Clonal growth of human articular cartilage and the functional role of the periosteum in chondrogenesis (original) (raw)
Related papers
Transplantation Proceedings, 2006
Background. Autologous chondrocyte transplantation (ACT) has been shown to heal cartilage defects under experimental and clinical conditions. However, the evaluation of successful transplantation still remains arbitrary and further research is required to establish objective criteria of treatment. The aim of the present study was to evaluate the criteria of successful ACT and to compare the results with those obtained following periosteal grafting (PG). Materials and Methods. Articular cartilage specimens were taken from the distal femur of 30 adolescent New Zealand rabbits and chondrocytes were obtained by collagenase digestion. The chondrocytes were identified by a functional assay, based on estimating procollagen type II mRNA by reverse-transcribed polymerase chain reaction. The cells cultured in vitro were transplanted under a periosteal flap into a full thickness defect (ICRS III 0). The quality of the repaired tissue was evaluated macroscopically according to a modified scale of Brittberg et al, and microscopically according to O'Driscoll et al. For comparative purposes animals treated with PG were used. Results. Cultured chondrocytes expressed procollagen type II and, upon transplantation into the defect, produced hyaline cartilage. To evaluate the results of transplantation, two categories of criteria were adopted-macroscopic analysis and microscopic examination. By all adopted criteria the results were significantly better in the ACT group (P Ͻ .05) than in the PG group. Conclusion. Prior to transplantation, assays for specialized functions of chondrocytes required semiquantitative evaluation of macroscopic and microscopic appearance of the repaired tissue, showing the benefit of autologous chondrocyte versus periosteal graft transplantation. M ODERN THERAPEUTIC METHODS including tissue engineering techniques have been used to treat mechanical damage to articular cartilage. Among a number of methods, recently developed autologous chondrocyte transplantation (ACT) seems promising. In this procedure, chondrocytes isolated from a cartilage fragment, taken from a non-weight-bearing part of the joint cultured in vitro, are subsequently transplanted into the damaged area. Several studies 1-4 have revealed that these cells are capable of producing in vivo an extracellular matrix of chemical composition and biomechanical properties similar to those of normal hyaline cartilage. The first application of this method by Brittberg et al 5 demonstrated that human chondrocytes, cultured in vitro and subsequently transplanted into a damaged area, retained their biological properties and were capable of reproducing hyalinelike articular cartilage. Transplantation of chondrocytes into
195 Preservation of the Chondrocytes Pericellular Matrix Improves Cell-Induced Cartilage Formation
Osteoarthritis and Cartilage, 2009
Purpose: Chondrocytes are often used for cartilage tissue engineering. However, in native cartilage, the chondrocytes are surrounded by a pericellular matrix, together forming the chondron. Since cells are influenced by their surroundings, we hypothesized that retaining the pericellular microenvironment would influence the synthetic capacity of the chondrocytes. Therefore the aim of this study was to investigate whether the pericellular matrix has an effect on cell-induced cartilage formation. Methods: Chondrocytes and chondrons isolated from nucleus pulposus (NP), annulus fibrosus (AF), and articular cartilage (AC) from goats, were cultured for 25 days in alginate beads. After 7, 18 and 25 days of culture, the amount of proteoglycans present in the alginate beads was measured and collagen was extracted from the beads. Immunoblotting for type II collagen was performed on the collagen extracted from the alginate beads. Protein expression of matrix metalloproteinase 2 (Mmp2) and Mmp9 was analyzed by zymography and gene expression levels of Mmp13 were measured by real-time PCR. Results: Chondrons and chondrocytes were successfully isolated from AC, AF, and NP. The amount of proteoglycans found in the alginate beads was significantly higher in the chondrons from AC and NP compared to the chondrocytes, but no differences were found between chondrons and chondrocytes from AF. The type II collagen that was extracted from the alginate beads containing the chondrons from all the cartilage sources was cross-linked, whereas the type II collagen produced by the chondrocytes consisted only of non-crosslinked alpha1 (II) chains. Both Mmp2 and Mmp9 expression were higher by the chondrocytes from AC and NP compared to the chondrons, no differences were found with the AF cells. At day 0 the gene expression levels of MMP13 were low in both chondrocytes and chondrons. However, after 18 and 25 days of culture, there was a significant increased expression by the chondrocytes and not by the chondrons. Conclusions: This study shows that maintaining the native chondrocytes pericellular matrix affects both anabolic and catabolic activities. The cross-links present in the type II collagen produced by the chondrons isolated from all the different tissues suggests that the pericellular matrix has an effect on the expression or the activity of enzymes involved in collagen cross-linking. The type II collagen produced by the chondrons does more resemble the collagen found in the native tissues. It is also likely that the altered cell-ECM interactions caused by removal of the pericellular matrix plays a role in the increased expression of the matrix metalloproteinases. Taken together, our data suggest that the extracellular matrix surrounding the chondrocytes is essential for maintaining its proper composition and that preserving the thin matrix layer surrounding the chondrocytes improves cell-induced hyaline cartilage formation.
