Tissue engineering: chondrocyte cultures on type I collagen support. Cytohistological and immunohistochemical study

Cartilaginous tissue has limited capacity for regeneration after damage, since the natural repair process leads to the formation of fibrocartilaginous tissue which does not have the resistance and capability of deformation under load, typical of hyaline cartilage which covers the articular surfaces. The possibility of transplanting human chondrocytes for cartilage reconstruction has been demonstrated in orthopaedics. The scope of our study was to evaluate the possibility of cultivating and expanding human chondrocytes seeded on a pure equine type I collagen support. Human articular cartilaginous cells multiplied and grew on a type I collagen substrate with production of extracellular matrix. This chondrocyte culture showed a correct morphology and phenotype as shown by alcian-PAS staining to indicate the presence of mucopolysaccharides and by immunohistochemical methods to identify type II collagen. The use of scaffolds may lead to improvement in the surgical technique, by making it possible to hold the cells physically in the area to be repaired and by allowing optimum spatial adaptation inside injuries of all shapes.     

Biomechanical regulation of type I collagen gene expression in ACLs in organ culture.

In this study, an ex vivo organ culture system that allows the application of controlled loads to the anterior cruciate ligament (ACL) was designed and used to characterize the influence of a step input in mechanical load on gene expression. A procedure for isolating bone-ACL-bone (B-ACL-B) complexes from rat knees was developed. After harvest and 24 hour culture, B-ACL-B complexes exhibited percentages of viability similar to that in intact ACLs (approximately 90%). Application of a physiologically relevant load of 5 N (superimposed on a I N tare load) resulted in changes in levels of mRNA encoding type I collagen. While levels of type I collagen mRNA significantly increased 32+/-13% (mean +/- standard errors of the mean (SEM)) over controls within the first hour of loading, levels decreased significantly to 44+/-9% of control after 2 h. Displacements induced by the 5 N load were measured by video dimensional analysis. Calculated axial strains of 0.141+/-0.034 were achieved rapidly during the first hour and remained essentially unchanged thereafter. These results demonstrate the feasibility of maintaining ligaments in organ culture and illustrate the time course expression of type I collagen following the application of a mechanical load.     

Gene expression and cell differentiation in matrix-associated chondrocyte transplantation grafts: a comparative study

Objective

Although scaffold composition and architecture are considered to be important parameters for tissue engineering, their influence on gene expression and cell differentiation is rarely investigated in scaffolds used for matrix-associated autologous chondrocyte transplantation (MACT). In this study we have therefore comparatively analyzed the gene expression of important chondrogenic markers in four clinical applied cell-graft systems with very different scaffold characteristics.

Methods

Residuals (n = 165) of four different transplant types (MACI^®, Hyalograft^®C, CaReS^® and Novocart^®3D) were collected during surgery and analyzed for Col1, Col2, aggrecan, versican, melanoma inhibitory activity (MIA) and IL-1β by real-time PCR. Scaffold and cell morphology were evaluated by histology and electron microscopy.

Results

Despite the cultivation on 3D scaffolds, the cell differentiation on all transplant types didn’t reach the levels of native cartilage. Gene expression highly differed between the transplant types. The highest differentiation of cells (Col2/Col1 ratio) was found in CaReS^®, followed by Novocart^®3D, Hyalograft^®C and MACI^®. IL-1β expression also exhibited high differences between the scaffolds showing low expression levels in Novocart^®3D and CaReS^® and higher expression levels in MACI^® and Hyalograft^®C.

Conclusions

Our data indicate that scaffold characteristics as well as culture conditions highly influence gene expression in cartilage transplants and that these parameters may have profound impact on the tissue regeneration after MACT.     

The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels.

Autologous chondrocyte implantation (ACI) is the most promising surgical treatment for large full thickness knee joint articular cartilage (AC) defects where cells from healthy non-weight bearing area AC are multiplied in vitro and implanted into such defects. In the routine surgical procedure for symptomatic knee full thickness AC defects, damaged AC surrounding the edge and the base of such defects is usually debrided and discarded. The purpose of this study was to examine if chondrocytes from this 'debrided' AC can proliferate, synthesize a cartilage specific matrix and thus can be used for ACI. METHODS: Biopsies were retrieved from 12 patients (debrided articular cartilage: DAC, aged 35-61) and from two autopsies (normal articular cartilage: NAC, aged 21 and 25). Chondrocytes were isolated, seeded at low density in type I collagen gels and as monolayer cultures for 4 weeks without passage. RESULTS: After 4 weeks cultures in type I collagen gels, cell proliferation from DAC (18.34 +/- 1.95 fold) was similar to cells from NAC (11.24 +/- 1.02 fold). Syntheses of proteoglycan and collagen in DAC were also similar to NAC. Newly synthesized matrices in gel cultures consisted predominantly of type II collagen as shown by immuno-labelling and SDS-PAGE followed by fluorography. Chondrocytes from 'debrided human AC' cultured at low density in type I collagen gels may be used for the ACI procedure as they provide sufficient viable cell numbers for ACI and maintain their chondrocyte phenotype as they synthesize a cartilage-like matrix.     

