No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus
Abstract
In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015.
© 2015 Wiley Periodicals, Inc.
... An increase in the density and a shrink of thickness of elastomer foams can be in Fig. 14(a and b), respectively. Biodegradable elastomeric scaffolds such as poly-e-caprolactone (PCL) [72][73][74][75][76] , or polyglycerol-sebacate (PGS) [77,78] are very special. Although they have similar fatigue behavior as conventional elastomers, their fatigue performance is also highly sensitive to the moisture content. ...
... [65] Elastomer Silicon rubber Compressive q : 650 kg/m 3 , constant load corresponding to 30 À 60% deformation based on stress-strain curve f ¼ 3 Hz. [69] EVA Impact ASTM F1614-99 [70,71] PCL Compressive e max ¼ 15%, e min ¼ 0 [75,76] PGS Compressive e max ¼ 15%, e min ¼ 0 [74,78] Polyacrylonitrile (PAN) Compressive, straincontrolled q : 7.69 kg/m 3 , e 0 : 3%, 5% f ¼ 5 Hz. [81] Polyvinylpyrrolidone (PVP) Compressive, straincontrolled q : 7.69 kg/m 3 , e 0 : 0-50% T @ À100-400 C. [82] Silicon dioxide Compressive, straincontrolled q : 14 kg/m 3 , T @ À100, 25, 300 C, e 0 : 1%, f ¼ 0.1 Hz. [83] processing methods. ...
... This matter has been investigated in a number of previous studies. The influence of cartilage formation on static and dynamic behaviour of PCL scaffolds has been studied by Panadero et al. [26] . PCL scaffolds were fabricated using porogen leaching method and filled with fibrin hydrogels. ...
Bone scaffolds play a crucial role in bone tissue engineering, providing mechanical support for the growth of new tissue while enduring static and fatigue loads. Although polymers possess favourable characteristics such as adjustable degradation rate, tissue-compatible stiffness, ease of fabrication, and low toxicity, their relatively low mechanical strength has limited their use in load-bearing applications. While numerous studies have focused on assessing the static strength of polymeric scaffolds, little has been conducted on their fatigue properties. The current review presents a comprehensive study on the fatigue behaviour of polymeric bone scaffolds. The fatigue failure in polymeric scaffolds is discussed and the impact of material properties, topological features, loading conditions, and environmental factors are also examined. The present review also provides insight into the fatigue damage evolution within polymeric scaffolds, drawing comparisons to the behaviour observed in natural bone. The effect of polymer microstructure, incorporating reinforcing materials, the introduction of topological features, and hydrodynamic/corrosive impact of body fluids in the fatigue life of scaffolds are also discussed. Understanding these parameters is crucial for enhancing the fatigue resistance of polymeric scaffolds and holds promise for expanding their application in clinical settings as structural biomaterials.
... It should be noted that the strain applied in this study was higher than the physiological percent strain and is equivalent to the macrofracture of tendons. Table 1 provides additional studies on mechanical stimulation and bioreactor used for stimulation [70][71][72][73]. ...
... PCL is an FDAapproved synthetic scaffold, which is widely used in TE because of its tunable mechanical strength. PCL can be prepared as an electrospun nanofibrous scaffold or porous scaffold depending on the desired application (Ding et al., 2014;Panadero et al., 2016). Ceramic scaffolds including tricalcium phosphate and hydroxyapatite are biocompatible and can promote osteoinduction but they are poorly absorbed and brittle (Ghassemi et al., 2018;Roddy et al., 2018). ...
Articular cartilage is composed of chondrocytes surrounded by a porous permeable extracellular matrix. It has a limited spontaneous healing capability post-injury which, if left untreated, can result in severe osteochondral disease. Currently, osteochondral (OC) defects are treated by bone marrow stimulation, artificial joint replacement, or transplantation of bone, cartilage, and periosteum, while autologous osteochondral transplantation is also an option; it carries the risk of donor site damage and is limited only to the treatment of small defects. Allografts may be used for larger defects; however, they have the potential to elicit an immune response. A possible alternative solution to treat osteochondral diseases involves the use of stromal/stem cells. Human adipose-derived stromal/stem cells (ASCs) can differentiate into cartilage and bone cells. The ASC can be combined with both natural and synthetic scaffolds to support cell delivery, growth, proliferation, migration, and differentiation. Combinations of both types of scaffolds along with ASCs and/or growth factors have shown promising results for the treatment of OC defects based on in vitro and in vivo experiments. Indeed, these findings have translated to several active clinical trials testing the use of ASC-scaffold composites on human subjects. The current review critically examines the literature describing ASC-scaffold composites as a potential alternative to conventional therapies for OC tissue regeneration.
... PCL is an FDA approved biodegradable aliphatic linear polyester and it is one of the most investigated polymers for tissue engineering applications due to its adjustable mechanical strength. PCL can be used to produce porous scaffolds as well as electrospun nanofibers (Zhu et al., 2014;Panadero et al., 2016). Visser et al. (2015) incorporated PCL microfibers into GelMA obtaining reinforced hydrogels with mechanical properties similar to articular cartilage. ...
