Biomedical subjects

M Alini

Publications and source records attributed to M Alini.

At least 19 recordsLinked to original sources

PubMed2006

Engineering of bone using bone marrow stromal cells and a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between bone formation and scaffold resorption.

Resorbable porous ceramic constructs, based on silicon-stabilized tricalcium phosphate, were implanted in critical-size defects of sheep tibias, either alone or after seeding with bone marrow stromal cells (BMSC). Only BMSC-loaded ceramics displayed a progressive scaffold resorption, coincident with new bone deposition. To investigate the coupled mechanisms of bone formation and scaffold resorption, X-ray computed microtomography (muCT) with synchrotron radiation was performed on BMSC-seeded ceramic cubes. These were analyzed before and after implantation in immunodeficient mice for 2 or 6 months. With increasing implantation time, scaffold thickness significantly decreased while bone thickness increased. The muCT data evidenced that all scaffolds showed a uniform density distribution before implantation. Areas of different segregated densities were instead observed, in the same scaffolds, once seeded with cells and implanted in vivo. A detailed muX-ray diffraction analysis revealed that only in the contact areas between deposited bone and scaffold, the TCP component of the biomaterial decreased much faster than the HA component. This event did not occur at areas away from the bone surface, highlighting coupling and cell-dependency of the resorption and matrix deposition mechanisms. Moreover, in scaffolds implanted without cells, both the ceramic density and the TCP:HA ratio remained unchanged with respect to the pre-implantation analysis.

Animals↗

PubMed2006

The involvement of aggrecan polymorphism in degeneration of human intervertebral disc and articular cartilage.

The functions of the intervertebral disc and of articular cartilage are intimately related to their aggrecan content. Aggrecan is a proteoglycan that interacts with hyaluronan to form large aggregates, which are responsible for the ability of the tissues to resist compressive loads. This function is related to the structure of aggrecan, and in particular to the large number of chondroitin sulphate chains present on its core protein. The chondroitin sulphate chains are present in two adjacent regions of the aggrecan core protein, termed the CS1 and CS2 domains. In the human, the region of the aggrecan gene encoding the CS1 domain exhibits size polymorphism, which can result in variation in the degree of chondroitin sulphate substitution of aggrecan in different individuals. This raises the possibility that the functional properties of aggrecan may vary between individuals, and that those individuals with an inferior aggrecan structure may be more susceptible to premature intervertebral disc or articular cartilage degeneration. Several studies have been performed to demonstrate such an association, but the results have been ambiguous. This review explains the relationship between aggrecan structure and function, describes the technique used to assess aggrecan polymorphism and the conclusions and limitations of the data obtained to date, and discusses the implications for tissue degeneration and clinical practice.

Aggrecans↗

PubMed2005

Stimulatory effects of creatine on metabolic activity, differentiation and mineralization of primary osteoblast-like cells in monolayer and micromass cell cultures.

The effects of creatine (Cr) supplementation on primary rat osteoblast-like cells cultured as monolayer and micromass were investigated. Cr was added to the medium at concentrations of either 10 or 20 mM. At various time points, the cell cultures were analyzed morphologically, metabolically and biochemically. The degree of differentiation of primary osteoblast-like cell cultures was higher in micromass cultures compared to monolayer cultures, as judged by alkaline phosphatase (ALP) activity and extent of mineralization. In both culture systems, Cr supplementation showed positive effects, which were dependent on the organizational level of the osteoblast-like cells in such a way that the cells in monolayer culture showed significantly increased metabolic activity, ALP activity and mineralization in the presence of Cr than without the supplement. In micromass cultures, Cr also significantly enhanced ALP activity and mineralization, without affecting metabolic activity. The effect of Cr on ALP activity was more pronounced at higher concentrations of Cr, but 20 mM Cr already showed some adverse effects on cell viability. In conclusion, chemically pure Cr added to low serum cell culture medium has a stimulatory effect on metabolic activity, differentiation and mineralization of osteoblast-like cells indicating that Cr supplementation could also be used as a potential clinical intervention to stimulate cell growth, differentiation and mineralization during bone repair in vivo.