New target genes for NOV/CCN3 in chondrocytes: TGF-beta2 and type X collagen.

We studied the involvement of NOV/CCN3, whose function is poorly understood, in chondrocyte differentiation. NOV was found to upregulate TGF-beta2 and type X collagen and to act as a downstream effector of TGF-beta1 in ATDC5 and primary chondrocytes. Thus, NOV is a positive modulator of chondrogenesis. INTRODUCTION: NOV/CCN3 is a matricellular protein that belongs to the CCN family. A growing body of evidence indicates that NOV could play a role in cell differentiation, particularly in chondrogenesis. During chick embryo development, NOV expression is tightly regulated in cartilage, and a high expression of NOV has been associated with cartilage differentiation in Wilms' tumors. However, a precise role for NOV and potential target genes of NOV in chondrogenesis are unknown. MATERIALS AND METHODS: ATDC5 cells and primary chondrocytes were either treated with NOV recombinant protein or transfected with a NOV-specific siRNA to determine, using quantitative RT-PCR, the effect of NOV on the expression of several molecules involved in chondrocyte differentiation. Stable ATDC5 clones expressing NOV were also established to show that NOV was a downstream effector of TGF-beta1. RESULTS: We established that NOV/CCN3 expression increases in ATDC5 cells at early stages of chondrogenic differentiation and precedes the appearance of TGF-beta2 and of several chondrocytic markers such as SOX9 or type X collagen. When exogenously administered, NOV recombinant protein up-regulates TGF-beta2 and type X collagen mRNA levels both in ATDC5 cells and in primary mouse chondrocytes but does not influence SOX9 expression. This regulation also occurs at the endogenous level because downregulation of NOV expression is correlated with an inhibition of TGF-beta2 and type X collagen in primary chondrocytes. Furthermore, we found that NOV expression is downregulated when chondrocytes are exposed to TGF-beta1-dedifferentiating treatment in chondrocytes, further providing evidence that NOV may counteract TGF-beta1 effects on chondrocytes. CONCLUSIONS: This study provides the first characterization of two new targets of NOV involved in chondrocyte differentiation, shows that NOV acts with TGF-beta1 in a cascade of gene regulation, and indicates that NOV is a positive modulator of chondrogenesis.     

Distribution of type VI collagen in chondrocyte microenvironment: study of chondrons isolated from human normal and degenerative articular cartilage and cultured chondrocytes

The chondron is the microanatomical unit composed of a chondrocyte and its pericellular microenvironment (PCME), including the pericellular matrix and capsule. In the present study, we extracted chondrons from human articular cartilages and investigated the relationship between the distribution of the matrix molecules, including type VI collagen, and the degeneration of articular cartilage. We also investigated the effects of interleukin-1 (IL-1) and transforming growth factor -1(TGF-₁) on the distribution of type VI collagen in cultured chondrocytes. Chondrons were extracted by low-speed homogenization from cartilage pieces obtained from forensic autopsies and from patients with knee osteoarthritis (OA) undergoing total knee arthroplasty. Cartilage sections were classified into three groups (normal, slight degeneration, and moderate degeneration) based on the degree of degeneration according to Mankins score. Extracted chondrons were immunostained, and the distribution of the matrix molecules, including type VI collagen, was investigated using a confocal laser scanning microscope (CLSM). The chondrocytes isolated by enzymic treatment were subjected to three-dimensional culture in agarose gel and then treated with IL-1 or TGF-₁. The distribution of newly synthesized type VI collagen in agarose gel was also investigated using the CLSM. Type VI collagen was localized specifically within the PCME of chondrons. The volume ratio of PCME to chondrocyte (P/C ratio) was significantly higher in the moderate degeneration group than in the other two groups. The accumulation of type VI collagen around a chondrocyte was obviously increased by the addition of TGF-₁. The P/C ratio significantly increased as the severity of the OA progressed, suggesting that type VI collagen distributed specifically in the PCME was playing a protective role for chondrocytes by maintaining the pericellular microenvironment in OA.     

Gene expression of collagen types IX and X in the lumbar disc

To study gene expression of collagen typesIX and X in human lumbar intervertebral discs duringaing and degeneration and to explore the role of collagentypes IX and X in disc degeneration.     