Articular cartilage is a highly specialised connective tissue of diarthrodial joints which provides a smooth, lubricated surface for joint articulation and plays a crucial role in the transmission of loads. In vivo cartilage is subjected to mechanical stimuli that are essential for cartilage development and the maintenance of a chondrocytic phenotype. Cartilage damage caused by traumatic injuries, ageing, or degradative diseases leads to impaired loading resistance and progressive degeneration of both the articular cartilage and the underlying subchondral bone. Since the tissue has limited self-repairing capacity due its avascular nature, restoration of its mechanical properties is still a major challenge. Tissue engineering techniques have the potential to heal osteochondral defects using a combination of stem cells, growth factors, and biomaterials that could produce a biomechanically functional tissue, representative of native hyaline cartilage. However, current clinical approaches fail to repair full-thickness defects that include the underlying subchondral bone. Moreover, when tested in vivo, current tissue-engineered grafts show limited capacity to regenerate the damaged tissue due to poor integration with host cartilage and the failure to retain structural integrity after insertion, resulting in reduced mechanical function. The aim of this review is to examine the optimal characteristics of osteochondral scaffolds. Additionally, an overview on the latest biomaterials potentially able to replicate the natural mechanical environment of articular cartilage and their role in maintaining mechanical cues to drive chondrogenesis will be detailed, as well as the overall mechanical performance of grafts engineered using different technologies.
... Panadero et al. have also reported similar strength for PCL based lyophilized scaffolds (~100 kPa at 12% strain). 29 Considering cyclic stress experienced by knee meniscus, dynamic mechanical properties have been studied for these scaffolds and compared with native human meniscus samples. Pereira et al. studied DMA in compression mode at frequency 0.1 to 10 Hz (Physiological condition -37 °C, pH 7.4) for 108 cylindrical human meniscal samples obtained from 15 donors. ...
We developed customizable biomolecule functionalized 3D poly-ε-caprolactone (PCL)scaffolds reinforced with carbon nanofibers (CNF) for human meniscal tissue engineering. 3D nanocomposite scaffolds exhibited commendable mechanical integrity and electrical properties with augmented cytocompatibility. Especially, the functionalized 3D (10wt%CNF) scaffolds showed ~363% increase in compressive moduli compared to the pristine PCL. In dynamic mechanical analysis, these scaffolds achieved highest value (~42MPa at 10Hz) among all tested scaffolds including pristine PCL and human menisci (33, 41, 56 years). In vitro results were well supported by the outcomes of cell proliferation analysis, microscopic images, Hoechst staining and extracellular-matrix estimation. Further, in vivo rabbit bio toxicity studies revealed, scaffolds non-toxicity and its future potential as a meniscus scaffold. This study also indicates that the incorporation of CNF in polymer matrix may be optimized based on mechanical properties of patient meniscus and it may help in developing the customized patient specific 3D constructs with improved multifunctional properties.
... Some preliminary works show that fatigue in scaffolds with in vitro generated ECM provides information that other common measurements miss. In a pioneering work [188], fatigue behavior is different for Poly-e-caprolactone (PCL) scaffolds with chondrogenic precursors seeded inside their pores -which produce ECM -that for the same scaffolds with other filler, despite that the initial elastic modulus is the same. It is not clear why this phenomenon happens, but it could be related with the boundaries between the filler, that can act also as different stress concentration points than other discontinuities. ...
Statement of significance:
It is well known the importance of biomechanical cues in chondrogenesis. This paper reviews the existing literature on the effect of mechanical stimulation on chondrogenic differentiation of mesenchymal stem cells in order to regenerate hyaline cartilage. Contradictory results found with respect to the effect of different modes of external loading can be explained by the different properties of the scaffolding system that holds the cells, which determine cell adhesion and morphology and spatial distribution of cells, as well as the stress transmission to the cells. Thus, this review seeks to provide an insight into the interplay between external loading program and scaffold properties during chondrogenic differentiation. The review of the literature reveals an important gap in the knowledge in this field and encourages new experimental studies. The main issue is that in each of the few cases in which the interplay is investigated, just two groups of scaffolds are compared, leaving intermediate adhesion conditions out of study. The authors propose broader studies implementing new high-throughput techniques for mechanical characterization of tissue engineering constructs and the inclusion of fatigue analysis as support methodology to more exhaustive mechanical characterization.
... The bioreactor was also evaluated under mechanical solicitation as described in (Panadero et al. 2015). Briefly, KUM5 mesenchymal cells were seeded in macroporous poly(εcaprolactone) scaffolds of the same dimensions-within experimental error- (Panadero et al. 2013), seeded in a fibrin suspension clotted inside the pores and cultured in chondrogenic differentiation medium for 2 weeks in static conditions. ...
Specific tissues, such as cartilage, undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation-amplitude and frequency-to three-dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.
Tissue engineering (TE) is a strongly expanding research area. TE approaches require biocompatible scaffolds, cells, and different applied stimuli, which altogether mimic the natural tissue microenvironment. Also, the extracellular matrix serves as a structural base for cells and as a source of growth factors and biophysical cues. The 3D characteristics of the microenvironment is one of the most recognized key factors for obtaining specific cell responses in vivo, being the physical cues increasingly investigated. Supporting those advances is the progress of smart and multifunctional materials design, whose properties improve the cell behavior control through the possibility of providing specific chemical and physical stimuli to the cellular environment. In this sense, a varying set of bioreactors that properly stimulate those materials and cells in vitro, creating an appropriate biomimetic microenvironment, is developed to obtain active bioreactors. This review provides a comprehensive overview on the important microenvironments of different cells and tissues, the smart materials type used for providing such microenvironments and the specific bioreactor technologies that allow subjecting the cells/tissues to the required biomimetic biochemical and biophysical cues. Further, it is shown that microfluidic bioreactors represent a growing and interesting field that hold great promise for achieving suitable TE strategies.