Alkaline Phosphatase↗

PubMed2004

Action of estradiol on epiphyseal growth plate chondrocytes.

Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion in both sexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17beta-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7-21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T3), (3) E2 concentrations ranging from 10(-13) M to 10(-7) M, or (4) a combination of T3 and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10(-7) M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T3 did not appreciably affect the response to T3 alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T3, suggesting that, in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate.

Alkaline Phosphatase↗

PubMed2002

A biological approach to treating disc degeneration: not for today, but maybe for tomorrow.

The intervertebral disc unites the vertebrae in the spine, providing the flexibility required for bending and twisting and resisting the compression inflicted by gravity when in an upright posture. The discs have a complex structure, with the outer annulus fibrosus having lamellae of organized collagen fibrils and the inner nucleus pulposus having a more random collagen organization and an abundance of aggregating proteoglycans. This composite nature endows the disc with both the tension-resisting properties of a ligament and the compression-resisting properties of articular cartilage. Unfortunately, disc structure and function does not remain optimal throughout life, but undergoes progressive degeneration, commencing in the young adult, and is particularly evident in the nucleus pulposus. With time, disc degeneration may result in clinical symptoms, such as low back pain, and require medical intervention. Such treatment may involve removal of the offending disc by surgery rather than its repair, which would be the preferred course of action. In the near future, current bioengineering techniques may offer the possibility of repairing the damaged disc, if an engineered tissue with the appropriate functional properties can be generated to augment the ailing disc. In this report, we summarized our recent results, in which disc cells were implanted into a scaffold of collagen and hyaluronan, or entrapped into a chitosan gel, and growth factors were used to modulate matrix synthesis in an attempt to produce a tissue with a similar molecular composition to native nucleus pulposus tissue.

Cell Transplantation↗

PubMed2002

The role of proteoglycans in aging, degeneration and repair of the intervertebral disc.

The ability of the nucleus pulposus of the intervertebral disc to resist compressive loads is due to its high content of the proteoglycan aggrecan. Degeneration of the intervertebral disc is preceded and accompanied by a loss of aggrecan due to proteolysis. Biological repair of intervertebral disc degeneration should strive to restore aggrecan content to its optimal functional level. One approach to such repair is to supplement the degenerate nucleus with cells that are capable of aggrecan synthesis. Such cells can be supported in a biomolecular scaffold, but it is essential that the scaffold is compatible with high aggrecan retention if a functional tissue is to be attained.

Aggrecans↗

PubMed2001

Elevated synthetic activity in the convex side of scoliotic intervertebral discs and endplates compared with normal tissues.

STUDY DESIGN: We measured concentrations of specific molecules reflecting matrix synthesis and degradation in normal and scoliotic intervertebral discs and endplates. OBJECTIVES: The aim of this work was to quantitate markers of matrix turnover in normal versus adolescent idiopathic scoliotic intervertebral discs and cartilaginous endplates. SUMMARY OF BACKGROUND DATA: Changes in the intervertebral disc and endplate composition have been implicated as possible etiologic factors in the pathogenesis of adolescent idiopathic scoliosis. To better understand this process, it is important to compare the turnover of matrix components in scoliotic and normal intervertebral disc and endplate tissues. This comparison may help to improve our understanding of the role that disc and endplate tissues may play in the induction and/or progression of idiopathic scoliosis. METHODS: Fifteen scoliotic and 17 normal intervertebral discs and endplates were analyzed for their water, collagen, proteoglycan, and protein content. In addition, newly synthesized aggrecan and collagen Types I and II were measured. Percent total denatured collagen was also determined. RESULTS: The total collagen content was significantly lower in the scoliotic anulus and endplate regions, whereas glycosaminoglycan (GAG) content was significantly lower in the scoliotic endplates and nucleus regions. Conversely, total protein content was significantly higher in scoliotic endplates and elevated in scoliotic nucleus regions. Water content was significantly lower in the scoliotic anulus and endplate regions. When comparing the concave and convex regions of scoliotic endplates, there was no significant difference in concentration of any matrix component. The major difference in the synthetic marker levels relates to the synthesis of Type II collagen, which was higher in the nucleus, anulus, and endplate regions of scoliotic discs than in the corresponding regions of normal tissues. By contrast, the percent total denatured collagen was significantly elevated in the nucleus of normal tissues compared with the scoliotic ones. CONCLUSIONS: The higher collagen Type II synthetic levels and increased total protein content with no matrix turnover suggest that scoliotic changes are due to an altered and ineffective synthetic response to a pathologic mechanical environment.

