PURPOSE: To evaluate the usefulness of three dimensional culture using alginate-fibrin beads on the chondrogenic differentiation of rabbit's perichondrial cells. MATERIALS AND METHODS: Rib perichondrial cells from rabbit expanded by monolayer culture were cultured in monolayer, in alginate bead, and in alginate-fibrin beads. Reverse transcription polymerase chain reaction (RT-PCR) for type I, II, X collagen, and aggrecan was performed at 3 weeks after culture. At that time, we removed the alginate component from the alginate-fibrin bead. Then, the cell-fibrin beads were transplanted into the partial physeal defect of proximal tibia, and some beads were cultured on for additional 3 weeks for RT-PCR. Histologic examination was performed at 2, 4, 8, 12, and 16 weeks after operation. RESULTS: At 3 weeks, type II collagen gene expression was maintained regardless of the culture system used. However, at 6 weeks it was maintained only in three-dimensional culture system using alginate beads and fibrin beads. Histologic examination showed that the implanted perichondrial cell-fibrin beads formed small nest composed of chondrocyte-like cells and matrix. CONCLUSION: These results suggest that alginate-fibrin beads may be used as a biodegradable scaffold for cartilage engineering using perichondrial cells.
Aggrecans ; Cartilage ; Collagen ; Collagen Type II ; Fibrin ; Gene Expression ; Polymerase Chain Reaction ; Reverse Transcription ; Ribs ; Tibia

OBJECTIVETo study the expression of Aggrecan and the relationship between the variable number of tandem repeats (VNTR) of Aggrecan and lumbar disc herniation (LDH).

METHODSThe disease group comprised of 74 patients already diagnosed with symptomatic LDH. The control group consisted of 15 patients restricted to spinal trauma and 113 healthy blood donors without symptoms of LDH who were not diagnosed with LDH. Disc tissue samples were obtained from surgical operations and blood samples were donated from all participants. The Aggrecan expression in isolated tissues was assessed by western blot using specific antibodies. The Aggrecan gene VNTR region was analyzed by PCR.

RESULTSThe Aggrecan expression positive rate of control group was statistically and significantly higher (control group:86.67%, disease group:13.51%;χ(2) = 34.83, P < 0.05) than that of the disease group. Moreover, there was a statistically significant higher frequency of Allele 25 or Allele 21 in disease group compared to controls (A25disease group = 22.97%, A25control group = 12.11%, χ(2)A25 = 8.20, PA25 = 0.004; A21disease group = 6.76%, A21control group = 0.39%, χ(2)A21 = 14.35, PA21 = 0.000). Compared to the participants with 2 Alleles>25 repeats, subjects with 1 or 2 Alleles ≤ 25 repeats statistically and significantly over represented the disease group without the expression of Aggrecan (χ(2) = 5.69, P = 0.017).

CONCLUSIONSThe findings suggest a relationship between Aggrecan and symptomatic LDH, where symptomatic LDH has a tendency of allele 21 and allele 25 repeats.In addition, an association between the distribution of Aggrecan gene VNTR polymorphism and the expression of Aggrecan is observed in symptomatic LDH.