Graphene foam (GF), a 3‐dimensional derivative of graphene, has received much attention recently for applications in tissue engineering due to its unique mechanical, electrical, and thermal properties. Although GF is an appealing material for cartilage tissue engineering, the mechanical properties of GF‐tissue composites under dynamic compressive loads have not yet been reported. The objective of this study is to measure the elastic and viscoelastic properties of GF and GF‐tissue composites under unconfined compression when quasi‐static and dynamic loads are applied at strain magnitudes below 20%. The mechanical tests demonstrate a 46% increase in the elastic modulus and a 29% increase in the equilibrium modulus after 28‐days of cell culture as compared to GF soaked in tissue culture medium for 24 h. There is no significant difference in the amount of stress relaxation, however, the phase shift demonstrates a significant increase between pure GF and GF that has been soaked in tissue culture medium for 24 h. Furthermore, the authors have shown that ATDC5 chondrocyte progenitor cells are viable on graphene foam and have identified the cellular contribution to the mechanical strength and viscoelastic properties of GF‐tissue composites, with important implications for cartilage tissue engineering.
Understanding materials’ analytical techniques is a key feature to ensure efficient material development and to fully evaluate their functional properties. Biobased materials are emergent materials that are attracting attention due to their outstanding properties, pushing forward their use in a wide range of applications going from biomedical and pharmacy to even food industry, among others. This chapter provides an educational resource with the most relevant characterization techniques that are applicable to the development and characterization of biobased materials. It discusses physical, thermal, and chemical characterization techniques that are able to provide information related to the microstructure and morphology, and the mechanical, thermal, and chemical properties of materials. Additionally, it provides cues and considerations for the characterization techniques, complemented with relevant case studies and examples from different biobased materials.
A modification of the Morrow and the Smith, Watson and Topper (SWT) mean stress correction models is proposed to account for the mean stress effect on fatigue life. The capability and accuracy of the proposed model are compared to those of the original Morrow and the SWT model using published mean stress fatigue test data. The proposed mean stress correction model was found to be superior to both the SWT and the Morrow model in the case of the Incoloy 901 superalloy and the ASTM A723 steel. On the other hand both the proposed and the original SWT model provided equally good correlation with experimental data in the case of 7075-T561 aluminium alloy and 1045 HRC 55 steel. The Morrow model was found to give the least accurate predictions for all four materials analysed.
The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP) in regulating chondrogenesis of mesenchymal stem cells (MSCs) and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker) found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.
Low-cycle fatigue (LCF) data of Sn-Ag eutectic solder (96.5Sn-3.5Ag) under various temperatures and frequencies has been described
using three different prediction models, i.e., Coffin-Manson model, Smith-Watson-Topper (SWT) model, and Morrow energy model.
The LCF behavior represented by the present prediction models showed temperature and frequency dependences, i.e., the fatigue
ductility coefficient increased with increasing frequency and decreasing temperature. In order to better correlate the LCF
data, a flow stress and/or frequency-dependent modifications were introduced to the Coffin-Manson and Morrow energy models.
The frequency-modified Coffin-Manson model could not describe the influence of temperature on LCF behavior, while the flow
stress-modified frequency-modified Morrow energy model, into which the metallurgical response (flow stress and frequency)
was introduced to account for the effect of temperature and frequency on LCF behavior, gave reasonable predictions of LCF
data under various temperatures and frequencies.
In vitro culturing and mechanical properties of three types of three-dimensional poly(caprolactone) scaffolds with interconnecting open-foam networks are reported. The scaffolds targeted bone tissue regeneration and were fabricated using twin screw extrusion and coextrusion techniques, for continuous mixing/shaping and formation of single or multilayers with distinct and tailorable porosities and pore sizes. Human fetal preosteoblastic cells, hFOB, were cultured on the extruded and coextruded scaffolds under osteogenic supplements and the samples of the resulting tissue constructs were removed and characterized for cell viability and proliferation using the MTS assay, differentiation, and mineralized matrix synthesis via the alkaline phosphatase, ALP, activity and Alizarin Red staining and cell migration using confocal microscopy and scanning electron microscopy. The hFOB cells formed a confluent lining on scaffold surfaces, migrated to the interior and generated abundant extracellular matrix after 2 weeks of culturing, indicative of the promise of such scaffolds for utilization in tissue engineering. The scaffolds and tissue constructs exhibited compressive fatigue behavior that was similar to that of cancellous bone, suggesting the suitability of their use as bone graft substitutes especially for repair of critical-sized defects or nonunion fractures.
Two series of 3D scaffolds based on ε-caprolactone were synthesized. The pore size and architecture (spherical interconnected pores) was the same in all the scaffolds. In one of the series of scaffolds, made of pure ε-polycaprolactone, the volume fraction of pores varied between 60% and 85% with the main consequence of varying the interconnectivity between pores since the pore size was kept constant. The other scaffolds were prepared with copolymers made of a ε-caprolactone-based hydrophobous monomer and hydroxyethyl acrylate, as the hydrophilic component. Thus, the hydrophilicity and, presumably, the adhesion properties varied monotonously in the copolymer series while porosity was kept constant. A suspension of human chondrocytes in culture medium was injected in the 3D scaffolds and cultured in static conditions up to 28 days. SEM and immunofluorescence assays allowed characterizing cells and extracellular matrix inside the scaffolds after different culture times. To do that, cross sections of the scaffolds were observed by SEM and confocal microscopy. The quantity of cells inside the scaffolds decreases with a decrease of the volume fraction of pores, due to the lack of interconnectivity between the cavities. The scaffolds up to a 30% of hydrophilicity behave in a similar way than the hydrophobous; a further increase of the hydrophilicity rapidly decreases cell viability. In all the experiments production of collagen type I, type II, and aggrecan was found, and some cells were Ki-67 positive, showing that some cells are adhered to the pore walls and maintain their dedifferentiated phenotype even when cultured in three-dimensional conditions.
