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Biomedical subjects

Karl J Jepsen

Publications and source records attributed to Karl J Jepsen.

16 recordsLinked to original sources
PubMed2006

Sexual dimorphism affects tibia size and shape but not tissue-level mechanical properties.

Understanding how growth influences adult bone morphology and tissue quality should provide important insight into why females show a greater incidence of stress fractures early in life and fragility fractures later in life compared to males. The objective of this study was to test whether females acquire similar tissue-level mechanical properties as males by the time peak bone properties are established. Standardized beams of bone were machined from the tibial diaphyses of 14 young, adult females ranging in age from 22 to 46 years. Data for males (n=17, age=17-46 years) were taken from a prior study. Measures of tissue-level mechanical properties, including stiffness, strength, ductility, toughness, and damageability, were compared between sexes using t-tests. The relationship between cross-sectional morphology and tissue-level mechanical properties was also examined. Males and females showed nearly identical tissue-level mechanical properties. Both sexes also showed similar age-related degradation of mechanical properties and a similar relationship between cross-sectional morphology and tissue quality. However, for all body sizes, female tibiae were smaller relative to body size (i.e., less robust) compared to males. The results indicated that sex-specific growth patterns affected transverse bone size, but did not affect tissue-level mechanical properties. This, combined with the observation that young, adult female long bones are undersized relative to body size, suggests that adult females would be expected to accumulate more damage under intense loading compared to males. This may be a contributing factor to the greater incidence of stress fractures observed for female military recruits.

Adolescent↗
PubMed2006

Three-dimensional reconstruction of fracture callus morphogenesis.

Rat and mouse femur and tibia fracture calluses were collected over various time increments of healing. Serial sections were produced at spatial segments across the fracture callus. Standard histological methods and in situ hybridization to col1a1 and col2a1 mRNAs were used to define areas of cartilage and bone formation as well as tissue areas undergoing remodeling. Computer-assisted reconstructions of histological sections were used to generate three-dimensional images of the spatial morphogenesis of the fracture calluses. Endochondral bone formation occurred in an asymmetrical manner in both the femur and tibia, with cartilage tissues seen primarily proximal or distal to the fractures in the respective calluses of these bones. Remodeling of the calcified cartilage proceeded from the edges of the callus inward toward the fracture producing an inner-supporting trabecular structure over which a thin outer cortical shell forms. These data suggest that the specific developmental mechanisms that control the asymmetrical pattern of endochondral bone formation in fracture healing recapitulated the original asymmetry of development of a given bone because femur and tibia grow predominantly from their respective distal and proximal physis. These data further show that remodeling of the calcified cartilage produces a trabecular bone structure unique to fracture healing that provides the rapid regain in weight-bearing capacity to the injured bone.

Animals↗
PubMed2006

Variability in tendon and knee joint biomechanics among inbred mouse strains.

Hereditary factors are thought to be responsible for impaired tendon function and joint laxity. The present study investigated the genotypic variability of knee laxity and stiffness and tendon mechanical and geometric properties among 16-week-old female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mice. In one group of mice, knee mechanics were quantified using a custom loading apparatus enabling translation of the tibia against a stationary femur. In a second group, flexor digitorum longus and Achilles tendons from the left hind limb underwent biomechanical testing, while those of the contralateral limb were analyzed histologically for determination of cross-sectional area. Our results demonstrate that tendon and joint mechanics varied significantly among the inbred mouse strains, indicating that biomechanical properties are genetically determined. A/J mouse knees exhibited greater laxity (p < 0.001) and lower stiffness (p < 0.001) compared to those of the B6 and C3H mice. The genotypic differences in whole joint properties were similar to those of the tendons' structural biomechanical traits. Although body mass did not differ (p > 0.2) among the three strains, significant genotypic differences were found at the whole tendon, material quality, and morphological levels of the tissue hierarchy. Furthermore, genetic regulation of tendon mechanical properties varied with anatomic site. Patterns of genotypic differences in tendon size were not consistent with those of biomechanical properties, suggesting that unique combinations of structural and compositional factors contribute to tendon growth, adaptation, and development. Therefore, the three inbred strains constitute a useful experimental model to elucidate genetic control of structure-function relationships in normal and healing tendons and ligaments.

Achilles Tendon↗
PubMed2006

Mice lacking cathepsin K maintain bone remodeling but develop bone fragility despite high bone mass.

