For tissue engineering of cartilage, chondrocytes can be seeded in a scaffold and stimulated to produce a cartilage-like matrix. In the present study, we investigated the effect of alginate as a ...chondrocyte-delivery substance for the construction of cartilage grafts. E210 (a non-woven fleece of polyglactin) was used as a scaffold.
When ‘bare’ E210 (without alginate and without chondrocytes) was implanted subcutaneously in nude mice for 8 weeks, the explanted tissue consisted of fat and fibrous tissue only. When E210 with alginate but without chondrocytes was implanted in nude mice, small areas of newly formed cartilage were found. Alginate seems to stimulate chondrogenesis of ingrowing cells. When chondrocytes were seeded in E210, large amounts of cartilage were found, independent of the use of alginate. This was expressed by a high concentration of glycosaminoglycans (30
μg/mg w.w.) and the presence of collagen type II (1.5
μg/mg w.w.). Macroscopically the grafts of E210 without alginate were shrunk and warped, whereas the grafts with alginate had kept their original shape during the 8 weeks of implantation. The use of alginate did not lead to inflammatory reactions nor increased capsule formation.
In conclusion, the use of alginate to seed chondrocytes in E210 does not influence the amount of cartilage matrix proteins produced per tissue wet weight. However, it provides retention of the graft shape.
To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess ...the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.
In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this ...study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.
In this study an animal model was developed for evaluation of the feasibility of cartilage grafts. In the cartilage of the external ear of the rabbit multiple holes, 6
mm in diameter, were punched, ...leaving the adherent skin intact. Different experimental groups were evaluated. First, the punch-hole model was validated under various conditions to study spontaneous or perichondrial initiated regeneration of the cartilage defect. When both cartilage and perichondrium was excised no spontaneous repair of the cartilage defect was observed. When perichondrium is present, variable patch-like closure of the punch hole was found. As ‘golden standard’ a punched out piece of cartilage was reimplanted directly. This condition showed adequate closure of the punch hole, however, no perfect integration of graft and surrounding cartilage was observed. Secondly, to evaluate the ‘punch-hole model’ a biomaterial ,trabecular demineralized bovine bone matrix (DBM), was implanted and tested as a scaffold for tissue engineering techniques in vivo and in vitro. Direct implantation of DBM did not lead to any cartilage formation to close the defect. In vivo engineered cartilage, generated by enveloping DBM in perichondrium for 3 weeks, could adequately close the punch hole. When DBM was seeded with isolated chondrocytes in vitro before implantation in the defect, a highly fragmented graft, with some islets of viable cells was seen. To promote an efficient and reliable evaluation of cartilage grafts a semi-quantitative grading system was developed. Items such as quality, quantity and integrity of the cartilage graft were included in a histomorphological grading system to provide information about the properties of a specific cartilage graft. To validate the grading system, all conditions were scored by two independent observers. An excellent reliability (
R=0.96) was seen between the observers. In summary, the rabbit pinna punch-hole model is a reliable and efficient method for first evaluation of cartilage grafts. The results can be easily analyzed using a semi-quantitative grading system.
Objective The ability of cartilage to regenerate following injury is limited, potentially leading to osteoarthritis. Integrative cartilage repair, necessary for durable restoration of cartilage ...lesions, can be regarded as a wound healing process. Little is known about the effects of growth factors regulating acute cartilage wound healingin vivo . In this study the temporal expression patterns of growth factors and proteoglycan content in cartilage wound edges in vivo were studied.
Design Cartilage wounds were created in rabbit ear cartilage using a 6mm biopsy punch. Specimens were subsequently harvested 1, 3, 7, 14 and 28 days after surgery. Paraffin sections were thionin stained to visualize proteoglycan loss and replacement. Immunohistochemical staining of TGFβ1, TGFβ3, IGF-1, IGF-II and FGF-2 was used to define growth factor expression at the cartilage wound sites.
Results Almost no effect of cartilage wounding was observed one day after surgery. A decrease of proteoglycan content, with a maximal loss at day 7, and a subsequent restoration was observed at the wound edges. Growth factor expression increased simultaneously. Maximal immunostaining for IGF1, IGFII, FGF2 and TGF-beta3 was observed at day 7, followed by a gradual decrease. Increased expression of TGFβ1 lasted from day 3 until day 14.