Cells interact with the surrounding environment by making tens to hundreds of thousands of nanoscale interactions with extracellular signals and features. The goal of nanoscale tissue engineering is to harness these interactions through nanoscale biomaterials engineering in order to study and direct cellular behavior. Here, we review two- and three-dimensional (2- and 3D) nanoscale tissue engineering technologies, and provide a holistic overview of the field. Techniques that can control the average spacing and clustering of cell adhesion ligands are well established and have been highly successful in describing cell adhesion and migration in 2D. Extension of these engineering tools to 3D biomaterials has created many new hydrogel and nanofiber scaffold technologies that are being used to design in vitro experiments with more physiologically relevant conditions. Researchers are beginning to study complex cell functions in 3D. However, there is a need for biomaterials systems that provide fine control over the nanoscale presentation of bioactive ligands in 3D. Additionally, there is a need for 2- and 3D techniques that can control the nanoscale presentation of multiple bioactive ligands and that can control the temporal changes in the cellular microenvironment.
This report describes a new method for simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. The method is based on the use of a reagent containing phenol and guanidine thiocyanate. A biological sample is homogenized in the reagent and the simultaneous isolation of RNA, DNA and proteins is accomplished in a single step by a liquid-phase separation. The isolation of RNA can be completed in about 1 h, and DNA and proteins in about 3 h. The simultaneously isolated RNA, DNA and proteins are ready for Northern, Southern and Western blotting. The complete recovery of DNA from samples used for the RNA and protein isolation makes it possible to normalize the results of gene expression studies based on DNA content instead of on the more variable total RNA, protein content or tissue weight.
Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and submitted to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long-term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behavior of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow's criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2014.
Fibrin is a protein that can be used as an ideal scaffolding material to promote tissue regeneration. In order to enhance its physical properties in the current study a natural crosslinker, genipin (GP), was employed with the aim to obtain a hydrogel with tuneable properties for tissue engineering applications. The fibrin gel was crosslinked by two different methods using four concentrations of GP to get a stable hydrogel network. Crosslinking density, mechanical properties, swelling and enzymatic degradation of the hydrogels were tested for each GP content and method employed. The method I: crosslinking after gel formation promotes a high crosslinking and retains the gel shape for long term whilst the method II: simultaneous gel formation and crosslinking improves the mechanical properties of the gel. This study confirms the use of GP at different concentrations as a suitable crosslinker of fibrin that promotes the cellular viability of L929 for 21 days of in vitro culture.
Polycaprolactone scaffolds modified with cross-linked hyaluronic acid were prepared in order to establish whether a more hydrophilic and biomimetic microenvironment benefits the progenitor cells arriving from bone marrow in a cell-free tissue-engineering approach. The polycaprolactone and polycaprolactone/hyaluronic acid scaffolds were characterized in terms of morphology and water absorption capacity. The polycaprolactone and polycaprolactone/hyaluronic acid samples were implanted in a chondral defect in rabbits; bleeding of the subchondral bone was provoked to generate a spontaneous healing response. Repair at 1, 4, 12, and 24 weeks was assessed macroscopically using the International Cartilage Repair Society score and the Oswestry Arthroscopy Score and microscopically using immunohistological staining for collagen type I and type II, and for Ki-67. The presence of hyaluronic acid improves scaffold performance, which supports a good repair response without biomaterial pre-seeding.
Tissue engineering applications rely on scaffolds that during its service life, either for in-vivo or in vitro applications, are under loading. The variation of the mechanical condition of the scaffold is strongly relevant for cell culture and has scarcely been addressed. The fatigue life cycle of poly-ε-caprolactone, PCL, scaffolds with and without fibrin as filler of the pore structure were characterized both dry and immersed in liquid water. It is observed that the there is a strong increase from 100 to 500 in the number of loading cycles before collapse in the samples tested in immersed conditions due to the more uniform stress distributions within the samples, the fibrin loading playing a minor role in the mechanical performance of the scaffolds.
A hybrid scaffold was obtained by the deposition of a thin network of submicron fibrin fibrils on the microporous walls of a macroporous poly(L-lactide) (PLLA) three-dimensional structure. The fibrin coating is homogeneous across the entire substrate and allowed the pore structure remain open in the hybrid scaffold. The elastic modulus of the hybrid scaffold (0.65 MPa) was increased up to twofold compared to the pure PLLA scaffold (0.29 MPa). Mouse pre-osteoblastic cells, MC3T3, were seeded on both pure PLLA and hybrid scaffolds, and cultured for 3, 6, and 24 h. The coating enhanced the cell colonization and proliferation and provided a more homogeneous distribution of cells within the scaffolds. In addition, the coating improved the scaffold adhesion properties by supplying new binding sites to the cells that modify the transmembrane receptors involved in initial cell adhesion mechanism. The expression of the β3 integrin was observed in cells cultured on fibrin-coated scaffolds instead of the α5 integrin, which was expressed in the uncoated scaffold. These hybrid PLLA/fibrin scaffolds have cell culture features suitable to promote early tissue regeneration.