UNLABELLED: Bone microstructural and biomechanical properties were analyzed in mice genetically lacking cathepsin K (CatK). CatK deficiency (CatK(-/-)) produced mild osteopetrosis, elevated numbers of osteoclasts, regions of disorganized bone microstructure, and increased bone fragility, showing how chronic alteration of enzyme activity during skeletal development dramatically affects bone organization and function. INTRODUCTION: Mouse models of CatK deficiency recapitulate the osteopetrosis of human pyknodysostosis and allow study of clinically relevant issues: how inhibition of this enzyme activity affects bone integrity structurally and biomechanically. To address these questions, we generated CatK-deficient mice by targeted disruption of the Ctsk gene and compared their bone structural and mechanical properties with wildtype (WT) controls. MATERIALS AND METHODS: Standard histomorphometric and biomechanical analyses were performed on femora from C57BL/6J male and female CatK(-/-), CatK(+/-), and WT mice. RESULTS: CatK(-/-) femora exhibited the mild metaphyseal osteopetrosis, a greater cortical bone area and thickness, normal bone strength, but a high degree of brittleness (nearly 50-70% decrease in postyield displacement versus WT) and a 30-40% reduction in the work-to-failure. In cancellous bone, osteoclast numbers and resorption surface were increased markedly (approximately 150% and 50%, respectively), despite the overall decrease in net bone resorption for CatK-deficient mice. Bone formation indices were altered in CatK(-/-) mice as well, with significant increases in mineral appositional rate, but not in bone formation surface; these data suggest difference in osteoblast work but not in their recruitment in CatK deficiency. CatK-deficient cortical bones had large areas of woven bone and intracortical resorption spaces within the disorganized tissue. Bone phenotype in CatK(-/-) was similar in males and females. CONCLUSIONS: Genetic CatK deficiency in mice results not only in the impairment of osteoclast function and osteopetrosis, but also altered osteoblast function, defective tissue organization, and very brittle bones. Whether this bone fragility in CatK deficiency results entirely from indirect effects of suppressed bone turnover because of impaired osteoclast function or perhaps represents a previously unappreciated more direct role for CatK in bone formation remains to be established.

Animals↗
PubMed2005

Genetic background influences cortical bone response to ovariectomy.

UNLABELLED: Peak bone mass is genetically determined, but little is known about the heritability of bone loss. Inbred mice were ovariectomized at 16 weeks of age and killed at three time-points after surgery. We found that the variation in estrogen deficit-related cortical bone loss is genetically determined. INTRODUCTION: Variability in adult bone morphology and composition among three inbred mouse strains-A/J, C57BL/6J (B6), and C3H/HeJ (C3H)-suggests that they gain bone in different ways during growth. In this study, we tested the hypothesis that these strains would also lose bone differently after estrogen deprivation. MATERIALS AND METHODS: Female A/J, B6, and C3H mice (N = 70/strain) were either ovariectomized (OVX) or sham-operated at 16 weeks of age and killed at 4, 8, and 16 weeks after surgery. Cortical bone histomorphometry was performed on right femoral mid-diaphyseal cross-sections. Mechanical properties were determined by loading left femoral mid-diaphyses to failure in four-point bending. RESULTS: Both OVX-A/J and OVX-B6 mice showed a 7-8% decrease in cortical area and width because of an 8-10% marrow expansion at 16 weeks after OVX. This bone loss did not affect mechanical properties in OVX-A/J femurs, but maximum load and stiffness in OVX-B6 decreased slightly (9%) at 4 and 8 weeks, and markedly (14-19%) at 16 weeks after OVX. In contrast, OVX-C3H showed a significant decrease in cortical area and width (6-7%) at 4 weeks after OVX and a slight decrease in the subperiosteal area (4%) at 8 weeks after OVX, although marrow area remained unchanged. Surprisingly, intracortical resorption spaces, which were present in sham-C3H mice, were greatly increased (+195%) in OVX-C3H mice at 8 weeks after OVX. Bone strength and stiffness in OVX-C3H mice decreased markedly (12-14%) at 4 weeks but slightly (8-10%) at 8 weeks after OVX. All indices except intracortical pore area in OVX-C3H mice returned to sham levels at 16 weeks after OVX. CONCLUSIONS: The magnitude, timing, and location of cortical bone loss after OVX varied significantly among A/J, B6, and C3H mice. The subsequent changes in mechanical properties after OVX depended on the variable bone patterns as well as the size and shape of the adult bone. Our results suggest that patterns of estrogen deficit-associated cortical bone loss are genetically determined.