Adolescent↗

PubMed2001

Changes in leucine-rich repeat proteoglycans during maturation of the bovine growth plate.

The primary growth plate of the fetal bovine tibia was studied in order to determine whether changes in the structure, abundance and expression of the leucine-rich repeat proteoglycans were occurring during tissue maturation from reserve cartilage to hypertrophic cartilage. The proteoglycans under study were decorin, biglycan, fibromodulin and lumican. Decorin was readily detectable in both the reserve and proliferating zones of the growth plate, but its abundance decreased markedly in the zones of maturation and hypertrophy where it could not be detected under the same conditions of analysis. In contrast to decorin, fibromodulin and biglycan could be detected throughout the growth plate, though their abundance was decreased in the proliferative and hypertrophic zones. Unlike the other proteoglycans, lumican could not be detected throughout the growth plate. At the message level, the expression of decorin shows a similar trend to that of protein abundance in the extracellular matrix, with its expression dropping markedly in the proliferative and hypertrophic zones. In the case of both biglycan and fibromodulin, message expression continued at a similar level throughout the growth plate. Thus, the leucine-rich repeat proteoglycans are different in the way they behave during growth plate maturation.

Animals↗

PubMed2000

Selective assembly and remodelling of collagens II and IX associated with expression of the chondrocyte hypertrophic phenotype.

The assembly and resorption of the extracellular matrix in the physis of the growth plate are poorly understood. By examining isolated fetal growth plate chondrocytes in culture and using immunochemical methods we show that type II collagen, proteoglycan aggrecan, and type IX collagen are assembled into a matrix that is initially enriched in type II collagen over proteoglycan and type IX collagen. When compared to the content of the COL2 domain in the alpha(1)(IX) chain it is evident that the majority ( 90%) of type IX molecules lack the NC4 domain unlike in articular cartilage. During matrix assembly the molar ratio of type II/COL2 of alpha(1)(IX) varied from 25:1 to 2.5:1. Following expression of the hypertrophic phenotype (initiation of type X collagen synthesis) there are parallel changes in both collagen and proteoglycan contents (inversely related to collagenase cleavage of type II collagen). The NC4 domain is then selectively, rapidly and irreversibly removed as mineralization is initiated, leaving the alpha(1)(IX) chain COL2 domain. Subsequently as mineralization progresses type II and type IX collagen (COL2 domain), but not the proteoglycan aggrecan, are resorbed coincident with a markedly increased cleavage of type II collagen by collagenase as mineral is deposited in the matrix. This study, therefore reveals a carefully orchestrated series of events in matrix assembly and resorption that prepares the extracellular matrix for mineralization.

Animals↗

PubMed2000

Collagen crosslinked N-telopeptides as markers for evaluating particulate osteolysis: a preliminary study.