Poly(l-lactide)/hydroxyapatite, PLLA/HA, composite membranes for bone regeneration with different concentrations of nanoparticles have been prepared and their physicochemical properties and bioactivity have been determined. Hydroxyapatite nanoparticles act as nucleating agent of the poly(l-lactide) crystals, as detected by DSC, and as reinforcing filler, as proven by the monotonous increase of the elastic modulus of the microporous membranes with increasing nano-filler content. The bioactivity, which regards to the use of these materials in bone regeneration, was tested by immersing the samples in a simulated body fluid, SBF. A faster deposition of a biomimetic apatite layer was observed as increases the content of hydroxyapatite nanoparticles, thus membranes with a 15% (w/w) of hydroxyapatite particles (relative to PLLA weight) present a homogeneous layer of hydroxyapatite on the surface of their pores after 7days of immersion in SBF. An especial emphasis has been made on the influence of a plasma treatment on the bioactivity of the membranes. With this aim, the membranes were submitted to a plasma treatment previously to their immersion in a simulated body fluid. It has been observed that the surface of a PLLA membrane after 21days of immersion in SBF is still not completely covered by hydroxyapatite whereas the same sample treated with plasma show a smooth layer of biomimetic hydroxyapatite. The increase of bioactivity achieved with this treatment was less important in high hydroxyapatite content composites.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Composites fabricated by biomimetic mineral precipitation on polymeric substrates are of interest for tissue engineering. As biological properties of such mineral layers vary with slight changes in composition, a good physical characterization is necessary in order to study their biological activity. In this work polycaprolactone sheets were subjected to air plasma treatment followed by nucleation of calcium phosphate seeds to activate the growth of an apatite-like coating when immersing in simulated body fluid. Two compositions of the SBF were prepared, one of them highly carbonated and the other with no carbonate or magnesium ions. Immersion of PCL in the high carbonate composition produced a low-crystallinity apatite-like layer while the absence of carbonate and magnesium ions yielded a high crystallinity apatite with low Ca/P ratio that is likely partially hydrolyzed octacalciumphosphate (OCP). The morphology, crystal structure and composition of both types of coatings were characterized; osteoblast-like cell adhesion behaviour on different surfaces was observed by fluorescence and electron microscopy.
Many factors are known to influence the mechanical fatigue life of rubber components. Four major categories of factors are reviewed here: the effects of mechanical loading history, environmental effects, effects of rubber formulation, and effects due to dissipative aspects of the constitutive response of rubber. For each category, primary factors are described, and existing literature is presented and reviewed. Rubber's fatigue behavior is extremely sensitive to both the maximum and minimum cyclic load limits. Other aspects of the mechanical load history are also discussed, including the effects of static loaded periods ("annealing"), load sequence, multiaxiality, frequency, and loading waveform. Environmental factors can affect both the short and long term fatigue behavior of rubber. The effects of temperature, oxy-gen, ozone, and static electrical charges are reviewed. A great range of behavior is available by proper manipulation of formulation and processing variables. Effects of elastomer type, filler type and volume fraction, antidegradants, curatives, and vulcanization are discussed. The role of dissipative constitutive behavior in the improvement of fatigue properties of rubber is also reviewed. Four distinct dissipative mechanisms are identified, and their effects on fatigue behavior are described.
Both hydrostatic pressure (HP) and cell-matrix interactions have independently been shown to regulate the chondrogenic differentiation of mesenchymal stem cells (MSCs). The objective of this study was to test the hypothesis that the response of MSCs to hydrostatic pressure will depend on the biomaterial within which the cells are encapsulated. Bone-marrow-derived MSCs were seeded into either agarose or fibrin hydrogels and exposed to 10 MPa of cyclic HP (1 Hz, 4 h per day, 5 days per week for 3 weeks) in the presence of either 1 or 10 ng ml(-1) of TGF-β3. Agarose hydrogels were found to support a spherical cellular morphology, while MSCs seeded into fibrin hydrogels attached and spread, with clear stress fiber formation. Hydrogel contraction was also observed in MSC-fibrin constructs. While agarose hydrogels better supported chondrogenesis of MSCs, HP only enhanced sulfated glycosaminoglycan (sGAG) accumulation in fibrin hydrogels, which correlated with a reduction in fibrin contraction. HP also reduced alkaline phosphatase activity in the media for both agarose and fibrin constructs, suggesting that this stimulus plays a role in the maintenance of the chondrogenic phenotype. This study demonstrates that a complex relationship exists between cell-matrix interactions and hydrostatic pressure, which plays a key role in regulating the chondrogenic differentiation of MSCs.