不同年龄肋软骨的组织形态学及Ⅱ型胶原蛋白含量变化的研究

目的 探讨肋软骨的组织形态学及II型胶原蛋白在不同年龄阶段的变化规律.方法 将2005年至2006年行耳廓再造的患者按年龄不同分为3组.5～10岁为儿奄组,11～17岁为青少年组,18～29岁为成人组,每组30例.观察不同年龄组肋软骨的组织形态学变化及Ⅱ型胶原免疫组织化学染色的定量分析,采用Motie Med6.0 A数码医学图像分析系统进行免疫组织化学图像定星测定,测量指标为平均积分光密度,对分析结果 进行统计学处理.结果 儿童组肋软骨膜血管最丰富,软骨基质染色均匀,软骨细胞数目最多,Ⅱ型胶原蛋白表达最活跃,平均积分光密度值最高;青少年组软骨膜内血管减少,软骨基质染色出现明显的不均质状,软骨陷窝体积变大,并呈分隔状,陷窝内软骨细胞数目减少,II型胶原蛋白表达较儿童组减弱;成人组软骨膜血管、细胞成分明显减少,软骨膜内的纤维成分明显玻璃样变,钙盐沉积较青少年组时明显增多,Ⅱ型胶原蛋白表达较青少年组减弱.经统计学分析,3组间差异有统计学意义(P＜0.01).结论 肋软骨的组织形态学随年龄增长发生变化,Ⅱ型胶原蛋白含量随年龄增长呈递减趋势.     

Autologous chondrocyte implantation for osteochondral lesions in the knee using a bilayer collagen membrane and bone graft: a two- to eight-year follow-up study

Matrix-induced autologous chondrocyte implantation (MACI) is an established technique used to treat osteochondral lesions in the knee. For larger osteochondral lesions (> 5 cm(2)) deeper than approximately 8 mm we have combined the use of two MACI membranes with impaction grafting of the subchondral bone. We report our results of 14 patients who underwent the 'bilayer collagen membrane' technique (BCMT) with a mean follow-up of 5.2?years (2 to 8). There were 12 men and two women with a mean age of 23.6 years (16 to 40). The mean size of the defect was 7.2 cm(2) (5.2 to 12?cm(2)) and were located on the medial (ten) or lateral (four) femoral condyles. The mean modified Cincinnati knee score improved from 45.1 (22 to 70) pre-operatively to 82.8 (34 to 98) at the most recent review (p < 0.05). The visual analogue pain score improved from 7.3 (4 to 10) to 1.7 (0 to 6) (p < 0.05). Twelve patients were considered to have a good or excellent clinical outcome. One graft failed at six?years. The BCMT resulted in excellent functional results and durable repair of large and deep osteochondral lesions without a high incidence of graft-related complications.     

Distribution of type X collagen in tibiotarsi of broiler chickens with vitamin D deficiency

Summary Type X collagen is a significant component of the extracellular matrix of the hypertrophic zone of physeal cartilage, but its precise role in endochondral ossification has not been determined. The concentration of type X collagen increases in physeal cartilage in chicks with vitamin D deficiency. The purpose of our study was to determine whether defective endochondral ossification due to vitamin D deficiency was associated with abnormalities in the distribution of type X collagen in the proximal tibiotarsus of chicks. To accomplish this, we induced vitamin D deficiency in broiler chicks and sequentially evaluated the pattern of type X collagen immunoreactivity in the proximal tibiotarsus using a monoclonal antibody specific for chicken type X collagen. Type X collagen immunoreactivity was present in the matrix of the prehypertrophic zone, hypertrophic zone, cartilage cores of the primary spongiosa, and within the chondrocytes of the prehypertrophic and early hypertrophic zones in vitamin D-deficient and D-replete chicks. However, rachitic chicks exhibited two consistent differences in type X collagen immunoreactivity: hypertrophic chondrocytes in the late hypertrophic zone and primary spongiosa contained intracellular type X collagen; and type X collagen was concentrated into laminated aggregates in the pericellular and territorial matrices in the late hypertrophic zone and primary spongiosa. We conclude from these findings that (1) normal serum concentrations of 1,25(OHD)₂D₃ and 25OHD₃ are not required for type X collagen production; (2) type X collagen production does not decrease in the most mature zones of the physes in chicks with vitamin D deficiency; and (3) newly secreted type X collagen accumulates in the pericellular and territorial matrices of the late hypertrophic zone and primary spongiosa of rachitic chicks, perhaps because it is not readily incorporated into the interterritorial matrix.     