Preservation of the chondrocyte's pericellular matrix improves cell‐induced cartilage formation
Journal of cellular …, 2010
The extracellular matrix surrounding chondrocytes within a chondron is likely to affect the metabolic activity of these cells. In this study we investigated this by analyzing protein synthesis by intact chondrons obtained from different types of cartilage and compared this with chondrocytes. Chondrons and chondrocytes from goats from different cartilage sources (articular cartilage, nucleus pulposus, and annulus fibrosus) were cultured for 0, 7, 18, and 25 days in alginate beads. Real-time polymerase chain reaction analyses indicated that the gene expression of Col2a1 was consistently higher by the chondrons compared with the chondrocytes and the Col1a1 gene expression was consistently lower. Western blotting revealed that Type II collagen extracted from the chondrons was cross-linked. No Type I collagen could be extracted. The amount of proteoglycans was higher for the chondrons from articular cartilage and nucleus pulposus compared with the chondrocytes, but no differences were found between chondrons and chondrocytes from annulus fibrosus. The expression of both Mmp2 and Mmp9 was higher by the chondrocytes from articular cartilage and nucleus pulposus compared with the chondrons, whereas no differences were found with the annulus fibrosus cells. Gene expression of Mmp13 increased strongly by the chondrocytes (>50-fold), but not by the chondrons. Taken together, our data suggest that preserving the pericellular matrix has a positive effect on cell-induced cartilage production.
FGF-2 enhances TGF-β1-induced periosteal chondrogenesis
Journal of Orthopaedic Research, 2004
The use of periosteum as a cell source for the in vitro engineering of grafts for articular cartilage repair requires the development of methods to obtain high viable cell numbers in the early stages of culture. In this study, we demonstrate that the addition of a mitogen, fibroblast growth factor-2 (FGF-2), during the early stage of the in vitro culture of periosteum in the presence of transforming growth factor-pl (TGF-PI), significantly enhances cell proliferation, which results in increased neo-cartilage formation at later stages. Periosteal explants were cultured in vitro within alginate or agarose based gels in the presence of either FGF-2 for the first week, TGF-P1 for the first 2 weeks, FGF-2 and TGF-PI for the first week and first 2 weeks respectively, or no added factors. Consistent with previous studies, periosteum derived neo-chondrogenesis occurred only in the presence of TGF-PI . The neo-cartilage was found to contain cartilage specific proteoglycans and Type-I1 collagen as determined by safranin-O and immunohistochemical staining respectively. Further medium supplementation with FGF-2 stimulated early cell proliferation (>3 fold higher total DNA content per explant at day lo). This resulted in a marked increase in the size of the cultured explants and in the total area of the explant staining positive for safranin-O (from around . %%I to 85%, (p < 0.05)) after 6 weeks culture. The ability to generate significant quantities of neo-cartilage within a biocompatible and biodegradable matrix such as alginate, which lacks the immunogenicity of agarose, could open new pathways to utilizing such constructs in articular cartilage tissue engineering applications.
Regenerative Therapy, 2018
Restoration of damaged cartilage tissue has been deemed futile with current treatments. Although there have been many studies on cartilage regeneration thus far, there is no report that chondrocytes were completely re-differentiated in vitro. The clarification of cellular composition and matrix production during cartilage regeneration must be elucidated to fabricate viable mature cartilage in vitro. In order to achieve this aim, the chondrocytes cultured on coverslips were transplanted into the peritoneal cavities of mice. At different time points post-transplantation, the cartilage maturation progression and cells composing the regeneration were examined. Cartilage regeneration was confirmed by hematoxylin & eosin (HE) and toluidine blue staining. The maturation progression was carefully examined further by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). At the first and second week time points, various cell shapes were observed using SEM. Chronologically, by the third week, the number of fibers increased, suggesting the progression of extracellular matrix (ECM) maturation. Observation through TEM revealed the chondrocytes located in close proximity to various cells including macrophage-like cells. On the second week, infiltration of lymphocytes and capillary vessels were observed, and after the third week, the chondrocytes had matured and were abundantly releasing extracellular matrix. Chronological observation of transplanted chondrocytes by electron microscopy revealed maturation of chondrocytes and accumulation of matrix during the re-differentiation process. Emerging patterns of host-derived cells such as macrophage-like cells and subsequent appearance of lymphocytes-like cells and angiogenesis were documented, providing crucial context for the identification of the cells responsible for in vivo cartilage regeneration.
Serum-free media for periosteal chondrogenesis in vitro
Journal of Orthopaedic Research, 2004
Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 pg/ml growth hormone), and/or ITS+ (2.08 pg/ml each of insulin, transferrin, and selenious acid, plus 1.78 pg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 100/0 FBS) was achieved simply by the addition of 2.0 pg/ml insulin in 0.10/0 BSA-containing medium (p < 0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.