Conclusion We have demonstrated the ability of chondrocytes to increase growth factor expression and to restore the rapid decrease in proteoglycan content in the initial phase following acute wounding. A temporal increase in intracellular growth factor expression suggests an autocrine and/or paracrine metabolic stimulation, which can be regarded a sign of chondrocytes repair capacity.
Objective: to study the histopathology of subglottic stenosis in children of different ages after treatment during different periods of time, with or without laser application. Partial resection of ...the anterior cricoid with adhering stenotic subglottic area in the live young patient provides unique material for studying wound healing and scarring processes.
Methods: 25 specimens obtained from partial cricotracheal resection (PCTR) in children, were histologically processed and stained with Haematoxylin and Eosin, Resorcin and Fuchsin (for elastic fibers), and immunohistochemical staining (for the presence of macrophages).
Results: all specimens were found to have severe and sclerotic scarring with squamous metaplasia of the epithelium, loss of glands and elastic mantle fibers (tunica elastica), and dilation of the remaining glands with formation of cysts. Also, the cricoid cartilage was affected on the internal and external side, with irreversible loss of perichondrium on the inside and resorption by macrophages of cartilage on both sides. Detrimental effects of laser therapy were demonstrated in four cases. The normal intercellular matrix was completely destroyed and the number of chondrocytes in the cartilage structure diminished.
Conclusion: wound healing after laryngeal injury is a process of intense restoration and reorganization of the various tissues involved. This process, however, does not guarantee complete repair. In the severe cases irreversible scarring has replaced normal tissues. There seems to be no direct relationship between the length of the post-lesional period, the age of the patient and the severity of the stenosis. When subglottic stenosis has developed and the majority of the tissues is replaced by dense fibrous tissue, PCTR is strongly indicated to achieve renewed patency of the airway.
This study was performed to determine the various processes involved in the behaviour of hyaline cartilage during the wound healing period after trauma or surgery of vulnerable structures like the ...nasal septal cartilage and the cricoid. The results of different procedures (perpendicular and parallel to the cartilage surface) in young and young-adult animals were analyzed: septal incision at different locations (young-old), cricoid split (young-old), suturing cartilage, closing defects with autologous cartilage (young), biomaterials (young) and newly engineered cartilage in 4- and 24-week-old rabbits (series of ten animals). Cartilage of the young rabbit and child have similar hyaline cartilage with a varying distribution in thickness. Thinner areas are more susceptible to malformations. Incisions through younger cartilage give rise to some new cartilage formation covered by a new layer of perichondrium; through older, differentiated cartilage the incision causes superficial but permanent necrosis. Edges of cut cartilage mostly do heal by formation of fibrous junctions. This forms a weak spot, sensitive to deviations. The same fate goes for the healing between the autologous graft and the surrounding pre-existent cartilage. Trauma parallel to the surface, leads to inconsistent quantity of neocartilage. With ageing the wound healing and regenerative capacities decrease. In general, biomaterials are less accepted by the surrounding tissues and would impede further growth. Only newly engineered, and thus less differentiated (younger) cartilage of hyaline nature, appeared to be well accepted at the interface with the edges of a cartilage defect. There are indications that the release of growth factors might play a role in cartilage wound healing. In the child as well as the adult, wound healing of hyaline cartilage structures is incomplete, and surgery remains `experimental' surgery. The clinical implications of gradual loss of the regenerative capacity of hyaline cartilage should be further investigated.
