Temporal assessment of ribose treatment on self-assembled articular cartilage constructs

Articular cartilage cannot repair itself in response to degradation from injury or osteoarthritis. As such, there is a substantial clinical need for replacements of damaged cartilage. Tissue engineering aims to fulfill this need by developing replacement tissues in vitro. A major goal of cartilage tissue engineering is to produce tissues with robust biochemical and biomechanical properties. One technique that has been proposed to improve these properties in engineered tissue is the use of non-enzymatic glycation to induce collagen crosslinking, an attractive solution that may avoid the risks of cytotoxicity posed by conventional crosslinking agents such as glutaraldehyde. The objectives of this study were (1) to determine whether continuous application of ribose would enhance biochemical and biomechanical properties of self-assembled articular cartilage constructs, and (2) to identify an optimal time window for continuous ribose treatment. Self-assembled constructs were grown for 4 weeks using a previously established method and were subjected to continuous 7-day treatment with 30 mM ribose during culture weeks 1, 2, 3, or 4, or for the entire 4-week culture. Control constructs were grown in parallel, and all groups were evaluated for gross morphology, histology, cellularity, collagen and sulfated glycosaminoglycan (GAG) content, and compressive and tensile mechanical properties. Compared to control constructs, it was found that treatment with ribose during week 2 and for the entire duration of culture resulted in significant 62% and 40% increases in compressive stiffness, respectively; significant 66% and 44% increases in tensile stiffness; and significant 50% and 126% increases in tensile strength. Similar statistically significant trends were observed for collagen and GAG. In contrast, constructs treated with ribose during week 1 had poorer biochemical and biomechanical properties, although they were significantly larger and more cellular than all other groups. We conclude that non-enzymatic glycation with ribose is an effective method for improving tissue engineered cartilage and that specific temporal intervention windows exist to achieve optimal functional properties.     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

Engineered cartilage covered ear implants for auricular cartilage reconstruction

Cartilage tissues are often required for auricular tissue reconstruction. Currently, alloplastic ear-shaped medical implants composed of silicon and polyethylene are being used clinically. However, the use of these implants is often associated with complications, including inflammation, infection, erosion, and dislodgement. To overcome these limitations, we propose a system in which tissue-engineered cartilage serves as a shell that entirely covers the alloplastic implants. This study investigated whether cartilage tissue, engineered with chondrocytes and a fibrin hydrogel, would provide adequate coverage of a commercially used medical implant. To demonstrate the in vivo stability of cell-fibrin constructs, we tested variations of fibrinogen and thrombin concentration as well as cell density. After implantation, the retrieved engineered cartilage tissue was evaluated by histo- and immunohistochemical, biochemical, and mechanical analyses. Histomorphological evaluations consistently showed cartilage formation over the medical implants with the maintenance of dimensional stability. An initial cell density was determined that is critical for the production of matrix components such as glycosaminoglycans (GAG), elastin, type II collagen, and for mechanical strength. This study shows that engineered cartilage tissues are able to serve as a shell that entirely covers the medical implant, which may minimize the morbidity associated with implant dislodgement.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Rapid prototyping of anatomically shaped, tissue-engineered implants for restoring congruent articulating surfaces in small joints

Background:  Preliminary studies investigated advanced scaffold design and tissue engineering approaches towards restoring congruent articulating surfaces in small joints.
Materials and methods:  Anatomical femoral and tibial cartilage constructs, fabricated by three-dimensional fibre deposition (3DF) or compression moulding/particulate leaching (CM), were evaluated in vitro and in vivo in an autologous rabbit model. Effects of scaffold pore architecture on rabbit chondrocyte differentiation and mechanical properties were evaluated following in vitro culture and subcutaneous implantation in nude mice. After femoral and tibial osteotomy and autologous implantation of tissue-engineered constructs in rabbit knee joints, implant fixation and joint articulation were evaluated.
Results:  Rapid prototyping of 3DF architectures with 100% interconnecting pores promoted homogeneous distribution of viable cells, glycosaminoglycan (GAG) and collagen type II; significantly greater GAG content and differentiation capacity (GAG/DNA) in vitro compared to CM architectures; and higher mechanical equilibrium modulus and dynamic stiffness (at 0.1 Hz). Six weeks after implantation, femoral and tibial constructs had integrated with rabbit bone and knee flexion/extension and partial load bearing were regained. Histology demonstrated articulating surfaces between femoral and tibial constructs for CM and 3DF architectures; however, repair tissue appeared fibrocartilage-like and did not resemble implanted cartilage.
Conclusions:  Anatomically shaped, tissue-engineered constructs with designed mechanical properties and internal pore architectures may offer alternatives for reconstruction or restoration of congruent articulating surfaces in small joints.     

