Keishi Marumo

Collagen cross-links as a determinant of bone quality: a possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus

Collagen cross-linking, a major post-translational modification of collagen, plays important roles in the biological and biomechanical features of bone. Collagen cross-links can be divided into lysyl hydroxylase and lysyl oxidase-mediated enzymatic immature divalent cross-links, mature trivalent pyridinoline and pyrrole cross-links, and glycation- or oxidation-induced non-enzymatic cross-links (advanced glycation end products) such as glucosepane and pentosidine. These types of cross-links differ in the mechanism of formation and in function. Material properties of newly synthesized collagen matrix may differ in tissue maturity and senescence from older matrix in terms of cross-link formation. Additionally, newly synthesized matrix in osteoporotic patients or diabetic patients may not necessarily be as well-made as age-matched healthy subjects. Data have accumulated that collagen cross-link formation affects not only the mineralization process but also microdamage formation. Consequently, collagen cross-linking is thought to affect the mechanical properties of bone. Furthermore, recent basic and clinical investigations of collagen cross-links seem to face a new era. For instance, serum or urine pentosidine levels are now being used to estimate future fracture risk in osteoporosis and diabetes. In this review, we describe age-related changes in collagen cross-links in bone and abnormalities of cross-links in osteoporosis and diabetes that have been reported in the literature.

Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis

The signaling molecule Wnt regulates bone homeostasis through β-catenin–dependent canonical and β-catenin–independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.

Degree of mineralization-related collagen crosslinking in the femoral neck cancellous bone in cases of hip fracture and controls

Based on the present definition of osteoporosis, both bone density and quality are important factors in the determination of bone strength. Collagen crosslinking is a determinant of bone quality. Cross-links can form enzymatically by the action of lysyl oxidase or non-enzymatically, resulting in advanced glycation end products. Collagen crosslinking is affected by tissue maturation as well as the degree of mineralization. Homocysteine and vitamin B6 (pyridoxal) are also regulatory factors of collagen crosslinking. We elucidate the relationship between the degree of mineralization and collagen cross-links in cancellous bone from hip fracture cases. We also determined plasma levels of homocysteine and pyridoxal. Twenty-five female intracapsular hip fracture cases (78 ± 6 years) and 25 age-matched postmortem controls (77 ± 6 years) were included in this study. Collagen crosslinking was analyzed after each bone specimen was fractionated into low (1.7–2.0 g/ml) and high (>2.0 g/ml) density fractions. The content of enzymatic (immature reducible and mature nonreducible cross-links) and nonenzymatic cross-link (pentosidine) were determined. In the controls, there was no difference in total enzymatic cross-links between low and high density bone, while pentosidine content was significantly higher in high density bone. In the fracture cases, not only reduced enzymatic cross-links in high density bone and increased pentosidine in both low and high density bone, but also higher plasma homocysteine and lower pyridoxal levels were evident compared with the controls. These results indicate that detrimental crosslinking in both low and high mineralized bone result in impaired bone quality in osteoporotic patients.

The “Ligamentization” Process in Human Anterior Cruciate Ligament Reconstruction With Autogenous Patellar and Hamstring Tendons A Biochemical Study

Background There is little information documenting whether the phenomenon of “ligamentization,” as proposed by Amiel, occurs in the human anterior cruciate ligament after clinically effective reconstruction. To clarify this point, we analyzed biochemical differences between the native anterior cruciate ligament; the patellar, semitendinosus, and gracilis tendons; and anterior cruciate ligaments reconstructed with autografts. Study Design Cohort study; Level of evidence, 2. Methods Fifty patients who underwent arthroscopically assisted anterior cruciate ligament reconstruction using either semi-tendinosus and gracilis tendon or bone-patellar tendon-bone autografts were selected for the study. Samples of grafted tissue were collected during arthroscopy and quantitatively analyzed for collagen content and the amount of reducible and nonreducible crosslinks at 4 to 6 postoperative months in patients with semitendinosus and gracilis tendon grafts and at 11 to 13 months in all patients with semitendinosus and gracilis tendon or bone-patellar tendon-bone grafts. Results The total collagen content and nonreducible/reducible crosslink ratios increased significantly during the postoperative period (P < .05). The dihydroxylysinonorleucine/hydroxylysinonorleucine ratio was 3.11 ± 0.56 in the native anterior cruciate ligament, 1.21 ± 0.47 in the patellar tendon, and 3.59 ± 1.58 in the anterior cruciate ligaments reconstructed with bone-patellar tendon-bone autografts 1 year after surgery. The dihydroxylysinonorleucine/hydroxylysinonorleucine ratio in both semitendinosus and gracilis tendons was less than 1.0. However, in anterior cruciate ligaments reconstructed with semitendinosus and gracilis tendon autografts, it was 2.34 ± 0.98 at 4 to 6 months and 3.43 ± 1.61 at 11 to 13 months after the operation. Conclusions After anterior cruciate ligament reconstruction with autografts, biochemical characteristics of the graft resembled those of the native anterior cruciate ligament. These findings suggest that, regarding the amount of collagen crosslinks and their architecture, the phenomenon of ligamentization occurs in the successfully reconstructed human anterior cruciate ligament within 1 year after operation.