A pedicled auricular perichondrial flap wrapped around trabecular demineralized bovine bone matrix can generate an autologous cartilage graft. In earlier experimental studies, it was demonstrated ...that this graft could be used for nasal and cricoid reconstruction. It was assumed that the vascularization of the perichondrial flap was obligatory, but it was never proven that the flap should be pedicled. Moreover, for clinical use, the dimensions of the auricle would set restrictions to the size of the graft generated. Therefore, the possibility to generate cartilage with a composite graft of a free perichondrial flap wrapped around demineralized bovine bone matrix, by using young New Zealand White rabbits, was studied. This composite graft was implanted at poorly (subcutaneously in the abdominal wall; n = 12), fairly (subcutaneously in the pinna; n = 12), and well-vascularized sites (quadriceps muscle; n = 12). As a control, trabecular demineralized bovine bone matrix was implanted without perichondrial cover. Half of these grafts (n = 6) were harvested after 3 weeks, and the remaining grafts (n = 6) after 6 weeks of implantation. In histologic sections of these grafts, the incidence of cartilage formation was scored. Furthermore, the amount of newly formed cartilage was calculated by computerized histomorphometry. Trabecular demineralized bovine bone matrix without perichondrial cover demonstrated early resorption; no cartilage or bone was formed. In demineralized bovine bone matrix wrapped in perichondrium, early cartilage formed after 3 weeks at well- and fairly vascularized sites. No cartilage could be detected in grafts placed at a poorly vascularized site after 3 weeks; minimal cartilage formed after 6 weeks. In summary, the highest incidence of cartilage formed when trabecular demineralized bovine bone matrix was wrapped either in a pedicled auricular perichondrial flap or in a free perichondrial flap, which was placed at a well-vascularized site. Second, a significantly higher percentage of the total area of the graft was cartilaginized at well-vascularized sites after 3 weeks. The newly generated cartilage contained collagen type II and proteoglycans with hyaluronic acid binding regions, whereas collagen type I was absent, indicating the presence of hyaline cartilage. This study demonstrates that new cartilage suitable for a graft can be generated by free perichondrial flaps, provided that the site of implantation is well vascularized. Consequently, the size of such a graft is no longer limited to the dimensions of the auricle.
Cartilage can be shaped by scoring. In an exploratory study in living adult animals, this phenomenon was demonstrated in cartilage of the nasal septum. Bending was observed immediately after ...superficial scoring of the cartilage surface, and the cartilage always warped in the direction away from the scored side. The scored piece of cartilage still showed its initially distorted shape 10 weeks after primary surgery. In ex vivo experiments, a clear relation between incision depth and bending of septal cartilage was observed. Under these controlled conditions, the variation between different septa was small. Deformation of the septal specimens was increased by introducing single superficial incisions deepening to half the thickness of the cartilage. A positive correlation between incision depth and bending was demonstrated. A model was used to accurately predict the degree of bending of the cartilage after making an incision of a particular depth. Hence, the effect of cartilage scoring can be predicted. Because the results of this controlled study showed excellent reproducibility for different septa, it is expected that this model can be extrapolated to human nasal septum cartilage. This would enable the surgeon to better predict the result of cartilage scoring, either preoperatively or perioperatively.
The effects of transforming growth factor-β (TGF-β) on proteoglycan synthesis of chondrocytes are controversial. The hypothesis that the differential effect of TGF-β is related to the differentiation ...stage of the chondrocytes is investigated in this study.
Rabbit auricular chondrocytes were cultured in alginate. When seeded in alginate immediately after isolation, cells keep their cartilaginous phenotype. When cells are first cultured in monolayer, they lose their cartilaginous phenotype and become dedifferentiated. We used three different cell populations: (1) Differentiated cells (PO: immediately after isolation); (2) partially (de)differentiated cells (P1: after one passage in monolayer); (3) dedifferentiated cells (P4: after four passages in monolayer). Cells were characterized by morphology using electron microscopy, amount of proteoglycans using the Farndale assay and type of collagen produced using immunohistochemistry.
The effects of addition of 10 ng/ml TGF-β2 for 7 days to P0, P1 and P4 cells were compared. TGF-β was added either directly from the start of the alginate culture, or after a preculture period of three weeks in alginate. The amount of proteoglycans was increased in all chondrocyte populations when TGF-β was added immediately after seeding in alginate, indicating that the effect of TGF-β on proteoglycan synthesis does not depend on the differentiation stage of cells. After preculture in alginate, stimulation of proteoglycan synthesis (as measured by amount of proteoglycans and
35S-sulfate incorporation) had vanished. This effect was independent of differentiation stage. A dose-response experiment with TGF-β (1, 10, 50 ng/ml) confirmed this differentiation-stage-independent effect of TGF-β on proteoglycan synthesis. Stimulation by TGF-β can be retained after enzymatic digestion of the pericellular matrix and reseeding of the cells in alginate, indicating the importance of pericellular matrix for the effect of TGF-β on matrix synthesis.
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