functional Restoration

Injury of articular cartilage due to trauma or pathological conditions is the major cause of disability worldwide, especially in North America. The increasing number of patients suffering from joint-related conditions leads to a concomitant increase in the economic burden. In this review article, we focus on strategies to repair and replace knee joint cartilage, since knee-associated disabilities are more prevalent than any other joint. Because of inadequacies associated with widely used approaches, the orthopedic community has an increasing tendency to develop biological strategies, which include transplantation of autologous (i.e., mosaicplasty) or allogeneic osteochondral grafts, autologous chondrocytes (autologous chondrocyte transplantation), or tissue-engineered cartilage substitutes. Tissue-engineered cartilage constructs represent a highly promising treatment option for knee injury as they mimic the biomechanical environment of the native cartilage and have superior integration capabilities. Currently, a wide range of tissue-engineering-based strategies are established and investigated clinically as an alternative to the routinely used techniques (i.e., knee replacement and autologous chondrocyte transplantation). Tissue-engineering-based strategies include implantation of autologous chondrocytes in combination with collagen I, collagen I/III (matrix-induced autologous chondrocyte implantation), HYAFF 11 (Hyalograft C), and fibrin glue (Tissucol) or implantation of minced cartilage in combination with copolymers of polyglycolic acid along with polycaprolactone (cartilage autograft implantation system), and fibrin glue (DeNovo NT graft). Tissue-engineered cartilage replacements show better clinical outcomes in the short term, and with advances that have been made in orthopedics they can be introduced arthroscopically in a minimally invasive fashion. Thus, the future is bright for this innovative approach to restore function.

Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part II: Biomimetic potential and applications

In order to evaluate the biomedical potential of three-dimensional chitinous scaffolds of poriferan origin, chondrocyte culturing experiments were performed. It was shown for the first time that freshly isolated chondrocytes attached well to the chitin scaffold and synthesized an extracellular matrix similar to that found in other cartilage tissue engineering constructs. Chitin scaffolds also supported deposition of a proteoglycan-rich extracellular matrix of chondrocytes seeded bioconstructs in an in vivo environment. We suggest that chitin sponge scaffolds, apart from the demonstrated biomedical applications, are highly optimized structures for use as filtering systems, templates for biomineralization as well as metallization in order to produce catalysts.

The evaluation of chrongenic potential for construction of tissue-engineered cartilage by combination of oscillationg seeding and ADSCs encapsulated with gels compound

Objective To analyze the osteogenic potential of tissue-engineered bone in vitro and in vivo, and to evaluate an effective way in constructing tissue-engineered bone. Methods Tissue-engineered cartilage was constructed in the following ways: ADSCs were encapsulated with gels compound and oscillating seeding ( group A ), ADSCs were encapsulated with gels compound and static culture ( group B), ADSCs were seeded with oscillating seeding (group C), and ADSCs were seeded with static seeding ( group D). There were 48 pieces of constructs for each group, and 6 piece of them in each group were examined at different periods. SEM and CLM were used to observe cellular pattern, survival rate, cell activity, and collagen type Ⅱ level and contents of DNA and GAG were detected at the 1st, 4th, 8th,12th, 16th, 20th, 24th, and 28th day. Results Under observation of SEM and CLM, new tissue showed more remarkable cell proliferation with abundant ECM in group A. As compared with other groups, the survival rate in group A was significantly higher ( 86. 39 ± 5.05, P < 0.05 ) and level of cell activity,contents of collagen type Ⅱ , DNA and GAG were significantly increased (0. 57 ± 0. 12, 71.30 ± 2.51,73.21 ± 1.38, 81.25 ± 1.29, P < 0. 05). Conclusion Gels encapsulation ADSCs combined with oscillating seeding promotes the cell augmentation and improves the quality of tissue-engineered cartilage. Key words: Adipose derived stromal cells; Gels; Culture; Tissue-engineered cartilage

Tailoring Thermoreversible Hyaluronan Hydrogels by “Click” Chemistry and RAFT Polymerization for Cell and Drug Therapy

