Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition

We investigated whether the post-expansion redifferentiation and cartilage tissue formation capacity of adult human nasal chondrocytes can be regulated by controlled modifications of scaffold composition and architecture. As a model system, we used poly(ethylene glycol)-terephthalate-poly(butylene)-terephthalate block copolymer scaffolds from two compositions (low or high PEG content, resulting in different wettability) and two architectures (generated by compression molding or three-dimensional (3D) fiber deposition) with similar porosity and mechanical properties, but different interconnecting pore architectures. Scaffolds were seeded with expanded human chondrocytes and the resulting constructs assessed immunohistochemically, biochemically and at the mRNA expression level following up to 4 weeks of static culture. For a given 3D architecture, the more hydrophilic scaffold enhanced cell redifferentiation and cartilaginous tissue formation after 4 weeks culture, as assessed by higher mRNA expression of collagen type II, increased deposition of glycosaminoglycan (GAG) and predominance of type II over type I collagen immunostain. The fiber-deposited scaffolds, with a more accessible pore volume and larger interconnecting pores, supported increased GAG deposition, but only if a more hydrophilic composition was used. By applying controlled and selective modifications of chemico-physical scaffold parameters, we demonstrate that both scaffold composition and architecture are instructive for expanded human chondrocytes in the generation of 3D cartilaginous tissues. The observed effects of composition and architecture were likely to have been mediated, respectively, by differential serum protein adsorption and efficiency of nutrient/waste exchange.     

The influence of scaffold architecture on chondrocyte distribution and behavior in matrix-associated chondrocyte transplantation grafts

Scaffold architecture and composition are important parameters in cartilage tissue engineering. In this in vitro study, we compared the morphology of four different cell-graft systems applied in clinical cartilage regeneration and analyzed the cell distribution (DAPI nuclei staining) and cell-scaffold interaction (SEM, TEM). Our investigations revealed major differences in cell distribution related to scaffold density, pore size and architecture. Material composition influenced the quantity of autogenous matrix used for cellular adhesion. Cell bonding was further influenced by the geometry of the scaffold subunits. On scaffolds with widely spaced fibers and a thickness less than the cell diameter, chondrocytes surrounded the scaffold fibers with cell extensions. On those fibers, chondrocytes were spherical, suggesting a differentiated phenotype. Fiber sizes smaller than chondrocyte size, and widely spaced, are therefore beneficial in terms of improved adhesion by cell shape adaptation. They also support the differentiated stage of chondrocytes by preventing the fibroblast-like and polygonal cell shape, at least briefly.     

The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage

A highly interconnecting and accessible pore network has been suggested as one of a number of prerequisites in the design of scaffolds for tissue engineering. In the present study, two processing techniques, compression-molding/particulate-leaching (CM), and 3D fiber deposition (3DF), were used to develop porous scaffolds from biodegradable poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) co-polymers with varying pore architectures. Three-dimensional micro-computed tomography (microCT) was used to characterize scaffold architectures and scaffolds were seeded with articular chondrocytes to evaluate tissue formation. Scaffold porosity ranged between 75% and 80%. Average pore size of tortuous CM scaffolds (182 microm) was lower than those of organized 3DF scaffolds (525 microm). The weight ratio of glycosaminoglycans (GAG)/DNA, as a measure of cartilage-like tissue formation, did not change after 14 days of culture whereas, following subcutaneous implantation, GAG/DNA increased significantly and was significantly higher in 3DF constructs than in CM constructs, whilst collagen type II was present within both constructs. In conclusion, 3DF PEGT/PBT scaffolds create an environment in vivo that enhances cartilaginous matrix deposition and hold particular promise for treatment of articular cartilage defects.     

Engineered cellular response to scaffold architecture in a rabbit trephine defect

Tight control of pore architecture in porous scaffolds for bone repair is critical for a fully elucidated tissue response. Solid freeform fabrication (SFF) enables construction of scaffolds with tightly controlled pore architecture. Four types of porous scaffolds were constructed using SFF and evaluated in an 8-mm rabbit trephine defect at 8 and 16 weeks (n = 6): a lactide/glycolide (50:50) copolymer scaffold with 20% w/w tri-calcium phosphate and random porous architecture (Group 1); another identical design made from poly(desaminotyrosyl-tyrosine ethyl ester carbonate) [poly(DTE carbonate)], a tyrosine-derived pseudo-polyamino acid (Group 2); and two poly(DTE carbonate) scaffolds containing 500 microm pores separated by 500-microm thick walls, one type with solid walls (Group 3), and one type with microporous walls (Group 4). A commercially available coralline scaffold (Interpore) with a 486-microm average pore size and empty defects were used as controls. There was no significant difference in the overall amount of bone ingrowth in any of the devices, as found by radiographic analysis, but patterns of bone formation matched the morphology of the scaffold. These results suggest that controlled scaffold architecture can be superimposed on biomaterial composition to design and construct scaffolds with improved fill time.     