Animals↗
PubMed2005

Genetic variation in bone growth patterns defines adult mouse bone fragility.

UNLABELLED: Femoral morphology and composition were determined for three inbred mouse strains between ages E18.5 and 1 year. Genotype-specific variation in postnatal, pubertal, and postpubertal growth patterns and mineral accrual explained differences in adult bone trait combinations and thus bone fragility. INTRODUCTION: Fracture risk is strongly regulated by genetic factors. However, this regulation is generally considered complex and polygenic. Therefore, the development of effective genetic-based diagnostic and treatment tools hinges on understanding how multiple genes and multiple cell types interact to create mechanically functional structures. The goal of this study was to connect variability in whole bone mechanical function, including measures of fragility, to variability in the biological processes underlying skeletal development. We accomplished this by testing for variation in bone morphology and composition among three inbred mouse strains from E18.5 to 1 year of age. MATERIALS AND METHODS: Mid-diaphyseal cross-sectional areas, diameters, moments of inertia, and ash content were determined for three strains of mice with widely differing adult whole bone femoral mechanical properties (A/J, C57BL/6J, and C3H/HeJ) at E18.5 and postnatal days 1, 7, 14, 28, 56, 112, 182, and 365 (n = 5-15 mice/strain/age). RESULTS: Significant differences in the magnitude and rate of change in morphological and compositional bone traits were observed among the three strains at each phase of growth, including prenatal, postnatal, pubertal, and adult ages. These genotype-specific variations in growth patterns mathematically determined how variation in adult bone trait combinations and mechanical properties arose. Furthermore, six bone traits were identified that characterize phenotypic variability in femoral growth. These include (1) bone size and shape at postnatal day 1, (2) periosteal and (3) endosteal expansion during early growth, (4) periosteal expansion and (5) endosteal contraction in later growth, and (6) ash content. These results show that genetic variability in adult bone traits arises from variation in biological processes at each phase of growth. CONCLUSIONS: Inbred mice achieve different combinations of adult bone traits through genotype-specific regulation of bone surface activity, growth patterns, and whole bone mineral accrual throughout femoral development. This study provides a systematic approach, which can be applied to the human skeleton, to uncover genetic control mechanisms influencing bone fragility.

Age Factors↗
PubMed2005

Relationship between bone morphology and bone quality in male tibias: implications for stress fracture risk.

UNLABELLED: Biomechanical properties were assessed from the tibias of 17 adult males 17-46 years of age. Tissue-level mechanical properties varied with bone size. Narrower tibias were comprised of tissue that was more brittle and more prone to accumulating damage compared with tissue from wider tibias. INTRODUCTION: A better understanding of the factors contributing to stress fractures is needed to identify new prevention strategies that will reduce fracture incidence. Having a narrow (i.e., more slender) tibia relative to body mass has been shown to be a major predictor of stress fracture risk and fragility in male military recruits and male athletes. The intriguing possibility that slender bones, like those shown in animal models, may be composed of more damageable material has not been considered in the human skeleton. MATERIALS AND METHODS: Polar moment of inertia, section modulus, and antero-posterior (AP) and medial-lateral (ML) widths were determined for tibial diaphyses from 17 male donors 17-46 years of age. A slenderness index was defined as the inverse ratio of the section modulus to tibia length and body weight. Eight prismatic cortical bone samples were generated from each tibia, and tissue-level mechanical properties including modulus, strength, total energy, postyield strain, and tissue damageability were measured by four-point bending from monotonic (n = 4/tibia) and damage accumulation (n = 4/tibia) test methods. Partial correlation coefficients were determined between each geometrical parameter and each tissue-level mechanical property while taking age into consideration. RESULTS: Significant correlations were observed between tibial morphology and the mechanical properties that characterized tissue brittleness and damageability. Positive correlations were observed between measures of bone size (AP width) and measures of tissue ductility (postyield strain, total energy), and negative correlations were observed between bone size (moment of inertia, section modulus) and tissue modulus. CONCLUSIONS: The correlation analysis suggested that bone morphology could be used as a predictor of tissue fragility and stress fracture risk. The average mechanical properties of cortical tissue varied as a function of the overall size of the bone. Therefore, under extreme loading conditions (e.g., military training), variation in bone quality parameters related to damageability may be a contributing factor to the increased risk of stress fracture for individuals with more slender bones.