Localization of type X collagen in canine growth plate and adult canine articular cartilage

Type X collagen was extracted from ends of canine growth plates by pepsin digestion after 4 M guanidine hydrochloride extraction, purified by stepwise salt precipitation (2.0 M NaCl in 0.5 M acetic acid), and chromatographed on a Bio-Gel A1.5 M column in 1.0 M CaCl2. Without reduction on sodium dodecyl sulfate (SDS) polyacrylamide gels, the preparation yielded a single, high-molecular-weight (mol wt) band; after reduction, a single band of relative mol wt 5.0 x 10(4) was found. Polyclonal sera were raised against the purified collagen and used in the immunolocalization of canine type X collagen. As expected, indirect immunoperoxidase (IP) or indirect immunofluorescent staining with the polyclonal sera demonstrated that most of the immunoreactivity was localized in the zone of provisional calcification of the growth plate and in cartilage remnants in the metaphyseal region of the physis. A progressive decrease in staining toward the diaphysis of the fetal canine long bone was apparent as the trabecular structures were remodeled to bone. Unexpectedly, type X collagen was also detected in the zone of calcified, mature articular cartilage. It was concentrated in the pericellular matrix of the chondrocytes, appeared at or just above the tidemark, and was expressed immediately before mineralization. Identification of type X collagen in both the canine growth plate and the zone of calcified articular cartilage suggests that cells in the deep layer of cartilage and in the zone of calcified cartilage in the adult animal retain some characteristics of a growth plate and may be involved in regulation of mineralization at this critical interface. The expression of growth plate-like properties would allow the deep chondrocytes of mature articular cartilage to play a role in remodeling of the joint with age and in the pathogenesis of osteoarthritis.     

Altered postnatal expression of insulin-like growth factor-I (IGF-I) and type X collagen preceding the Perthes' disease-like lesion of a rat model.

The spontaneously hypertensive rat (SHR) is a widely used animal model for the study of hypertension. It also exhibits an osteonecrosis of the femoral epiphysis that resembles the clinical features of Perthes' disease in humans. In this rat model, occlusion of the epiphyseal vessels occurs as a result of a breakdown of the mechanically vulnerable epiphysis. The postnatal development of the epiphysis recapitulates the serial events of the endochondral ossification (i.e., cartilage formation), chondrocyte hypertrophy, cartilage mineralization, vascularization, and introduction of osteoblasts that form the secondary ossification center within the epiphysis. In the present study, a detailed radiographic and histological analysis demonstrates that the osteonecrosis is preceded by a disturbance of the cartilage mineralization and a disturbance of the ossification, despite a normal hypertrophy of the epiphyseal cartilage. These observations suggest that abnormal development of the femoral epiphysis occurs much earlier than manifestation of the osteonecrosis. They lead us to a hypothesis that yet-unclarified transitional events between the cartilage hypertrophy and the cartilage mineralization may be affected in SHRs. Type X collagen is a developmentally regulated matrix molecule that is implicated in the mineralization of the hypertrophied chondrocytes. We show that the expression of type X collagen during epiphyseal ossification is delayed in SHRs (vs. normal controls), suggesting disturbed growth and/or differentiation of the epiphyseal chondrocytes. Postnatal growth and differentiation of the chondrocytes at least partly depend on insulin-like growth factor-I (IGF-I), which is produced by the chondrocytes in response to the pituitary growth hormone and stimulates cartilage growth in situ. The present study demonstrates an altered IGF-I expression during early postnatal life in SHRs and suggests that the altered IGF-I expression as well as the following delay in upregulation of type X collagen may cause the mechanical vulnerability of the femoral epiphysis in SHRs.     

Coordinate inhibition of alkaline phosphatase and type X collagen syntheses by 1,25-dihydroxyvitamin D3 in primary cultured hypertrophic chondrocytes

Summary The effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) (2.3×10^-12-1.4×10^-6 [M]) on alkaline phosphatase, collagen, and cell proliferation were examined in primary cultured hypertrophic chondrocytes prepared from the distal epiphyseal growth plate of the tibias of 12-day chick embryos. 1,25(OH)₂D₃ showed time- and dose-dependent inhibitory effects on the alkaline phosphatase and collagen levels. The inhibition of alkaline phosphatase activity became detectable at 2×10^-11 [M] and reached 10% of control at 10^-7 [M]. The concentration of 1,25(OH)₂D₃ giving a 50% inhibition of the enzyme level was approximately 3×10^-10 [M]. Of the two extracellular collagen pools, a cell-associated matrix pool showed a more dramatic decrease (to 10% of control) than a culture medium pool (to 50% of control) at increased 1,25(OH)₂D₃ concentrations. The degree of inhibition was different for each type of chondrocyte-specific collagen (types II, IX, X, and XI). Types II and IX were inhibited in a parallel manner to only 60–80% of control. On the other hand, types X and XI were more greatly reduced up to 10% of control, and their dose-dependent inhibitory curves were similar to that of alkaline phosphatase. On cell proliferation, 1,25(OH)₂D₃ had a biphasic effect: stimulation at 10^-10–10^-8 [M] and inhibition at higher levels. The results revealed the significant involvement of 1,25(OH)₂D₃ in the metabolism of two probable calcification-related products, alkaline phosphatase and type X collagen.