The purpose of this study was to determine whether a marker of bone resorption could be used noninvasively to diagnose and assess treatment of periprosthetic osteolysis. The crosslinked N-telopeptide marker of osteoclast-mediated bone resorption potentially has the sensitivity to detect periprosthetic osteolysis. Second-morning urine specimens were obtained from (a) seven age-matched controls, (b) eight patients who had a hip arthroplasty, hybrid implants at least 1 year after surgery, and no osteolysis, (c) 11 patients who had a hip arthroplasty and osteolysis, and (d) 10 patients who had a hip arthroplasty and with osteolysis before and after 6 weeks of oral Fosamax (alendronate) treatment. The Fosamax treatment consisted of one 10-mg dose per day for 6 weeks. Men and young women (less than 40 years old) were chosen for this study to avoid bone resorption enhanced after menopause as a possible confounder. An enzyme-linked immunosorbent assay technique for quantifying crosslinked N-telopeptides of type-I collagen was performed on all specimens. The patients with osteolysis had significantly elevated levels of N-telopeptide compared with the implant control group. In addition, levels of N-telopeptide were significantly lowered after Fosamax treatment. These findings indicate that a systemic bone-resorption marker (N-telopeptide) can be used to evaluate local particulate-induced osteolysis and its treatment. The study also provides clinical evidence that osteolysis is associated with increased osteoclast-mediated bone resorption and that this locally induced bone resorption can be suppressed by certain bisphosphonates (Fosamax). These insights have potential value in the understanding and clinical management of aseptic loosening.

Adult↗

PubMed1999

Age-related changes in fibromodulin and lumican in human intervertebral discs.

STUDY DESIGN: An analysis of proteoglycans of the intervertebral disc using immunoblotting of tissue extracts. OBJECTIVES: To investigate the changes in structure and abundance of fibromodulin and lumican in human intervertebral discs during aging and degeneration. SUMMARY OF BACKGROUND DATA: Fibromodulin and lumican are keratan sulfate proteoglycan constituents of the disc's extracellular matrix, whose interaction with collagen fibrils may contribute to the mechanical properties of the tissue. Changes in their abundance and/or structure that occur with aging and degeneration therefore may have an impact on disc function. METHODS: Lumbar intervertebral discs were obtained from individuals of different ages, and extracts of anulus fibrosus and nucleus pulposus were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for fibromodulin and lumican. RESULTS: The major changes in abundance observed with age were a decrease in fibromodulin in the adult nucleus pulposus and an increase in lumican in anulus fibrosus during early juvenile development. In addition, fibromodulin in the anulus fibrosus exhibited a structural change with increasing age, characterized by a shift toward the predominance of its glycoprotein form lacking keratan sulfate. Fibromodulin was more abundant in the anulus fibrosus than in nucleus pulposus at all ages, whereas lumican was much more abundant in nucleus pulposus than in anulus fibrosus in the young juvenile; in the adult, however, lumican was present in comparable levels in both tissues. With increasing degrees of degeneration, fibromodulin exhibited an increase in abundance. CONCLUSIONS: Growth, aging, and degeneration of the intervertebral disc are associated with changes in the abundance and structure of fibromodulin and lumican, which presumably influence the functional properties of the tissue.

Adolescent↗

PubMed1999

Compression-induced changes in intervertebral disc properties in a rat tail model.