Thermoreversible hydrogels are promising matrices for tissue-engineered cartilage and spine constructs. They require specific properties during all the stages of a cell therapy (e.g., cell expansion, recovery, injection, delivery). Thermoreversible hyaluronan-poly(N-isopropylacrylamide) (HA-PNIPAM) hydrogels with well-defined molecular architecture and properties were synthesized through RAFT polymerization and "click" chemistry. The effect of PNIPAM grafting length and density on HA-PNIPAM properties was evaluated by methods relevant for a cell therapy. It was found that reversibility of the PNIPAM gelling process was improved in the presence of HA. Increasing M(n) of PNIPAM decreased the viscosity at 20 degrees C and led to high G' at T > 30 degrees C; however, higher grafting density led to lower mechanical properties. Water uptake of the hydrogels was mainly dependent on PNIPAM M(n). All of the hydrogels and their degradation products were cytocompatible to hTERT-BJ1 fibroblasts. A composition with properties ideal for cell encapsulation was identified and characterized by a low viscosity at 20 degrees C, rapid gelling at 37 degrees C, absence of volume change upon gelling, and G' of 140 Pa at 37 degrees C.

Protocol Development for Vitrification of Tissue-Engineered Cartilage.

The success of tissue-engineered cartilage constructs (TECCs) as treatment options for healing cartilage defects can only be achieved if suitable preservation methods are found that can maintain their viability and function. Simply lowering the temperature of cells and tissues to below their freezing point invariably destroys them due to ice crystals that form in the water-laden cells and tissues. In addition, high salt concentrations that result from removal of water due to ice formation create a toxic imbalance. If the formation of ice crystals can be minimized while still halting metabolic activity of cells at low temperatures, then the viability and functionality of the preserved tissue may be maintained. Several important variables have been studied that affect cryopreservation of cells and tissues to maximize cell survival and tissue functionality: cryoprotectant agents, cryoprotectant concentrations, methods of introduction and elution, cooling and warming rates, and storage temperatures.[1–5] In 1949, Polge et al.[6] discovered a significant breakthrough in the role of glycerol as an intracellular cryoprotectant, which permeates cells to minimize intracellular ice formation. Later on, other intracellular cryoprotectants were discovered such as dimethylsulfoxide (Me2SO)[7] and extracellular cryoprotectants such as starches and sucrose, which protect cell membranes or act as osmotic buffers.[2,8] Formamide, acetamide and other such chemicals reduce the toxicity of cryoprotectants used in high concentration. Amides, in general, are weak cryoprotectants but improve cell viability when combined with cryoprotectants.[9] Combinations of intracellular and extracellular cryoprotectants can have additive or synergistic effects on cell viability upon rewarming of cryopreserved samples.[1] There are two main approaches to cryopreservation for cells, tissues, and organs. The first is the conventional freezing technique where up to 30% of cell water is substituted by a cryoprotective compound, usually Me2SO, permitting the storage of many types of cells in vitrified channels within ice.[1] Conventional freezing relies on slow cooling and warming rates. In contrast, vitrification uses high concentrations of cryoprotectants resulting in greater than 50% replacement of water in the cell or tissue. When combined with fast cooling and rewarming rates, both intracellular and extracellular ice formation is avoided. This method minimizes or prevents formation of ice, creating a glass-like material, while still halting metabolic activity and maintaining an osmotic balance between cells and their environment. The major limitation of vitrification, however, is the potential cytotoxicity of the high cryoprotectant concentrations employed.[1] Vitrification has been shown to provide effective preservation for a number of tissues including oocytes, early embryos, cartilage, skin, blood vessels (natural and engineered), and heart valves.[10–19] Furthermore, vitrified arterial blood vessels and tissue-engineered vascular grafts have been shown to maintain viscoelastic properties similar to fresh vascular tissues while demonstrating superior biomechanical performance when compared with frozen cryopreserved specimens.[15–17] Song et al. achieved significantly better viability by employing vitrification (80%) with a 55% (w/v) vitrification solution (VS), known as VS55, than with conventional freezing (12.8%) employing 0.6 mm thick rabbit articular cartilage.[20] Guan et al. demonstrated that 51% viability occurred when bovine cartilage was vitrified, and only 5% viability when bovine cartilage was preserved without cryoprotectants.[21] Subsequently, we found that VS55 was inadequate for preservation of thicker, large mammal cartilage and that better preservation was obtained using a more concentrated 83% vitrification formulation.[22, 23] Kuleshova et al. demonstrated the need to consider different approaches in the preservation of engineered tissues due to potential interactions between the scaffold material and tissue matrix.[24] There are three main variables affecting cell viability during cryopreservation by vitrification (cooling/warming rates, effective tissue permeation, and cytotoxicity) and protocols that work for one tissue type do not necessarily apply for other tissues or, in the case of cartilage, even the same tissue type from different species. The investigation of long-term storage is an important field in tissue engineering. The main reason for this study was to determine whether vitrification protocols developed for native articular cartilage were effective for tissue-engineered cartilage constructs. TECCs were harvested from a bioreactor, vitrified employing different protocols, and cell viability was assessed after rewarming and cryoprotectant removal. The end goal of this study was to employ a bioreactor, the perfusion concentric cylinder bioreactor, to aid in the cryopreservation of tissue-engineered cartilage.