The objective of this study was to determine cartilage strains near, and in apposition to, a focal defect during patello-femoral articulation. Bovine osteochondral blocks from the trochlea (TRO) and patella (PAT) were apposed, compressed 12%, and subjected to sliding under video microscopy. Samples, lubricated with synovial fluid, were tested intact and then with a full-thickness defect in PAT cartilage. Shear (E(xz)), axial (E(zz)), and lateral (E(xx)) strains were determined locally for TRO and PAT cartilage. For articulation with a focal defect, the strain amplitudes of PAT cartilage near the surface were ∼2-8× lower in E(xz) and ∼1.4× higher in -E(zz) than intact PAT cartilage. At 20% depth, E(xz) and E(xx) for PAT cartilage with a focal defect were ∼2× and ∼10-25× higher than intact PAT, respectively. For TRO articulating against a focal defect, E(xz) and -E(zz) near the surface and at 20% depth were ∼2-4× lower than that for articulation against intact cartilage. The results elucidate dramatic region-specific changes in strain due to lateral motion. In these regions, such altered cartilage mechanics during knee movement may cause focal defects to extend by induction of damaging levels of strain to bordering regions of cartilage.
Cartilage tissue engineering using synthetic scaffolds allows maintaining mechanical integrity and withstanding stress loads in the body, as well as providing a temporary substrate to which transplanted cells can adhere.
This study evaluates the use of polycaprolactone (PCL) scaffolds for the regeneration of articular cartilage in a rabbit model.
Controlled laboratory study.
Five conditions were tested to attempt cartilage repair. To compare spontaneous healing (from subchondral plate bleeding) and healing due to tissue engineering, the experiment considered the use of osteochondral defects (to allow blood flow into the defect site) alone or filled with bare PCL scaffold and the use of PCL-chondrocytes constructs in chondral defects. For the latter condition, 1 series of PCL scaffolds was seeded in vitro with rabbit chondrocytes for 7 days and the cell/scaffold constructs were transplanted into rabbits' articular defects, avoiding compromising the subchondral bone. Cell pellets and bare scaffolds were implanted as controls in a chondral defect.
After 3 months with PCL scaffolds or cells/PCL constructs, defects were filled with white cartilaginous tissue; integration into the surrounding native cartilage was much better than control (cell pellet). The engineered constructs showed histologically good integration to the subchondral bone and surrounding cartilage with accumulation of extracellular matrix including type II collagen and glycosaminoglycan. The elastic modulus measured in the zone of the defect with the PCL/cells constructs was very similar to that of native cartilage, while that of the pellet-repaired cartilage was much smaller than native cartilage.
The results are quite promising with respect to the use of PCL scaffolds as aids for the regeneration of articular cartilage using tissue engineering techniques.
In recent years, there has been a great deal of interest in the development of regenerative approaches to produce hyaline cartilage ex vivo that can be utilized for the repair or replacement of damaged or diseased tissue. It is clinically imperative that cartilage engineered in vitro mimics the molecular composition and organization of and exhibits biomechanical properties similar to persistent hyaline cartilage in vivo. Experimentally, much of our current knowledge pertaining to the regulation of cartilage formation, or chondrogenesis, has been acquired in vitro utilizing high-density cultures of undifferentiated chondroprogenitor cells stimulated to differentiate into chondrocytes. In this review, we describe the extracellular matrix molecules, nuclear transcription factors, cytoplasmic protein kinases, cytoskeletal components, and plasma membrane receptors that characterize cells undergoing chondrogenesis in vitro and regulate the progression of these cells through the chondrogenic differentiation program. We also provide an extensive list of growth factors and other extracellular signaling molecules, as well as chromatin remodeling proteins such as histone deacetylases, known to regulate chondrogenic differentiation in culture. In addition, we selectively highlight experiments that demonstrate how an understanding of normal hyaline cartilage formation can lead to the development of novel cartilage tissue engineering strategies. Finally, we present directions for future studies that may yield information applicable to the in vitro generation of hyaline cartilage that more closely resembles native tissue.
In this review, we outline seminal and recent work highlighting the potential of mesenchymal stem cells (MSCs) in producing cartilage-like tissue equivalents. Specific focus is placed on the mechanical properties of engineered MSC-based cartilage and how these properties relate to that of engineered cartilage based on primary chondrocytes and to native tissue properties. We discuss current limitations and/or concerns that must be addressed for the clinical realization of MSC-based cartilage therapeutics, and provide some insight into potential underpinnings for the observed deviations from chondrocyte-based engineered constructs. We posit that these differences reveal specific deficits in terms of our description of chondrogenesis, and suggest that new benchmarks must be developed towards this end. Further, we describe the growing body of literature on the mechanobiology of MSC-based cartilage, highlighting positive findings with regards to the furtherance of the chondrogenic phenotype. We likewise discuss the failure of early molecular changes to translate directly into engineered constructs with improved mechanical properties. Finally, we highlight recent work from our group and others that may point to new strategies for enhancing the formation of engineered cartilage based on MSCs.
Cartilage repair is a very successful pioneering area of regenerative medicine in which techniques of in situ regeneration and cell and tissue transplantation dominate over cell-free approaches to generate durable neocartilage. This review concentrates on advantages and limitations of mesenchymal stem cell (MSC)-based cartilage repair strategies induced by marrow stimulation. Detailed knowledge on the biology of MSC will be discussed in light of the requirements for MSC recruitment, retention, proliferation and chondrogenic differentiation. An improved microenvironment with timely correlated signals from biomaterials, growth factors, proteases, adjacent cartilage and subchondral bone may be key to a third generation of techniques to regenerate hyaline cartilage.