Matrix development in self-assembly of articular cartilage

Background

Articular cartilage is a highly functional tissue which covers the ends of long bones and serves to ensure proper joint movement. A tissue engineering approach that recapitulates the developmental characteristics of articular cartilage can be used to examine the maturation and degeneration of cartilage and produce fully functional neotissue replacements for diseased tissue.

Methodology/Principal Findings

This study examined the development of articular cartilage neotissue within a self-assembling process in two phases. In the first phase, articular cartilage constructs were examined at 1, 4, 7, 10, 14, 28, 42, and 56 days immunohistochemically, histologically, and through biochemical analysis for total collagen and glycosaminoglycan (GAG) content. Based on statistical changes in GAG and collagen levels, four time points from the first phase (7, 14, 28, and 56 days) were chosen to carry into the second phase, where the constructs were studied in terms of their mechanical characteristics, relative amounts of collagen types II and VI, and specific GAG types (chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronan). Collagen type VI was present in initial abundance and then localized to a pericellular distribution at 4 wks. N-cadherin activity also spiked at early stages of neotissue development, suggesting that self-assembly is mediated through a minimization of free energy. The percentage of collagen type II to total collagen significantly increased over time, while the proportion of collagen type VI to total collagen decreased between 1 and 2 wks. The chondroitin 6- to 4- sulfate ratio decreased steadily during construct maturation. In addition, the compressive properties reached a plateau and tensile characteristics peaked at 4 wks.

Conclusions/Significance

The indices of cartilage formation examined in this study suggest that tissue maturation in self-assembled articular cartilage mirrors known developmental processes for native tissue. In terms of tissue engineering, it is suggested that exogenous stimulation may be necessary after 4 wks to further augment the functionality of developing constructs.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Chondrogenesis and developments in our understanding

Conditions affecting cartilage through damage or age-related degeneration pose significant challenges to individual patients and their healthcare systems. The disease burden will rise in the future as life expectancy increases. This has resulted in vigorous efforts to develop novel therapies to meet current and future needs. Due to the limited regenerative capacity of cartilage, in vitro tissue engineering techniques have emerged as the favoured technique by which to develop replacements. Tissue engineering is mainly concerned with developing cartilage replacements in the form of chondrocyte suspensions and three-dimensional scaffolds seeded with chondrocytes. One major limiting factor in the development of clinically useful cartilage constructs is our understanding of the process by which cartilage is formed, chondrogenesis. For example, techniques of culturing chondrocytes in vitro have been used for decades, resulting in chondrocyte-like cells which produce an extracellular matrix of similar composition to native cartilage, but with inferior physical properties. It has now been realised that one aspect of chondrogenesis which had been ignored was the physical context in which cartilage exists in vivo. This has resulted in the development of bioreactor systems which aim to introduce various physical stresses to engineered cartilage in a controlled environment. This has resulted in some improvements in the quality of tissue engineered cartilage. This is but one example of how the knowledge of chondrogenesis has been translated into research practice. This paper aims to review what is currently known about the process of chondrogenesis and discusses how this knowledge can be applied to tissue engineering.     

Microgravity tissue engineering

Summary Tissue engineering studies were done using isolated cells, three-dimensional polymer scaffolds, and rotating bioreactors operated under conditions of simulated microgravity. In particular, vessel rotation speed was adjusted such that 10 mm diameter × 2 mm thick cell-polymer constructs were cultivated in a state of continuous free-fall. Feasibility was demonstrated for two different cell types: cartilage and heart. Conditions of simulated microgravity promoted the formation of cartilaginous constructs consisting of round cells, collagen and glycosaminoglycan (GAG), and cardiac tissue constructs consisting of elongated cells that contracted spontaneously and synchronously. Potential advantages of using a simulated microgravity environment for tissue engineering were demonstrated by comparing the compositions of cartilaginous constructs grown under four different in vitro culture conditions: simulated microgravity in rotating bioreactors, solid body rotation in rotating bioreactors, turbulent mixing in spinner flasks, and orbital mixing in petri dishes. Constructs grown in simulated microgravity contained the highest fractions of total regenerated tissue (as a percent of construct dry weight) and of GAG, the component required for cartilage to withstand compressive force.     

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue^1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz^1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue^4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.     