Adolescent↗
PubMed2004

Genetic variation in structure-function relationships for the inbred mouse lumbar vertebral body.

UNLABELLED: Structure-function relationships were determined for L(5) vertebral bodies from three inbred mouse strains. Genetic variability in whole bone mechanical properties could be explained by a combination of the traits specifying the amount, distribution, and quality of the cortical and trabecular bone tissue. INTRODUCTION: Although phenotypically correlated with fracture, BMD may be disadvantageous to use in genetic and biomechanical analyses because BMD does not distinguish the contributions of the underlying morphological and compositional bone traits. Developing functional relationships between the underlying bone traits and whole bone mechanical properties should further our understanding of the genetics of bone fragility. MATERIALS AND METHODS: Microarchitecture and composition of L(5) vertebral bodies (n = 10/strain) from A/J, C57BL/6J, and C3H/HeJ inbred mouse strains were determined using muCT with an isotropic voxel size of 16 mum(3). Failure load, stiffness, and total deformation as a measure of ductility were measured in compression using a noncontact strain extensometer imaging system. A correlation analysis related morphological and compositional bone traits to whole bone mechanical properties. A multivariate analysis identified structure-function relationships for each genotype. RESULTS: No single bone trait accurately explained the genetic variation in mechanical properties. However, a combination of traits describing the amount, distribution, and quality of cortical and trabecular bone tissue explained >70% of the variation in vertebral mechanical properties. Importantly, structure-function relationships were unique among genotypes. CONCLUSIONS: Different genetic backgrounds use different combinations of underlying bone traits to create mechanically functional structures. Using a single complex trait such as BMD or BV/TV as the sole phenotypic marker in genetic analyses may prove to be disadvantageous because of the complex relationship between mechanical properties and the underlying bone traits. Therefore, considering multiple bone traits and the interaction among these bone traits is necessary to understand the relationship between genetic background and complex whole bone mechanical properties.

Analysis of Variance↗
PubMed2004

Long-term disuse osteoporosis seems less sensitive to bisphosphonate treatment than other osteoporosis.

UNLABELLED: We sought to determine whether risedronate can preserve cortical bone mass and mechanical properties during long-term disuse in dogs, assessed by histomorphometry and biomechanics on metacarpal diaphyses. Risedronate slowed cortical thinning and partially preserved mechanical properties, but it was unable to suppress bone loss to the degree seen in other osteoporoses. INTRODUCTION: Disuse induces dramatic bone loss resulting from greatly elevated osteoclastic resorption. Targeting osteoclasts with antiresorptive agents, such as bisphosphonates, should be an effective countermeasure for preventing disuse osteoporosis. MATERIALS AND METHODS: Single forelimbs from beagles (5-7 years old, n = 28) were immobilized (IM) for 12 months. Age-matched, non-IM dogs served as controls. One-half the animals received either risedronate (RIS, 1 mg/kg) or vehicle daily. Histomorphometry was performed on second metacarpal mid-diaphyses. Cortical mechanical properties were determined by testing third metacarpal diaphyses in four-point bending. RESULTS: IM caused marked reduction in cortical area (-42%) and cortical thinning (-40%) through endocortical resorption, extensive intracortical tunneling, and periosteal resorption; both bone resorption and formation were significantly elevated over control levels on all envelopes. IM also decreased maximum load and stiffness by approximately 80% compared with controls. RIS reduced both periosteal bone loss and marrow cavity expansion; however, cortical area remained significantly lower in RIS-treated IM animals than in untreated non-IM controls (-16%). RIS also increased resorption indices in all envelopes compared with nontreated IM, indicating that RIS suppressed osteoclast activity but not osteoclast recruitment. RIS did not affect bone formation. RIS treatment conserved some whole bone mechanical properties, but they were still significantly lower than in controls. There were no significant differences in tissue level material properties among the groups. CONCLUSION: RIS treatment reduces cortical bone loss at periosteal and endocortical surfaces caused by long-term immobilization, thus partially conserving tissue mechanical properties. This modest effect contrasts with more dramatic actions of the bisphosphonate in other osteoporoses. Our results suggest that risedronate impairs osteoclastic function but cannot completely overcome the intense stimulus for osteoclast recruitment during prolonged disuse.

Animals↗
PubMed2004

Targeted overexpression of androgen receptor in osteoblasts: unexpected complex bone phenotype in growing animals.