In this study, human bone marrow mesenchymal stem cell (hMSC)-seeded fibrin-polyurethane composite scaffolds were subjected to various mechanical load protocols to determine the effect of compression and surface rotation frequency and axial compression magnitude on the induction of chondrocyte-specific gene expression and protein synthesis. After 7 days of preculture and 7 days of load, application of dynamic compression and surface shear 1 h daily enhanced chondrogenesis of hMSCs compared with the nonloaded control samples. Higher load frequency and higher compression amplitude induced higher glycosaminoglycan synthesis, higher chondrocytic gene expression, higher chondrocytic:fibroblastic, chondrocytic:hypertrophic, and chondrocytic:osteoblastic gene expression ratios, as well as higher transforming growth factor beta1 (TGFB1) and TGFB3 gene expression. The chondrogenesis level of hMSCs was positively related to the TGFB1 and TGFB3 gene expression level, which was determined by various load regimes. In conclusion, chondrogenesis of human MSCs induced by mechanical stimulation can be further enhanced by modifying frequency and compression.
Polymer-ceramic composites are favourite candidates when aiming to replace bone tissue. We present here scaffolds made of polycaprolactone-hydroxyapatite (PCL-HAp) composites, and investigate in vitro mineralisation of the scaffolds in SBF after or without a nucleation treatment. In vitro bioactivity is enhanced by HAp incorporation as well as by nucleation treatment, as demonstrated by simulated body fluid (SBF) mineralization. Surprisingly, we obtained a hybrid interconnected organic-inorganic structure, as a result of micropore invasion by biomimetic apatite, which results in a mechanical strengthening of the material after two weeks of immersion in SBF92. The presented scaffolds, due to their multiple qualities, are expected to be valuable supports for bone tissue engineering.
The objective of this study was to investigate the influence of dynamic compressive loading on chondrogenesis of mesenchymal stem cells (MSCs) in the presence of TGF-beta3. Isolated porcine MSCs were suspended in 2% agarose and subjected to intermittent dynamic compression (10% strain) for a period of 42 days in a dynamic compression bioreactor. After 42 days in culture, the free-swelling specimens exhibited more intense alcian blue staining for proteoglycans, while immunohistochemical analysis revealed increased collagen type II immunoreactivity. Glycosaminoglycan (GAG) content increased with time for both free-swelling and dynamically loaded constructs, and by day 42 it was significantly higher in both the core (2.5+/-0.21%w/w vs. 0.94+/-0.03%w/w) and annulus (1.09+/-0.09%w/w vs. 0.59+/-0.08%w/w) of free-swelling constructs compared to dynamically loaded constructs. This result suggests that further optimization is required in controlling the biomechanical and/or the biochemical environment if such stimuli are to have beneficial effects in generating functional cartilaginous tissue.
Osteoarthritis (OA) resulting from trauma, degenerative or age-related disease presents a major clinical challenge due to the limited repair capacity of articular cartilage. This poor self-repair capacity of osteochondral defects has resulted in the development of a wide variety of new treatment approaches. Although the use of chondrocytes in applications of cartilage tissue engineering is still prevalent, concerns associated with donor-site morbidity, cell de-differentiation and the limited lifespan of these cells have brought the use of mesenchymal stem cells (MSCs) to the forefront of such applications. Therefore, in the last two decades MSCs have come into the focus of connective tissue engineering and regenerative medicine and have become increasingly sought after as an alternative cell source for improving well-established methods of osteochondrotic cartilage defect repair such as the Autologous Chondrocyte Transplantation method, but are also being tested as an ideal cell source in combination with newly developed implantable scaffolds or as a target/carrier cell in other new concepts of regenerative medicine. However, up to now, although in animal models MSCs have already shown significant potential for cartilage repair and novel approaches using MSCs as an alternative cell source to patient-derived chondrocytes are being tested, much more research is needed before feasible clinical application of MSCs becomes reality.
It is well accepted that mechanical forces can modulate the metabolic activity of chondrocytes, although the specific mechanisms of mechanical signal transduction in articular cartilage are still unknown. One proposed pathway through which chondrocytes may perceive changes in their mechanical environment is directly through cellular deformation. An important step toward understanding the role of chondrocyte deformation in signal transduction is to determine the changes in the shape and volume of chondrocytes during applied compression of the tissue. Recently, a technique was developed for quantitative morphometry of viable chondrocytes within the extracellular matrix using three-dimensional confocal scanning laser microscopy. In the present study, this method was used to quantify changes in chondrocyte morphology and local tissue deformation in the surface, middle, and deep zones in explants of canine articular cartilage subjected to physiological levels of matrix deformation. The results indicated that at 15% surface-to-surface equilibrium strain in the tissue, a similar magnitude of local tissue strain occurs in the middle and deep zones. In the surface zone, local strains of 19% were observed, indicating that the compressive stiffness of the surface zone is significantly less than that of the middle and deep zones. With this degree of tissue deformation, significant decreases in cellular height of 26, 19, and 20% and in cell volume of 22, 16, and 17% were observed in the surface, middle, and deep zones, respectively. The deformation of chondrocytes in the surface zone was anisotropic, with significant lateral expansion occurring in the direction perpendicular to the local split-line pattern. When compression was removed, there was complete recovery of cellular morphology in all cases. These observations support the hypothesis that deformation of chondrocytes or a change in their volume may occur during in vivo joint loading and may have a role in the mechanical signal transduction pathway of articular cartilage.
Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage.