STUDY DESIGN: An Ilizarov-type apparatus was applied to the tails of rats to assess the influence of immobilization, chronically applied compression, and sham intervention on intervertebral discs of mature rats. OBJECTIVES: To test the hypothesis that chronically applied compressive forces and immobilization cause changes in the biomechanical behavior and biochemical composition of rat tail intervertebral discs. SUMMARY OF BACKGROUND DATA: Mechanical factors are associated with degenerative disc disease and low back pain, yet there have been few controlled studies in which the effects of compressive forces on the structure and function of the disc have been isolated. METHODS: The tails of 16 Sprague-Dawley rats were instrumented with an Ilizarov-type apparatus. Animals were separated into sham, immobilization, and compression groups based on the mechanical conditions imposed. In vivo biomechanical measurements of disc thickness, angular laxity, and axial and angular compliance were made at 14-day intervals during the course of the 56-day experiment, after which discs were harvested for measurement of water, proteoglycan, and collagen contents. RESULTS: Application of pins and rings alone (sham group) resulted in relatively small changes of in vivo biomechanical behavior. Immobilization resulted in decreased disc thickness, axial compliance, and angular laxity. Chronically applied compression had effects similar to those of immobilization alone but induced those changes earlier and in larger magnitudes. Application of external compressive forces also caused an increase in proteoglycan content of the intervertebral discs. CONCLUSIONS: The well-controlled loading environment applied to the discs in this model provides a means of isolating the influence of joint-loading conditions on the response of the intervertebral disc. Results indicate that chronically applied compressive forces, in the absence of any disease process, caused changes in mechanical properties and composition of tail discs. These changes have similarities and differences in comparison with human spinal disc degeneration.

Animals↗

PubMed1998

Quantitative magnetic resonance imaging in the assessment of degenerative disc disease.

Understanding degenerative intervertebral disc diseases hinges on the ability to objectively and noninvasively assess the disc matrix composition and integrity. The potential of quantitative magnetic resonance imaging to meet these demands was evaluated. Analyzing the T1 and T2 signal patterns in the nucleus pulposus with increasing Thompson morphological grade revealed a significant reduction related to grade. This significant decrease in T1 and T2 in the nucleus pulposus with grade 4 degeneration and the corresponding low correlation coefficients with respect to the content of individual matrix molecules suggest that matrix integrity plays an important and distinct role in determining T1 and T2 signal. Similarly, the significant increase in magnetization transfer in the nucleus pulposus observed in grade 4 degeneration did not correlate with the changes in molecular content in these highly degenerated discs. Again, this lack of correlation clearly indicates that the tissue integrity and matrix composition independently contribute to the magnetization transfer signal. This study presents the first clear evidence that quantitative magnetic resonance analysis reflects not only the disc matrix composition, but also the structural integrity of the matrix of the disc.

Adult↗

PubMed1997

Aggrecan degradation in human intervertebral disc and articular cartilage.

Aggrecan degradation in human intervertebral disc and articular cartilage has been studied by using anti-neoepitope antibodies specific for the N-terminal degradation products generated by cleavage within the interglobular domain at the metalloproteinase and aggrecanase sites. Immunoblot analysis of extracts of annulus fibrosus, nucleus pulposus and articular cartilage demonstrated age-related patterns in the abundance of both degradation products. In all three tissues the metalloproteinase-generated fragment was present at very low levels in young individuals but increased in abundance with age. In the disc tissues, the abundance of this degradation product levelled off in the juvenile; for cartilage this occurred in early adulthood. Despite these temporal differences, the levels attained in adults were comparable for the three tissues. In contrast, the aggrecanase-generated degradation product exhibited tissue-specific differences in the variation of its abundance with age. Whereas this degradation product increased with age in annulus fibrosus and articular cartilage and had levelled off by adulthood, in nucleus pulposus it was present in greatest abundance in young individuals and decreased to very low levels with age. Examination of discs exhibiting various degrees of degeneration did not reveal any differences in the levels of the metalloproteinase and aggrecanase-generated cleavage products that could not be accounted for by differences in age. In adults the product of aggrecanase action was much more abundant in articular cartilage than in either of the disc tissues, despite the age-related increase also observed for annulus fibrosus. Analysis of tissue extracts with an antibody recognizing the G1 domain of aggrecan identified two major degradation products whose abundance and size were correlated with the fragments detected by the anti-neoepitope antibodies. Taken together, these results indicate that cleavage at the metalloproteinase and aggrecanase sites are quantitatively important events in aggrecan catabolism in both articular cartilage and intervertebral disc in vivo. Moreover the two enzyme systems act independently and exhibit differences in the degree to which they contribute to aggrecan degradation in these tissues.

Adolescent↗

PubMed1996

The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration.