Over the past few years, considerable progress has been achieved about the extracellular elements and intracellular regulatory molecules that are involved in the regulation of chondrogenesis. However, little is known about the molecular mechanism of how these molecules influence the gene activities during cartilage differentiation. Recently we isolated a Chicken Cartilage derived Matrix Protein (CCMP 10), a novel protein, from chicken prechondrogenic mesenchyme. To further understand the function of CCMP-10 in cartilage development, we investigated the expression of CCMP-10 during the prechondrocyte differentiation in chick embryos and micromass cultured prechondrogenic cells, using a variety of methods such as transient transfection of CCMP 10, immunohistochemical localization, northern analysis, and western analysis. When transiently transfected, CCMP 10 was expressed in both nucleus and cytoplasm, with stronger intensity in the nucleus. In an immunohistochemical study, CCMP 10 was expressed in prechondrogeinc mesenchymal cell, perichondrium, and resting and proliferative zone of the growth plate of long bone, while no expression of CCMP 10 was observed in upper mature chondrocytes and hypertrophic chondrocytes. Northern analysis of micromass cultured prechondrogenic cells showed the expression of CCMP-10 mRNA for first 2 days, while Col 2a1, aggrecan, and CMP mRNAs, known genes to express in mature chondrocyte, initiated the expression at day 2 and continued to express by day 5. In western analysis, CCMP-10 was detected at initial stage and continued to express by day 3, while Col 2al protein began to express only one day after, and continued to express. Taken together, our data suggest that CCMP-10 may play a significant role in the early cartilage development.
Aggrecans ; Animals ; Cartilage* ; Chick Embryo ; Chickens* ; Chondrocytes ; Chondrogenesis* ; Cytoplasm ; Growth Plate ; Mesoderm ; RNA, Messenger ; Transfection

Experimental findings have suggested that the metabolic activities of articular cartilage can be influenced by mechanical stimuli. Our recent mathematical analysis predicted that cyclic compressive loading may create periods of intermittent negative hydrostatic pressure within the cartilage extracellular matrix. Therefore, we hypothesize that intermittent negative hydrostatic pressure, created in the cartilage extracellular matrix during dynamic compression, has a stimulative effect on the biosynthesis of chondrocytes. In order to test this hypothesis, the present study developed a custom designed negative pressure generator to subject a monolayer culture of chondrocytes to an intermittent negative pressure. It was found that the intermittent negative pressure produced a 40% increase in proteoglycan and a l7% increase in non-collagenous protein synthesis during the pressurization period(p (0.05). The collagenous protein synthesis was not affected by the intermittent negative pressure regimen used in this study. After the intermittent negative pressurization, the metabolic activities of the chondrocytes returned to normal(control level). The intermittent negative pressure also produced an increase in the mRNA signals for aggrecan. Therefore, we conclude that intermittent negative pressure may be one of the major mechanical stimulators of chondrocytes in articular cartilage during dynamic compression.
Aggrecans ; Cartilage ; Cartilage, Articular ; Chondrocytes* ; Collagen ; Extracellular Matrix ; Hydrostatic Pressure* ; Metabolism* ; Proteoglycans ; RNA, Messenger

Cartilage repair is substantially intractable due to poor self-healing ability. Porous microspheres can be a fascinating three-dimensional matrix for cell culture and injectable carrier in cartilage engineering. In this study, we assessed the feasible use of porous biopolymer microspheres for chondrocyte carriers. When seeded onto the blended biopolymer microspheres and followed by a dynamic spinner flask culture, the chondrocytes showed robust growth behaviors during the culture period. The gene expressions of SOX9, type II collagen, and aggrecan were significantly upregulated after 2-week of culture. Furthermore, immunolocalization of type II collagen and secretion of glycosaminolglycan became prominent. The results suggest the feasible usefulness of the porous microspheres as the cell culture matrix and the subsequent delivery into cartilage defects.
Aggrecans ; Biopolymers ; Cartilage* ; Cell Culture Techniques ; Chondrocytes* ; Collagen Type II ; Feasibility Studies* ; Gene Expression ; Microspheres

OBJECTIVE: To analyze the clinical and genetic characteristics of five Chinese pedigrees affected with short stature. METHODS: A retrospective analysis was carried out for the clinical data and results of genetic testing for the probands. A literature search was also conducted. RESULTS: The five probands have all featured short stature with a family history. Genetic testing has revealed that they have harbored variants of the ACAN gene, including p.Val2042Argfs*6, p.Val1597del, c.630-1G>A, c.23delT and c.2026+1G>A(previously reported). CONCLUSION Except for short stature, children harboring heterozygous variants of the ACAN gene may have no involvement of other systems. Some of these children may response to short-term growth hormone treatment.
Aggrecans/genetics* ; Body Height/genetics* ; Child ; China ; Genetic Testing ; Humans ; Pedigree ; Retrospective Studies