Effects of different embedding gels on periosteal chondrogenesis in vitro
Apmis Acta Pathologica Microbiologica Et Immunologica Scandinavica, 2002
To develop a more useful organ culture model for periosteal chondrogenesis in vitro, we compared the effects of the embedding of explants in agarose versus collagen gels. Chondrogenic differentiation was examined by means of histological observation and in terms of the expression of mRNA encoding two cartilage markers, Type II collagen and Aggrecan. Periosteal explants were derived from the tibiae of rabbits. These explants were embedded in either agarose gel or collagen gel and cultured for 6 weeks. Histological examinations revealed that in the agarose gel, cells were neither present in the explants nor in the gel. In the collagen gel, cells migrated from the explanted tissues into the gel, and some cells were round. However, no explants showed safranin-O staining. Only 10% of 10 surviving explants in the agarose gel expressed the Type II collagen gene and the Aggrecan gene. On the other hand, of 25 surviving explants in the collagen gel, the expression of the Type II collagen gene was detected in 18 explants (72%) and that of the Aggrecan gene was detected in 21 explants (84%). In conclusion, we demonstrated that the periosteum exerts chondrogenesis in certain circumstances and its chondrogenesis is closely related to the culture material.
Journal of Veterinary Medical Science, 2004
To evaluate the effects of chondrocytes transplantation on the regeneration of cartilage by intraarticular injection or injection into blood clots at cartilage defects, eight full-thickness cartilage defects were created surgically on the articular surface o f each femoral trochlea of two calves. Autologous chondrocytes were isolated individually from the cartilage pieces collected at the creation of defects. And isolated cells were cultured in monolayers for proliferation. Cells were injected into synovial fluid (Group 2, n=11) or into the blood clots at the cartilage defects (Group 3, n=5) of the left femoropatellar joint on weeks 2 and 3, respectively after the operation. The defects (Group 1, n=16) of right femoropatellar joint were left untreated in the control group. After 14 weeks, repaired tissues were evaluated based on gross and histological examinations. In Group 3, more repaired tissues and a better interface between the repaired tissue and host cartilage were observed compared with the results for Groups 1 and 2. Moreover, cartilaginous tissue were observed more in defects of Group 3 than in defects of other groups. In conclusion, the present study suggests that the injection of cells into the blood clot at a cartilage defect might be applicable for the regeneration of damaged cartilage.
Behavior of Human Articular Chondrocytes During In Vivo Culture in Closed, Permeable Chambers
Cell Medicine, 2012
The exact contribution of transplanted chondrocytes for cartilage tissue repair prior expansion in monolayer cultures remains undetermined. At our laboratory, we have created a new permeable chamber to study the chondrogenesis of dedifferentiated cells implanted ectopically in a closed and controlled environment. The behavior of chondrocytes has been studied in settings frequently used in clinical approaches during transplantation, namely injection of autologous chondrocyte cells in suspension (ACI), cells soaked in collagen membranes (MACI), and cells applied in a polymer gel (fibrin). As controls, we have tested the redifferentiation of chondrocytes in cell aggregates, and we have checked the proper functionality of chambers both in vitro and in vivo. After retrieval, firmed tissue-like shapes were recovered only from chambers containing cells seeded in membranes. Histomorphological, immunohistochemical, and ultrastructural analyses revealed synthesis of fibrous-like tissue, characterized by low-density collagen fibers, low collagen type II, abundant collagen type I, and low amounts of proteoglycans. Additionally, neither the collagen membranes nor the fibrin gel was reabsorbed by cells. In summary, our results show that the newly developed permeable chambers function correctly, allowing proper cell feeding and preventing cell leakage or host cell invasion. Additionally, our results suggest that, under these circumstances, chondrocytes are not able to orchestrate formation of hyaline cartilage and have little capacity to degrade artificial membranes or carrier gels such as fibrin. These are interesting observations that should be considered for understanding what role the transplanted chondrocytes play during restoration of articular cartilage after implantation.
Rabbit Articular Cartilage Defects Treated With Autologous Cultured Chondrocytes
Clinical Orthopaedics and Related Research, 1996
Adult New Zealand rabbits were used to transplant autologously harvested and in vitro cultured chondrocytes into patellar chondral lesions that had been made previously and were 3 mm in diameter, extending down to the calcified zone. Healing of the defects was assessed by gross examination, light microscope, and histological-histochemical scoring at 8, 12, and 52 weeks. Chondrocyte transplantation significantly increased the amount of newly formed repair tissue compared to that found in control knees in which the lesion was solely covered by a periosteal flap. In another experiment, carbon fiber pads seeded with chondrocytes were used as scaffolds, and repair significantly increased at both 12 and 52 weeks compared to knees in which scaffolds without chondrocytes were implanted. The histologic quality scores of the repair tissue were significantly better in all knees in which defects were treated with chondrocytes compared to knees treated with periosteum alone and better at 52