Modulation of the mechanical properties of tissue engineered cartilage

Cartilaginous constructs have been grown in vitro using chondrocytes, biodegradable polymer scaffolds, and tissue culture bioreactors. In the present work, we studied how the composition and mechanical properties of engineered cartilage can be modulated by the conditions and duration of in vitro cultivation, using three different environments: static flasks, mixed flasks, and rotating vessels. After 4-6 weeks, static culture yielded small and fragile constructs, while turbulent flow in mixed flasks induced the formation of an outer fibrous capsule; both environments resulted in constructs with poor mechanical properties. The constructs that were cultured freely suspended in a dynamic laminar flow field in rotating vessels had the highest fractions of glycosaminoglycans and collagen (respectively 75% and 39% of levels measured in native cartilage), and the best mechanical properties (equilibrium modulus, hydraulic permeability, dynamic stiffness, and streaming potential were all about 20% of values measured in native cartilage). Chondrocytes in cartilaginous constructs remained metabolically active and phenotypically stable over prolonged cultivation in rotating bioreactors. The wet weight fraction of glycosaminoglycans and equilibrium modulus of 7 month constructs reached or exceeded the corresponding values measured from freshly explanted native cartilage. Taken together, these findings suggest that functional equivalents of native cartilage can be engineered by optimizing the hydrodynamic conditions in tissue culture bioreactors and the duration of tissue cultivation.     

A high throughput mechanical screening device for cartilage tissue engineering

Articular cartilage enables efficient and near-frictionless load transmission, but suffers from poor inherent healing capacity. As such, cartilage tissue engineering strategies have focused on mimicking both compositional and mechanical properties of native tissue in order to provide effective repair materials for the treatment of damaged or degenerated joint surfaces. However, given the large number design parameters available (e.g. cell sources, scaffold designs, and growth factors), it is difficult to conduct combinatorial experiments of engineered cartilage. This is particularly exacerbated when mechanical properties are a primary outcome, given the long time required for testing of individual samples. High throughput screening is utilized widely in the pharmaceutical industry to rapidly and cost-effectively assess the effects of thousands of compounds for therapeutic discovery. Here we adapted this approach to develop a high throughput mechanical screening (HTMS) system capable of measuring the mechanical properties of up to 48 materials simultaneously. The HTMS device was validated by testing various biomaterials and engineered cartilage constructs and by comparing the HTMS results to those derived from conventional single sample compression tests. Further evaluation showed that the HTMS system was capable of distinguishing and identifying ‘hits’, or factors that influence the degree of tissue maturation. Future iterations of this device will focus on reducing data variability, increasing force sensitivity and range, as well as scaling-up to even larger (96-well) formats. This HTMS device provides a novel tool for cartilage tissue engineering, freeing experimental design from the limitations of mechanical testing throughput.     

In Vivo Evaluation of a Novel Oriented Scaffold-BMSC Construct for Enhancing Full-Thickness Articular Cartilage Repair in a Rabbit Model

Tissue engineering (TE) has been proven usefulness in cartilage defect repair. For effective cartilage repair, the structural orientation of the cartilage scaffold should mimic that of native articular cartilage, as this orientation is closely linked to cartilage mechanical functions. Using thermal-induced phase separation (TIPS) technology, we have fabricated an oriented cartilage extracellular matrix (ECM)-derived scaffold with a Young''s modulus value 3 times higher than that of a random scaffold. In this study, we test the effectiveness of bone mesenchymal stem cell (BMSC)-scaffold constructs (cell-oriented and random) in repairing full-thickness articular cartilage defects in rabbits. While histological and immunohistochemical analyses revealed efficient cartilage regeneration and cartilaginous matrix secretion at 6 and 12 weeks after transplantation in both groups, the biochemical properties (levels of DNA, GAG, and collagen) and biomechanical values in the oriented scaffold group were higher than that in random group at early time points after implantation. While these differences were not evident at 24 weeks, the biochemical and biomechanical properties of the regenerated cartilage in the oriented scaffold-BMSC construct group were similar to that of native cartilage. These results demonstrate that an oriented scaffold, in combination with differentiated BMSCs can successfully repair full-thickness articular cartilage defects in rabbits, and produce cartilage enhanced biomechanical properties.     

In vitro culture increases mechanical stability of human tissue engineered cartilage constructs by prevention of microscale scaffold buckling

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling.     

Effects of co‐cultures of meniscus cells and articular chondrocytes on PLLA scaffolds