OBJECTIVE: To analyze the genetic etiology of a Chinese pedigree affected with short stature. METHODS: A child with familial short stature (FSS) who had presented at the Ningbo Women and Children's Hospital in July 2020 and his parents and paternal and maternal grandparents were selected as the study subject. Clinical data of the pedigree was collected, and the proband was subjected to routine growth and development assessment. Peripheral blood samples were collected. The proband was subjected to whole exome sequencing (WES), and the proband, his parents and grandparents were subjected to chromosomal microarray analysis (CMA). RESULTS: The height of the proband and his father was 87.7cm (-3 s) and 152 cm (-3.39 s) respectively. Both of them were found to harbor a 15q25.3-q26.1 microdeletion, which has encompassed the whole of the ACAN gene which is closely associated with short stature. The CMA results of his mother and grandparents were all negative, and above deletion has not been included in population database and related literature, and was rated as pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). After 14 months of rhGH treatment, the height of the proband has increased to 98.5 cm (-2.07 s). CONCLUSION The 15q25.3-q26.1 microdeletion probably underlay the FSS, in this pedigree. Short-term rhGH treatment can effectively improve the height of the affected individuals.
Child ; Female ; Humans ; Male ; Aggrecans/genetics* ; Dwarfism/genetics* ; East Asian People ; Mutation ; Pedigree

OBJECTIVE: To develop a simple, reproducible model of disc degeneration in rabbits through percutaneous annular puncture and to confirm the degree of degeneration over time. METHODS: Fifteen New Zealand white rabbits (4 to 5 months old and weighing approximately 3 to 3.5 kg each) underwent annular puncture of the L2-L3, L3-L4, and L4-L5 discs. Rabbits were sacrificed at 4, 8, or 20 weeks after puncture. For a longitudinal study to assess changes in disc height over time, serial X-rays were performed at 0, 2, 4, 8, and 20 weeks for rabbits in the 20-week group. Upon sacrifice, the whole spinal column and discs were extracted and analyzed with magnetic resonance imaging (MRI), real time reverse transcriptase-polymerase chain reaction, and histological staining. RESULTS: The X-rays showed a slow, progressive decrease in disc height over time. Significant disc space narrowing compared to preoperative disc height was observed during the time period (p<0.001). The MRI grade, aggrecan, and matrix metalloprotease-13 mRNA expression and hematoxylin and eosin/safranin O/anti-collagen II staining were consistently indicative of degeneration, supporting the results of the X-ray data. CONCLUSION: Percutaneous annular puncture resulted in slow, reproducible disc degeneration that was confirmed by radiology, biochemistry, and histology. This in vivo model can be used to study and evaluate the safety and efficacy of biologic treatments for degenerative disc disease.
Aggrecans ; Biochemistry ; Gene Expression ; Hematoxylin ; Intervertebral Disc Degeneration ; Longitudinal Studies ; Magnetic Resonance Imaging ; Models, Animal ; Punctures ; Rabbits ; RNA, Messenger ; Spine