Intracellular Na+ and Ca2+ modulation increases the tensile properties of developing engineered articular cartilage

Significant collagen content and tensile properties are difficult to achieve in tissue-engineered articular cartilage. The aim of this study was to investigate whether treating developing tissue-engineered cartilage constructs with modulators of intracellular Na(+) or Ca(2+) could increase collagen concentration and construct tensile properties. Inhibitors of Na(+) ion transporters and stimulators of intracellular Ca(2+) were investigated for their ability to affect articular cartilage development in a scaffoldless, 3-dimensional chondrocyte culture. Using a systematic approach, we applied ouabain (Na(+)/K(+)-ATPase inhibitor), bumetanide (Na(+)/K(+)/2Cl(-) tritransporter inhibitor), histamine (cAMP activator), and ionomycin (a Ca(2+) ionophore) to tissue-engineered constructs for 1 hour daily on days 10-14 of culture and examined the constructs at 2 weeks or 4 weeks. The gross morphology, biochemical content, and compressive and tensile mechanical properties of the constructs were assayed. The results of these experiments showed that 20 microM ouabain, 0.3 microM ionomycin, or their combination increased the tensile modulus by 40-95% compared with untreated controls and resulted in an increased amount of collagen normalized to construct wet weight. In constructs exposed to ouabain, the increased percentage of collagen per construct wet weight was secondary to decreased glycosaminoglycan production on a per-cell basis. Treatment with 20 microM ouabain also increased the ultimate tensile strength of neo-tissue by 56-86% at 4 weeks. Other construct properties, such as construct growth and type I collagen production, were affected differently by Na(+) modulation with ouabain versus Ca(2+) modulation with ionomycin. These data are the first to show that treatments known to alter intracellular ion concentrations are a viable method for increasing the mechanical properties of engineered articular cartilage and identifying potentially important relationships to hydrostatic pressure mechanotransduction. Ouabain and ionomycin may be useful pharmacologic agents for increasing tensile integrity and directing construct maturation.

Construction of tissue-engineered cartilage using human placenta-derived stem cells

Human placenta-derived stem cells (hPDSCs) were isolated by trypsinization and further induced into cartilage cells in vitro. The engineered cartilage was constructed by combining hPDSCs with collagen sponge and the cartilage formation was observed by implantation into nude mice. Results showed that hPDSCs featured mesenchymal stem cells and maintained proliferation in vitro for over 30 passages while remaining undifferentiated. All results indicated that hPDSCs have the potential to differentiate into functional cartilage cells in vitro when combined with collagen sponge, which provided experimental evidence for prospective clinical application.