The effect of mechanical stimulation on the maturation of TDSCs-poly(L-lactide-co-e-caprolactone)/collagen scaffold constructs for tendon tissue engineering

Mechanical stimulation plays an important role in the development and remodeling of tendons. Tendon-derived stem cells (TDSCs) are an attractive cell source for tendon injury and tendon tissue engineering. However, these cells have not yet been fully explored for tendon tissue engineering application, and there is also lack of understanding to the effect of mechanical stimulation on the maturation of TDSCs-scaffold construct for tendon tissue engineering. In this study, we assessed the efficacy of TDSCs in a poly(L-lactide-co-ε-caprolactone)/collagen (P(LLA-CL)/Col) scaffold under mechanical stimulation for tendon tissue engineering both in vitro and in vivo, and evaluated the utility of the transplanted TDSCs-scaffold construct to promote rabbit patellar tendon defect regeneration. TDSCs displayed good proliferation and positive expressed tendon-related extracellular matrix (ECM) genes and proteins under mechanical stimulation in vitro. After implanting into the nude mice, the fluorescence imaging indicated that TDSCs had long-term survival, and the macroscopic evaluation, histology and immunohistochemistry examinations showed high-quality neo-tendon formation under mechanical stimulation in vivo. Furthermore, the histology, immunohistochemistry, collagen content assay and biomechanical testing data indicated that dynamically cultured TDSCs-scaffold construct could significantly contributed to tendon regeneration in a rabbit patellar tendon window defect model. TDSCs have significant potential to be used as seeded cells in the development of tissue-engineered tendons, which can be successfully fabricated through seeding of TDSCs in a P(LLA-CL)/Col scaffold followed by mechanical stimulation.     

Respiratory response to mechanical stimulation of the gallbladder

Since stimuli from abdominal or pelvic viscera can affect respiratory muscle function, we hypothesized that mechanical stimulation of the gallbladder would result in inhibition of motor activity to the diaphragm and to upper airway muscles. We studied 12 decerebrate, vagotomized, paralyzed, artificially ventilated cats and recorded hypoglossal (HG) and phrenic (PHR) nerve activities while applying 600-1000 g of traction on the gallbladder during four respiratory cycles. Traction resulted in an initial reduction of PHR activity to 87.6+/-15.0% (mean+/-S.D.% of its baseline value), a reduction of HG activity to 74.2+/-27.5% and a lengthening of expiratory time to 178.8+/-81.0%. Subsequently, PHR activity and expiratory time returned toward control values, while HG remained diminished, at 66.4+/-19.1%. Our results show that mechanical stimulation of the gallbladder results in a respiratory inhibition with a disproportionate reduction in HG activity relative to PHR discharge. We speculate that gallbladder stimulation by contractions or surgery may compromise breathing by inhibition of phrenic discharge and upper airway obstruction.     

The effect of scaffold architecture on odontogenic differentiation of human dental pulp stem cells

Previous studies have shown the superiority of nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffolds in supporting the osteogenic differentiation of a few cell types and bone regeneration. The aim of the current study was to investigate whether NF-PLLA scaffolds are advantageous for the odontogenic differentiation and mineralization of human dental pulp stem cells (DPSCs) over solid-walled (SW) PLLA scaffolds. The in?vitro studies demonstrated that, compared with SW scaffolds, NF scaffolds enhanced attachment and proliferation as well as odontogenic differentiation of human DPSCs. The alkaline phosphatase (ALP) activity and the expression of odontogenic genes of human DPSCs were increased on NF scaffolds compared with that on SW scaffolds. In addition, more mineral deposition was observed on the NF scaffolds, as demonstrated by von Kossa staining, calcium content measurement and scanning electron microscopy. Consistent with the in?vitro studies, NF scaffolds promoted odontogenic differentiation and hard tissue formation compared with SW scaffolds after 8 weeks of ectopic transplantation in nude mice, as confirmed by von Kossa staining, Masson's trichrome staining and immunohistochemical staining for dentin sialoprotein. In conclusion, NF-PLLA scaffolds enhanced the odontogenic differentiation of human DPSCs and mineralization both in?vitro and in?vivo, and are promising scaffolds for dentin regeneration.     

Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture

Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical deformation and recovery. This loading is essential for chondrocytes to perform their normal function of maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional (3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected by culture. Further experiments were performed to test the effects of load on the cytoskeleton. Explants were placed in culture and immediately subjected to static uniaxial radially unconfined compressive loads of 0.5, 1, 2 or 4 MPa for 1 h using a pneumatic loading device. Loads greater than 0.5 MPa maintained the vimentin organisation over the culture period. At 0.5 MPa, the chondrocytes within the explant behaved as in free-swelling culture. The rapid change in vimentin organisation probably relates to rapid swelling of the explants—under free-swelling conditions, these reached their maximum swollen size in just 15 min of culture. The chondrocytes' response to change in tissue dimensions, and thus to their relationship to their immediate environment, was to disassemble their vimentin networks. Loading probably counteracts the swelling pressure of the tissue. Overall, this work suggests that chondrocytes maintain their actin cytoskeleton and modify their vimentin cytoskeleton in response to changing mechanical conditions.     

Electrical and mechanical response of arteries to stimulation of sympathetic nerves

1. When common carotid arteries of sheep were studied in vitro by the sucrose-gap method, application of acetylcholine or nicotine caused small irregular spikes of depolarization. The discharge was prevented by hexamethonium, Hydergine, phentolamine, or chronic denervation, indicating that it represented electrical activity of groups of smooth muscle cells induced by the stimulation of sympathetic nerve fibres.2. The size of spikes produced by acetylcholine or nicotine, together with counts of the total number of smooth muscle cells in cross-sections of the arterial strips, indicated that the larger groups of smooth muscle cells activated by one sympathetic nerve fibre contained approximately 1300 cells.3. Sections of arteries treated with hot formaldehyde vapour contained numerous fluorescent fibres which were intensified by previous injection of noradrenaline into the animal and were scanty or absent after chronic sympathetic denervation. They are therefore believed to be post-ganglionic sympathetic nerve fibres.4. Most of these fibres ran circularly in the outer (1/2)-(3/4) of the media. A few ran longitudinally in the adventitia. There were none in the inner (1/4)-(1/2) of the media.5. Electrical stimulation of the cervical sympathetic nerve of anaesthetized sheep caused large contractions of the common carotid artery of the same side, reducing its external diameter by 30-39%.     

The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes

Primary bovine chondrocytes and PEG-based hydrogels were used to investigate the effects of scaffold composition and architecture on the cellular response to large dynamic compressive strain stimulation. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic scaffolds. Second passage articular chondrocytes were encapsulated into four different scaffold compositions: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only and subjected to 15% dynamic compressive strain at 1-Hz frequency. Cellular response was evaluated in terms of cell number, glycosaminoglycans (GAGs), collagen type II and collagen type I accumulation in the constructs following 24h and 28 days of stimulated and static culture. Stimulation of the constructs resulted in an increase in the cell number in all scaffolds, with no statistical difference measured among them. Dynamic stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the GAGs by 33%, 53.4%, 240.5%, and 284.5%, compared to their static controls. The permissive PEG and PA scaffolds showed a significantly larger relative increase in the GAGs in comparison to the other scaffolds tested. Collagen type II content in the PF, PA and PEG constructs increased by 78%, 1266% and 896% respectively, compared to their static controls. Permissive constructs showed a significantly larger relative increase and final absolute values of GAGs and type II collagen, compared to the PF constructs. Immunostaining for collagen type I, an indicator for chondrocyte de-differentiation, indicated that stimulation inhibited its production. Correlation maps between scaffold properties highlighted the major differences between permissive and instructive scaffolds. These results support the hypothesis that both compressive strain and scaffold bioactivity have an important effect on the chondrocyte metabolic response to mechanical stimulation, and that the 3-D environment surrounding chondrocytes can actively participate in translating mechanical stimulation to the resident cells.     