The androgen receptor (AR), as a classic steroid receptor, generally mediates biologic responses to androgens. In bone tissue, both AR and the estrogen receptor (ER) are expressed in a variety of cell types. Because androgens can be converted into estrogen via aromatase activity, the specific role of the AR in maintenance of skeletal homoeostasis remains controversial. The goal of this study was to use skeletally targeted overexpression of AR as a means of elucidating the specific role(s) for AR transactivation in bone homeostasis. Rat AR cDNA was cloned downstream of a 3.6-kb alpha1(I)-collagen promoter fragment and used to create AR-transgenic mice. AR-transgenic males gain less weight and body and femur length is shorter than wild-type controls, whereas females are not different. AR-transgenic males also demonstrate thickened calvaria and increased periosteal but reduced endosteal labeling by fluorescent labeling and reduced osteocalcin levels. High-resolution micro-computed tomography shows normal mineral content in both male and female AR-transgenic mice, but male AR-transgenics reveal a reduction in cortical area and moment of inertia. Male AR-transgenics also demonstrate an altered trabecular morphology with bulging at the metaphysis. Histomorphometric analysis of trabecular bone parameters confirmed the increased bone volume comprised of more trabeculae that are closer together but not thicker. Biomechanical analysis of the skeletal phenotype demonstrate reduced stiffness, maximum load, post-yield deflection, and work-to-failure in male AR-transgenic mice. Steady-state levels of selected osteoblastic and osteoclastic genes are reduced in tibia from both male and female transgenics, with the exception of increased osteoprotegerin expression in male AR-transgenic mice. These results indicate that AR action is important in the development of a sexually dimorphic skeleton and argue for a direct role for androgen transactivation of AR in osteoblasts in modulating skeletal development and homeostasis.

Animals↗
PubMed2004

The Collaborative Cross, a community resource for the genetic analysis of complex traits.

The goal of the Complex Trait Consortium is to promote the development of resources that can be used to understand, treat and ultimately prevent pervasive human diseases. Existing and proposed mouse resources that are optimized to study the actions of isolated genetic loci on a fixed background are less effective for studying intact polygenic networks and interactions among genes, environments, pathogens and other factors. The Collaborative Cross will provide a common reference panel specifically designed for the integrative analysis of complex systems and will change the way we approach human health and disease.

Animals↗
PubMed2003

Relationship between damage accumulation and mechanical property degradation in cortical bone: microcrack orientation is important.

The accumulation of damage and the associated degradation of the mechanical properties of cortical bone are postulated to contribute to age-, disease-, overuse-, and disuse-related skeletal fragilities. Therefore, gaining insight into the relationship between damage and degradation processes is essential in understanding the etiology of skeletal fractures. In investigating this relationship, the damage measure ideally needs to account for the size, the distribution density, and the orientation of microcracks. Existing measures of damage address the size and distribution density of microcracks; however, the orientation of cracks has not been well-investigated. Because the overall orientation of microcracks determines the material axis along which the greatest degradation will be experienced, we hypothesized that the incorporation of the relative orientation between microcracks and loading direction will improve the significance of the relationship between damage accumulation and material property degradation. A three-cycle damage protocol was used to induce tensile damage and to quantify the degradation of the elastic modulus of specimens from human donor femoral cortical bone (a 24-year-old and a 72-year-old man). Microcracks were evaluated by en bloc basic fuchsin staining of specimens after testing. The length (L(i)) and the orientation with respect to the loading direction (beta(i)) of each crack were quantified by a video microscopy system. Three damage measures were quantified for each specimen: the number of linear microcracks (Cr #), the sum of the crack lengths (SigmaL(i)) accounting for the microcrack size alone, and the sum of the projected crack length [SigmaL(Pi) = SigmaL(i)cos(beta(i))] accounting for both crack size and orientation. Inclusion of the orientation parameter improved the coefficient of determination between damage accumulation and the degradation of the elastic modulus: the coefficient of determination of the sum of the projected crack length (R(2) = 0.239) was 60% greater than that of the sum the projected crack length (R(2) = 0.149) and 33% greater than that of the number of linear microcracks (R(2) = 0.180). We conclude that microcrack orientation is an essential physical variable in the relationship between damage accumulation and degradation of mechanical properties of cortical bone tissue.

Adult↗
PubMed2003

Hierarchical relationship between bone traits and mechanical properties in inbred mice.