The applications of a wide range of hydrogels in tissue engineering were presented. The design parameters of hydrogels required for them to be useful, regardless of their origin from natural resources or synthetic creation were discussed. The use of polymers in promoting blood vessel network formation in the tissue was described. The problems encountered in the design and engineering of various sequences of polypeptides with known functions were also studied.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
The histomorphogenesis of articular cartilage is regulated during skeletal development by the intermittent forces and motions imposed at diarthrodial joints. A key feature in this development is the formation of the superficial, transitional, radial, and calcified cartilage zones through the cartilage thickness. The histomorphological, biological, and mechanical characteristics of these zones can be correlated with the distributions of pressures, deformations, and pressure-induced fluid flow that are created in vivo. In a mature joint, cyclic loads produce cyclic hydrostatic fluid pressure through the entire cartilage thickness that is comparable in magnitude to the applied joint pressure. Prolonged physical activity can cause the total cartilage thickness to decrease about 5%, although the consolidation strains vary tremendously in the different zones. The superficial zone can experience significant fluid exudation and consolidation (compressive strains) in the range of 60% while the radial zone experiences relatively little fluid flow and consolidation. The topological variation in the histomorphologic appearance of articular cartilage is influenced by the local mechanical loading of chondrocytes in the different zones. Patterns of stress, strain, and fluid flow created in the joint result in spatial and temporal changes in the rates of synthesis and degradation of matrix proteins. When viewed over the course of a lifetime, even subtle difference in these cellular processes can affect the micro- and macro-morphology of articular cartilage. This hypothesis is supported by in vivo and ex vivo experiments where load-induced changes in matrix synthesis and catabolism, gene expression, and signal transduction pathways have been observed.
Recent works have shown that mechanical loading can alter the metabolic activity of chondrocytes cultured in 3D scaffolds. In this study we determined whether the stage of development of engineered cartilaginous constructs (expanded adult human articular chondrocytes/Polyactive foams) regulates the effect of dynamic compression on glycosaminoglycan (GAG) metabolism. Construct maturation depended on the culture time (3-14 days) and the donor (4 individuals). When dynamic compression was subsequently applied for 3 days, changes in GAG synthesized, accumulated, and released were significantly positively correlated to the GAG content of the constructs prior to loading, and resulted in stimulation of GAG formation only in the most developed tissues. Conversely, none of these changes were correlated with the expression of collagen type II mRNA, indicating that the response of chondrocytes to dynamic compression does not depend directly upon the stage of cell differentiation, but rather on the extracellular matrix surrounding the cells.
What is it that defines a bone marrow-derived chondrocyte? We attempted to identify marrow-derived cells with chondrogenic nature and immortality without transformation, defining "immortality" simply as indefinite cell division. KUM5 mesenchymal cells, a marrow stromal cell line, generated hyaline cartilage in vivo and exhibited enchondral ossification at a later stage after implantation. Selection of KUM5 chondroblasts based on the activity of the chondrocyte-specific cis-regulatory element of the collagen alpha2(XI) gene resulted in enhancement of their chondrogenic nature. Gene chip analysis revealed that OP9 cells, another marrow stromal cell line, derived from macrophage colony-stimulating factor-deficient osteopetrotic mice and also known to be niche-constituting cells for hematopoietic stem cells expressed chondrocyte-specific or -associated genes such as type II collagen alpha1, Sox9, and cartilage oligomeric matrix protein at an extremely high level, as did KUM5 cells. After cultured OP9 micromasses exposed to TGF-beta3 and BMP2 were implanted in mice, they produced abundant metachromatic matrix with the toluidine blue stain and formed type II collagen-positive hyaline cartilage within 2 weeks in vivo. Hierarchical clustering and principal component analysis based on microarray data of the expression of cell surface markers and cell-type-specific genes resulted in grouping of KUM5 and OP9 cells into the same subcategory of "chondroblast," that is, a distinct cell type group. We here show that these two cell lines exhibit the unique characteristics of hyaline cartilage formation and enchondral ossification in vitro and in vivo.
Damage to and degeneration of articular cartilage is a major health issue in industrialized nations. Articular cartilage has a particularly limited capacity for auto regeneration. At present, there is no established therapy for a sufficiently reliable and durable replacement of damaged articular cartilage. In this, as well as in other areas of regenerative medicine, tissue engineering methods are considered to be a promising therapeutic component. Nevertheless, there remain obstacles to the establishment of tissue-engineered cartilage as a part of the routine therapy for cartilage defects. One necessary aspect of potential tissue engineering-based therapies for cartilage damage that requires both elucidation and progress toward practical solutions is the reliable, cost effective cultivation of suitable tissue. Bioreactors and associated methods and equipment are the tools with which it is hoped that such a supply of tissue-engineered cartilage can be provided. The fact that in vivo adaptive physical stimulation influences chondrocyte function by affecting mechanotransduction leads to the development of specifically designed bioreactor devices that transmit forces like shear, hydrostatic pressure, compression, and combinations thereof to articular and artificial cartilage in vitro. This review summarizes the basic knowledge of chondrocyte biology and cartilage dynamics together with the exploration of the various biophysical principles of cause and effect that have been integrated into bioreactor systems for the cultivation and stimulation of chondrocytes.
Internal Friction Damping and Cyclic Plasticity. Philadelphia: ASTM-STP 378l
- Morrowjd
Morrow JD. Internal Friction, Damping, and Cyclic Plasticity. Philadelphia: ASTM-STP 378l 1965.
Submitted for publication
J Biomed Mater Res B Appl Biomater 2014. Submitted for publication. DOI: 10.1002/jbm.b.33276.