The effect of PEGT/PBT scaffold architecture on oxygen gradients in tissue engineered cartilaginous constructs

Repair of articular cartilage defects using tissue engineered constructs composed of a scaffold and cultured autologous cells holds promise for future treatments. However, nutrient limitation (e.g. oxygen) has been suggested as a cause of the onset of chondrogenesis solely within the peripheral boundaries of larger constructs. In the present study, oxygen gradients were evaluated by microelectrode measurements in two porous polyethylene glycol terephthalate/polybutylene terephthalate (PEGT/PBT) scaffold architectures, a compression-molded and particle-leached sponge (CM) and a 3D-deposited fiber (3DF) scaffold. During the first 14 days in vitro, gradients intensified, after which a gradual decrease of the gradients was observed in vitro. In vivo, however, gradients changed instantly and became less pronounced. Although similar gradients were observed regardless of scaffold type, significantly more cells were present in the center of 3DF constructs after 2 weeks of in vivo culture. Our results stress the importance of a rationally designed scaffold for tissue-engineering applications. Organized structures, such as the 3DF PEGT/PBT polymer scaffolds, offer possibilities for regulation of nutrient supply and, therefore, hold promise for clinical approaches for cartilage repair.     

The effect of sintering temperature on the microstructure and mechanical properties of a bioceramic bone scaffold

Micro and nanostructural properties are believed to play a critical role in the osteoinductive capacity of bioceramic bone scaffolds. Physical characteristics also play an important role for optimum biological performance, including osteoconductivity and strength. In this study microstructural and nano-mechanical properties of a bioceramic bone scaffold were investigated as a function of the sintering temperature in the range of 950-1150?°C, through the use of scanning electron microscopy (SEM), X-ray diffraction (XRD) and nanoindentation testing. Although the samples presented the same crystallographic phase, an increase in sintering temperature resulted in increased grain size, density and crystallite size. The intrinsic mechanical properties were measured by nanoindentation testing and analyzed with the Oliver-Pharr method. The nanoindentation tests consisted of a series of fourteen partial unload tests (n=14 per treatment) of twelve steps ranging from 1?to 12?mN. Statistically significant increases in hardness and elastic modulus were measured for increasing sintering temperature. These results support the development of clinically successful bioceramic scaffolds with mechanical properties that encourage bone ingrowth and provide structural integrity.     

The dosage dependence of VEGF stimulation on scaffold neovascularisation

Growth factors are often used in tissue regeneration to stimulate vascularisation of polymeric scaffolds, with vascular endothelial growth factor (VEGF) having been extensively studied for short-term vessel ingrowth. We have therefore evaluated the effect of different concentrations of VEGF on the vascularisation of a porous scaffold in the short-, intermediate- and long-term, by delivering 15, 150 and 1500ng VEGF/day to polyurethane scaffolds by osmotic pumps for up to 6 weeks. An increased vascularisation months after termination of VEGF delivery was only achieved with 150ng/day (46%, p<0.05). This dosage consistently showed elevated levels of vascularisation (144, 125, 160 and 60% above PBS controls at 10, 20, 30 and 42 days, respectively, p<0.05), whilst the vessels induced by the highest dosage, though initially maximally elevated (265 and 270% at 10 and 20 days, p<0.05) tended to regress after 20 days of VEGF delivery. Pericyte coverage was decreased at 20 days for the highest dosage (30%, p<0.05). Lectin perfusion demonstrated that vessels within the scaffold were connected to the host vasculature at all time points and perfusion was substantially raised by VEGF delivery at day 20. These results suggest concentration of VEGF plays a critical role in the nature and persistence of vasculature formed in a tissue regenerative scaffold.     

Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression

To investigate the potential utility of mechanical loading in articular cartilage tissue engineering, porous type II collagen scaffolds seeded with adult canine passaged chondrocytes were subjected to static and dynamic compressions of varying magnitudes (0-50% static strain) and durations (1-24 h), and at different times during culture (2-30 days postseeding). The effects of mechanical compression on the biosynthetic activity of the chondrocytes were evaluated by measuring the amount of (3)H-proline-labeled proteins and (35)S-sulfate-labeled proteoglycans that accumulated in the cell-scaffold construct and was released to the medium during the loading period. Similar to published results on loading of articular cartilage explants, static compression decreased protein and proteoglycan biosynthesis in a time- and dose-dependent manner (each p < 0.005), and selected dynamic compression protocols were able to increase rates of biosynthesis (p < 0.05). The main difference between the results seen for this tissue engineering system and cartilage explants was in the amount of newly synthesized matrix molecules that accumulated within the construct under dynamic loading, with less accumulating in the type II collagen scaffold. In summary, the general biosynthetic response of passaged chondrocytes in the porous type II collagen scaffolds is similar to that seen for chondrocytes in their native environment. Future work needs to be directed to modifications of the cell-seeded construct to allow for the capture of the newly synthesized matrix molecules by the scaffold.     