Osteoporotic fracture incidence and underlying risk factors like low peak bone mass are heritable, but the genetic basis of osteoporosis remains poorly understood. Based on beam theory, stating that mechanical properties of a structure depend on both the amount and quality of the constituent materials, we investigated the relationship between whole bone mechanical properties and a set of morphological and compositional traits in femurs of eight inbred mouse strains. K-means cluster analysis revealed that individual femora could be classified reliably according to genotype based on the combination of bone area (tissue amount), moment of inertia (tissue distribution), and ash content (tissue quality). This trait combination explained 66-88% of the inter-strain variability in four whole-bone mechanical properties that describe all aspects of the failure process, including measures of brittleness. Stiffness and maximum load were functionally associated with cortical area, while measures of brittleness were associated with ash content. In contrast, work-to-failure was not directly related to a single trait but depended on a combination of trait magnitudes. From these findings, which were entirely consistent with established mechanical theory, we developed a hierarchical paradigm relating the mechanical properties that define bone fragility with readily measurable phenotypic traits that exhibit strong heritability. This paradigm will help guide the search for genes that underlie fracture susceptibility and osteoporosis. Moreover, because the traits we examined are measurable with non-invasive means, this approach may also prove directly applicable to osteoporosis risk assessment.

Animals↗
PubMed2002

A syndrome of joint laxity and impaired tendon integrity in lumican- and fibromodulin-deficient mice.

Lumican and fibromodulin regulate the assembly of collagens into higher order fibrils in connective tissues. Here, we show that mice deficient in both of these proteoglycans manifest several clinical features of Ehlers-Danlos syndrome. The Lum(-/-)Fmod(-/-) mice are smaller than their wild type littermates and display gait abnormality, joint laxity, and age-dependent osteoarthritis. Misaligned knee patella, severe knee dysmorphogenesis, and extreme tendon weakness are the likely causes for joint laxity in the double-nulls. Fibromodulin deficiency alone leads to significant reduction in tendon stiffness in the Lum(+/+)Fmod(-/-) mice, with further loss in stiffness in a Lum gene dose-dependent way. At the protein level, we show marked increase of lumican in Fmod(-/-) tendons, which may partially rescue the tendon phenotype in this genotype. These results establish fibromodulin as a key regulator and lumican as a modulator of tendon strength. A disproportionate increase in small diameter immature collagen fibrils and a lack of progression to mature, large diameter fibrils in the Fmod(-/-) background may constitute the underlying cause of tendon weakness and suggest that fibromodulin aids fibril maturation. This study demonstrates that the collagen fibril-modifying proteoglycans, lumican and fibromodulin, are candidate genes and key players in the pathogenesis of certain types of Ehlers-Danlos syndrome and other connective tissue disorders.

Animals↗
PubMed2002

Hydrostatic pressure induces apoptosis in human chondrocytes from osteoarthritic cartilage through up-regulation of tumor necrosis factor-alpha, inducible nitric oxide synthase, p53, c-myc, and bax-alpha, and suppression of bcl-2.

Hydrostatic pressure (HP) is thought to increase within cartilage extracellular matrix as a consequence of fluid flow inhibition. The biosynthetic response of human articular chondrocytes to HP in vitro varies with the load magnitude, load frequency, as well as duration of loading. We found that continuous cyclic HP (5 MegaPascals (MPa) for 4 h; 1 Hz frequency) induced apoptosis in human chondrocytes derived from osteoarthritic cartilage in vitro as evidenced by reduced chondrocyte viability which was independent of initial cell densities ranging from 8.1 x 10(4) to 1.3 x 10(6) cells ml(-1). HP resulted in internucleosomal DNA fragmentation, activation of caspase-3, and cleavage of poly-ADP-ribose polymerase (PARP). At the molecular level, induction of apoptosis by HP was characterized by up-regulation of p53, c-myc, and bax-alpha after 4 h with concomitant down-regulation of bcl-2 after 2 h at 5 MPa as measured by RT-PCR. In contrast, beta-actin expression was unchanged. Real-time quantitative RT-PCR confirmed a HP-induced (5 MPa) 1.3-2.6 log-fold decrease in bcl-2 mRNA copy number after 2 and 4 h, respectively, and a significant increase (1.9-2.5 log-fold) in tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) mRNA copy number after 2 and 4 h, respectively. The up-regulation of p53 and c-myc, and the down-regulation of bcl-2 caused by HP were confirmed at the protein level by Western blotting. These results indicated that HP is a strong inducer of apoptosis in osteoarthritic human chondrocytes in vitro.

Apoptosis↗