Very little is known about the turnover of extracellular matrix in the human intervertebral disc. We measured concentrations of specific molecules reflecting matrix synthesis and degradation in predetermined regions of 121 human lumbar intervertebral discs and correlated them with ageing and Thompson grade of degeneration. Synthesis in intervertebral discs, measured by immunoassay of the content of a putative aggrecan biosynthesis marker (846) and the content of types I and II procollagen markers, is highest in the neonatal and 2-5-yr age groups. The contents of these epitopes/molecules progressively diminished with increasing age. However, in the oldest age group (60-80 yr) and in highly degenerated discs, the type I procollagen epitope level increased significantly. The percentage of denatured type II collagen, assessed by the presence of an epitope that is exposed with cleavage of type II collagen, increased twofold from the neonatal discs to the young 2-5-yr age group. Thereafter, the percentage progressively decreased with increasing age; however, it increased significantly in the oldest group and in highly degenerate discs. We identified three matrix turnover phases. Phase I (growth) is characterized by active synthesis of matrix molecules and active denaturation of type II collagen. Phase II (maturation and ageing) is distinguished by a progressive drop in synthetic activity and a progressive reduction in denaturation of type 11 collagen. Phase III (degeneration and fibrotic) is illustrated by evidence for a lack of increased synthesis of aggrecan and type II procollagen, but also by an increase in collagen type II denaturation and type I procollagen synthesis, both dependent on age and grade of tissue degeneration.

Adult↗

PubMed1996

A novel angiogenic molecule produced at the time of chondrocyte hypertrophy during endochondral bone formation.

Angiogenesis is a pivotal event in endochondral ossification. Vessels grow into the hypertrophic cartilage and erode it to produce a scaffold on which osteoblasts settle to produce woven bone. A new culture system was used to determine whether growth-plate chondrocytes produce an angiogenic molecule. Chondrocytes from primary growth plates of bovine fetuses were separated into maturationally distinct subpopulations. When cultured these cells produce an extensive extracellular matrix and the prehypertrophic cells mature to express the hypertrophic phenotype defined by the synthesis of type X collagen and matrix calcification. The culture medium collected from the hypertrophic cells contains a chemoattractant, nonmitogenic molecule for bovine endothelial cells which can induce angiogenesis in vivo in the rabbit cornea model. This molecule has a Mr of approximately 120 x 10(3). The production of this molecule by hypertrophic cells is enhanced by both 1,25-(OH)2 vitamin D3 and 24,25-(OH)2 vitamin D3 at 10(-8)-10(-12) M, but only in pre- and early hypertrophic cells. In contrast, these metabolites have either no effect or an inhibitory effect on the more mature hypertrophic cells. These results describe for the first time the production of an angiogenic molecule by hypertrophic chondrocytes. They demonstrate an important role for vitamin-D3 metabolites in regulating hypertrophy and angiogenesis during normal skeletal growth and differentiation. Thus, a defective regulation of these processes, due to the lack of vitamin-D metabolites, may explain the observed enlargement of the hypertrophic zone and impairment of skeletal growth in rickets which is induced clinically and experimentally by a deficiency of vitamin D.

24,25-Dihydroxyvitamin D 3↗

PubMed1996

The human lumbar endplate. Evidence of changes in biosynthesis and denaturation of the extracellular matrix with growth, maturation, aging, and degeneration.