The knee meniscus, a fibrocartilaginous tissue located in the knee joint, is characterized by heterogeneity in extracellular matrix and biomechanical properties. To recreate these properties using a tissue engineering approach, co‐cultures of meniscus cells (MCs) and articular chondrocytes (ACs) were seeded in varying ratios (100:0, 75:25, 50:50, 25:75, and 0:100) on poly‐L ‐lactic acid (PLLA) scaffolds and cultured in serum‐free medium for 4 weeks. Histological, biochemical, and biomechanical tests were used to assess constructs at the end time point. Strong staining for collagen and glycosaminoglycan (GAG) was observed in all groups. Constructs with 100% MCs were positive for collagen I and constructs cultured with 100% ACs were positive for collagen II, while a mixture of collagen I and II was observed in other co‐culture groups. Total collagen and GAG per construct increased as the percentage of ACs increased (27 ± 8 µg, 0% AC to 45 ± 8 µg, 100% ACs for collagen and 12 ± 4 µg, 0% ACs to 40 ± 5 µg, 100% ACs for GAG). Compressive modulus (instantaneous and relaxation modulus) of the constructs was significantly higher in the 100% ACs group (63 ± 12 and 22 ± 9 kPa, respectively) when compared to groups with higher percentage of MCs. No differences in tensile properties were noted among groups. Specific co‐culture ratios were identified mimicking the GAG/DW of the inner (0:100, 25:75, and 50:50) and outer regions (100:0) of the meniscus. Overall, it was demonstrated that co‐culturing MCs and ACs on PLLA scaffolds results in functional tissue engineered meniscus constructs with a spectrum of biochemical and biomechanical properties. Biotechnol. Bioeng. 2009;103: 808–816. © 2009 Wiley Periodicals, Inc.     

Influence of perfusion on metabolism and matrix production by bovine articular chondrocytes in hydrogel scaffolds

Articular cartilage has a limited capacity for self-repair after damage. Engineered cartilage is a promising treatment to replace or repair damaged tissue. The growth of engineered cartilage is sensitive to the extracellular culture environment. Chondrocytes were seeded into alginate beads and agarose scaffolds at 4 millions/mL, and the response to static and perfusion culture was examined over period of up to 12 days. For both types of scaffolds, the chondrocytes kept their differentiated morphology over 12 days in all culture conditions. In alginate beads, more glycosaminoglycans (GAGs) were produced in perfusion culture than in static conditions. GAG distribution in alginate constructs was more uniform in perfusion culture than in static culture. However, in agarose constructs there was no significant difference in GAG production between static culture and perfusion culture. Under perfusion culture, the retention rate of GAG in alginate was higher than in agarsoe. It is suggested that the positive effect of perfusion culture only can be achieved by an appropriate choice of other factors such as scaffold materials.     

The role of bioreactors in cartilage tissue engineering

Cartilage tissue engineering is concerned with developing in vitro cartilage implants that closely match the properties of native cartilage, for eventual implantation to replace damaged cartilage. The three components to cartilage tissue engineering are cell source, such as in vitro expanded autologous chondrocytes or mesenchymal progenitor cells, a scaffold onto which the cells are seeded and a bioreactor which attempts to recreate the in vivo physicochemical conditions in which cartilage develops. Although much progress has been made towards the goal of developing clinically useful cartilage constructs, current constructs have inferior physicochemical properties than native cartilage. One of the reasons for this is the neglect of mechanical forces in cartilage culture. Bioreactors have been defined as devices in which biological or biochemical processes can be re-enacted under controlled conditions e.g. pH, temperature, nutrient supply, O2 tension and waste removal. The purpose of this review is to detail the role of bioreactors in the engineering of cartilage, including a discussion of bioreactor designs, current state of the art and future perspectives.     

Composite scaffolds for cartilage tissue engineering

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.     

Chondrogenesis on BMP‐6 loaded chitosan scaffolds in stationary and dynamic cultures

We originally investigated the suitability of chitosan scaffolds loaded with bone morphogenetic protein 6 (BMP‐6) in both stationary and dynamic conditions for cartilage tissue engineering. In the first part of the present study, ATDC5 murine chondrogenic cells were seeded in chitosan and BMP‐6 loaded chitosan scaffolds and cultured for 28 days under static conditions. In the following part, we examined the influence of dynamic cultivation conditions over BMP‐6 loaded chitosan scaffolds by using rotating bioreactor with perfusion (RCMW?). Tissue engineered constructs were characterized by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazoliumbromide (MTT) assay, scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and biochemical assays for glycosaminoglycans (GAG) deoxyribonucleic acid (DNA) and collagen Type II quantification. At the end of 4 weeks static incubation period high levels of GAG (21.22 mg/g dry weight), DNA amounts (1.37 mg/g dry weight) and collagen Type II amounts (1.94 µg/g dry weight) were achieved for BMP‐6 loaded chitosan scaffolds compared to chitosan scaffolds. However, the results obtained from morphological observations suggested hypertrophic differentiation of ATDC5 cells in the presence of BMP‐6 under stationary conditions. The influence of mechanical stimulation appeared significantly with differentiated cells, cultured under dynamic conditions, showing the effect of retaining their phenotypes without hypertrophy. Biotechnol. Bioeng. 2009; 104: 601–610 © 2009 Wiley Periodicals, Inc.