PURPOSE: Earlier work suggested that two cytokines inhibit synthesis of type II collagen and of aggrecans by chondrocytes and they depress chondrocyte proliferation, but there was little report how the chondrocyte is modulated by culture conditions such as the joint fluids of the rheumatoid arthritis and that of the osteoarthritis. The purpose of this investigation was to determine whether RA(rheumatic arthritis) or OA(Osteoarthritis) joint fluid influence proliferation and differentiation in cultured human articular chondrocytes. MATERIALS AND METHODS: Human chondrocytes were cultured in a standard media (DMEM and 10% FBS), RA and OA joint fluid were added to media at the concentration of 20, 40 and 60% respectively for 1, 3 and 6days. 3H-thymidine and 3H-uridine uptake of cultured chondrocytes were measured as indicators of cell proliferation. Synthesis of human collagen type I, II was estimated by the RT-PCR procedures. RESULTS: 3H-thymidine uptake of the chondrocyte cultured in RA SF(synovial fluid) medium at 2 and 4 days; its uptake in the group treated by RA SF 20%, 40%, 60% increased more significantly than that in control group (P<0.05). 3H-thyrnidine uptake of the chondrocyte cultured in OA SF medium at 2 days; its uptake of the group treated in OA SF 60%(P<0.05), but there was no significant difference of its uptake between in the control group & the group treated in OA SF 20%, 40% (P<0.05). 3H-thymidine uptake of the chondrocyte cultured in OA SF medium at 4 days; there was no significant difference of its uptake between control group & OASF treated group(P>0.05). 3H-uridine uptake of the chondrocyte cultured in RA SF medium at 2 and 4 days; its uptake of the group treated by RA SF 20%, 40%, 60% increased more significantly than that of control group (P<0.05). 3H-uridine uptake of the chondrocyte cultured in OA SF medium at 2 days; its uptake of the group treated by OA SF 20%, 40%, 60% increased more significantly than that of control group (P<0.05). 3H-uridine uptake of the chondrocyte cultured in OA SF medium at 4 days; its uptake of the group treated by OA SF 20%, 60% increased more significantly than that of control group (P<0.05), but there was no significant difference of its uptake between control group & OA SF 40% treated group(P>0.05). Human type I collagen mRNA expressions of the chondrocyte markedly increased in RA and OA SF mixed groups. Human type II collagen mRNA expressions of the chondrocyte were reduced in RA and OA SF mixed groups, especially RA SF 60% mixed groups. CONCLUSION: RA and OA SF increased the proliferation of the articular chondrocyte, but its decreased the differentiation of the chondrocyte. RA and OA SF may change the phenotype of the articular chondrocyte and this phenomenon was more outstanding in RA SF.
Aggrecans ; Arthritis, Rheumatoid ; Cell Proliferation ; Chondrocytes* ; Collagen Type I ; Collagen Type II ; Cytokines ; Humans ; Joints* ; Osteoarthritis ; Phenotype ; RNA, Messenger

PURPOSE: We intended to check the growth rates and phenotypic markers of chondrocytes in the dedifferentiated cells cultivated in various conditions in order to establish the ideal culture system for implantation. MATERIALS AND METHODS: Culturing rabbit chondrocytes from proximal tibia, we checked the phenotypes at first, second, and third week. Then we cultured the chondrocytes in different circumstances such as monolayer or three dimensional gel in the presence or abscence of TGF-B1, and checked the growth rates and phenotypic markers. RESULTS: There was no difference in growth rates and mRNA level of type I, type II collagen and aggrecan between the cells cultured in monolayer and three dimensional gel of collagen. However, the responses of the cells to TGF-B1, were quite different between these two groups. In monolayer culture, the expression of type I collagen was depressed by TGF-B1 while the growth rate was markedly increased. Oppositely in three dimensional culture, the mRNA level of type I collagen was markedly increased and the growth rate was completely suppressed by TGF-B1. The expression of type II collagen could be detected only in TGF-B1-treated cells cultured in three dimensional gel for 4 or more days. The mRNA level of aggrecan was also increased by TGF-B1, in the cells cultured in three dimensional gel. CONCLUSIONS: These results suggest that the number of chondrocytes can be efficiently expanded by culturing the cells in monolayer and the phenotypes of chondrocyte can be restored by culturing the cells in three dimensional gel containing TGF-B1. The application of semi-solid gel containing differentiated chondrocytes in physeal implantation should be further evaluated
Aggrecans ; Chondrocytes* ; Collagen ; Collagen Type I ; Collagen Type II ; Phenotype ; RNA, Messenger ; Tibia ; Transforming Growth Factor beta1*