STUDY DESIGN: The authors measured concentrations of specific molecules reflecting matrix synthesis and degradation in 121 human endplates and correlated them with aging and grade of degeneration. OBJECTIVES: Abnormal endplate development has been implicated in many spinal abnormalities, yet little is known about endplate matrix component turnover. SUMMARY OF BACKGROUND DATA: Techniques are available to perform an in situ investigation of matrix component turnover with aging and degeneration. METHODS: Newly synthesized aggrecan and Type I and Type II procollagens were measured with recently developed immunoassays. Percentage of denatured Type II collagen was assessed with a new enzyme-linked immunosorbent inhibition assay. RESULTS: Synthesis in endplates, measured by content of an aggrecan marker (846) and content of Types I and II procollagen markers (CPI and CPII), is highest in the neonatal and 2- to 5-year age groups and steadily diminishes with increasing age. However, in the oldest age group and in highly degenerated discs, the CPI epitope level increased significantly. Percentage of denatured Type II collagen, assessed by the presence of an epitope exposed with the cleavage of Type II collagen, increased from the neonatal to the 2- to 5-year age groups. The percentage progressively decreased with increasing age. However, it significantly increased in endplates from highly degenerated discs. CONCLUSIONS: The authors identified three matrix turnover phases, related to age and grade of degeneration. Phase I (growth) is characterized by active synthesis of matrix molecules and active denaturation of Type II collagen. Phase II (aging and maturation) is distinguished by a drop in synthetic activity and a reduction in denaturation of Type II collagen. Phase III (degenerative) is illustrated by an increase in Type II collagen denaturation and Type I procollagen synthesis, both related to grade of tissue degeneration.

Adolescent↗

PubMed1996

In serum-free culture thyroid hormones can induce full expression of chondrocyte hypertrophy leading to matrix calcification.

A serum-free culture system has been developed to examine the biologic factors involved in the regulation of cellular maturation, extracellular matrix assembly, and calcification in the physis of the bovine fetal growth plate. Isolated prehypertrophic chondrocytes in high density culture undergo a process of cellular maturation whereby full expression of the hypertrophic phenotype is characterized first by type X collagen synthesis followed by matrix calcification. Using this culture system, we compared the capacity of tri-iodothyronine (T3) with thyroxine (T4) to stimulate expression of the hypertrophic phenotype and matrix calcification in three (B, C, and D) maturationally distinct prehypertrophic chondrocyte subpopulations. The B cell subpopulation was the most mature followed by C and D subpopulations in order of decreasing maturity. Comparisons were made to cultures in fetal calf serum (FCS). In Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, and selenium, both hormones (T3/T4) separately induced, in a dose-dependent manner, chondrocyte maturation to the hypertrophic phenotype characterized by increased type X collagen mRNA and induction of protein synthesis of this molecule, together with increased alkaline phosphatase activity, and eventually calcification of the extracellular matrix. Such cellular maturation to the hypertrophic phenotype was not observed in the absence of T3 or T4 with subpopulations C and D. Only in older fetuses (> 210 days) was this observed and then only in the B subpopulation. Furthermore, T3 was at least 50-fold more potent than T4. The effects of T3 were most pronounced with the most immature cells (subpopulations C and D) where, in the case of the subpopulation C, in contrast to 0.5 nM T3 50 nM T4 was unable to induce expression of the hypertrophic phenotype. Alkaline phosphatase activity was also increased in the C cell subpopulation treated with 1 nM T3 (35.5 U/micrograms of DNA) over that supplemented with 50 nM T4 (7.8 U/micrograms of DNA). Furthermore, matrix calcification, measured by the incorporation of 45Ca2+ into the cell layer, always occurred earlier in cells cultured with T3 compared with T4. Cellular maturation to the hypertrophic phenotype was not accompanied by significant changes in DNA content; this ordinarily increases during culture in the presence of serum. Compared with cells cultured in the presence of serum, either thyroid hormone more potently induced cellular maturation. This study demonstrates that the most immature chondrocytes at the prehypertrophic stage are direct targets for T3 and T4 and, to a much a lesser degree, that either hormone is able to induce full chondrocyte hypertrophy from an early maturational stage leading to matrix calcification. But T3 is much more potent than T4. These studies also offer a new serum-free chemically defined medium containing T3 or T4 for the culture of defined prehypertrophic chondrocytes that supports matrix assembly, hypertrophic expression, followed by matrix calcification.

Animals↗