Influence of scaffold thickness and scaffold composition on bioartificial graft survival

Biological scaffolds exhibit advantageous properties for tissue engineering of small diameter vessels. The influence of their extracellular matrix (ECM) components during in vivo repopulation is unknown. We implanted different xenogenic vascular matrices in a rat model to determine the influence of scaffold-thickness and ECM composition on in vivo repopulation. Decellularized ovine jugular vein (JV, n=42), carotid artery (CA, n=42) and aorta (AO, n=42) were implanted subcutaneously in the neck of adult male rats. Animals were sacrificed 2, 4 and 8 weeks after implantation. Cell and matrix morphology of explanted scaffolds were characterized by hematoxylin-eosin and pentachrome staining. Monoclonal anti-rat-CD31 was used to identify revascularization. Quantification of cell density was done by DNA-isolation.THICKNESS OF IMPLANTED XENOGENIC SCAFFOLDS VARIED ACCORDING TO THE MATERIAL USED (AO: 3.0-3.8mm; CA: 0.7-0.88mm; JV: 0.35-0.61mm). Immunohistology revealed complete repopulation of AO, CA, and JV scaffolds with endothelial cells and myofibroblasts within 2 weeks. After 8 weeks of implantation, AO scaffolds were completely covered by an endothelial monolayer and showed signs of a central matrix degeneration. JV scaffolds were completely degenerated at this stage. In contrast, CA scaffolds showed preserved ECM with a normal myofibroblast population and endothelial cell coverage.     

The effect of acidaemia on the response to stimulation of the autonomic nerves to the heart

1. The effects are described of an acidaemia produced either by an inhalation of carbon dioxide or by an infusion of hydrochloric acid on the response of the heart to stimulation of the ansae subclaviae and right vagus nerve in anaesthetized dogs.2. The results show that during an acidaemia (pH 6.95) of up to 2 hr duration the response to stimulation of the right vagus nerve was enhanced and that the inotropic response to stimulation of sympathetic nerves was not changed; the chronotropic response was depressed during acidaemia only at the low end of the range of responses, from 0 to 40 beats/min. The importance of preventing acidaemia when investigating reflex heart rate responses is discussed.3. It is suggested that in the intact animal with an innervated heart the response of the heart to stimulation of the sympathetic nerves is unaltered in acidaemia and that the reported effects of changed cardiovascular response during acidaemia may in part be explained by the enhanced response to vagal stimulation and an altered response of the peripheral vessels.     

Parotid salivary flow in response to mechanical and gustatory stimulation in man

To examine further the role of the oral receptors in the masticatory-salivary reflex, a study with eight subjects was performed. The influence on mean parotid salivation of combined alterations in frequency and force of chewing and length of the chewing object was evaluated by group comparison. Salivary flow rate was recorded using a sensitive micromanometer, and the frequency (12, 60 and 90 cycles min-1) and force of chewing (10 and 40% of maximum) were controlled by a metronome and masseter muscle EMG, respectively. The maximum instantaneous flow and the latency of the masticatory-salivary reflex were examined in three subjects. For comparison with mean salivation rate during chewing, gustatory stimulation was performed with 0.5 or 5.0% citric acid. The masticatory-salivary reflex was mainly ipsilateral, and depended upon having an object between the teeth. Salivation increased with increases in frequency and force of chewing and with the number of teeth involved, each parameter of chewing having the greatest influence when increased from a low level of action. The salivation response to chewing showed two phases; the first, presumably due to contraction of the myoepithelial cells, had a latency of 0.2-0.4 s, while the second phase occurred about 1 s later. Our results support the hypothesis that the periodontal mechanoreceptors have a major role in the parotid response to chewing. Application of 0.5 and 5.0% citric acid on the back of the tongue induced dose-dependent parotid secretions, significantly higher than those of chewing. A negative correlation was found between the maximum fluid outputs during chewing and 5.0% citric acid stimulation.