Cytoplasmic superoxide causes bone fragility owing to low‐turnover osteoporosis and impaired collagen cross‐linking

The aging process correlates with the accumulation of cellular and tissue damage caused by oxidative stress. Although previous studies have suggested that oxidative stress plays a pathologic role in the development of bone fragility, little direct evidence has been found. In order to investigate the pathologic significance of oxidative stress in bones, we analyzed the bone tissue of mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn‐SOD, encoded by the Sod1 gene; Sod1−/−). In this study, we showed for the first time that in vivo cytoplasmic superoxide caused a distinct weakness in bone stiffness and decreased BMD, aging‐like changes in collagen cross‐linking, and transcriptional alterations in the genes associated with osteogenesis. We also showed that the surface areas of osteoblasts and osteoclasts were decreased significantly in the lumbar vertebrae of Sod1−/− mice, indicating the occurrence of low‐turnover osteopenia. In vitro experiments demonstrated that intracellular oxidative stress induced cell death and reduced the proliferation in primary osteoblasts but not in osteoclasts, indicating that impaired osteoblast viability caused the decrease in osteoblast number and suppressed RANKL/M‐CSF osteoclastogenic signaling in bone. Furthermore, treatment with an antioxidant, vitamin C, effectively improved bone fragility and osteoblastic survival. These results imply that intracellular redox imbalance caused by SOD1 deficiency plays a pivotal role in the development and progression of bone fragility both in vivo and in vitro. We herein present a valuable model for investigating the effects of oxidative stress on bone fragility in order to develop suitable therapeutic interventions.

Diabetes, Collagen, and Bone Quality

Diabetes increases risk of fracture, although type 2 diabetes is characterized by normal or high bone mineral density (BMD) compared with the patients without diabetes. The fracture risk of type 1 diabetes as well as type 2 diabetes increases beyond an explained by a decrease of BMD. Thus, diabetes may reduce bone strength without change in BMD. Whole bone strength is determined by bone density, structure, and quality, which encompass the micro-structural and tissue material properties. Recent literature showed that diabetes reduces bone material properties rather than BMD. Collagen intermolecular cross-linking plays an important role in the expression of bone strength. Collagen cross-links can be divided into beneficial enzymatic immature divalent and mature trivalent cross-links and disadvantageous nonenzymatic cross-links (Advanced glycation end products: AGEs) induced by glycation and oxidation. The formation pathway and biological function are quite different. Not only hyperglycemia, but also oxidative stress induces the reduction in enzymatic cross-links and the formation of AGEs. In this review, we describe the mechanism of low bone quality in diabetes and the usefulness of the measurement of plasma or urinary level of AGEs for estimation of fracture risk.

Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction

Hyaluronan is a major molecule in joint fluid and plays a crucial role in joint motion and the maintenance of joint homeostasis. The concentration and average molecular weight of hyaluronan in the joint fluids are reduced in osteoarthritis and rheumatoid arthritis. To elucidate the underlying mechanism, we analyzed the message expression of three isoforms of hyaluronan synthase and hyaluronidase from knee synovium, using real-time reverse transcriptase polymerase chain reaction. Synovia were obtained from 17 patients with osteoarthritis, 14 patients with rheumatoid arthritis, and 20 healthy control donors. The message expression of hyaluronan synthase-1 and -2 in the synovium of both types of arthritis was significantly less than in the control synovium, whereas that of hyaluronidase-2 in the synovium of both arthritides was significantly greater than in the control synovium. The decreased expression of the messages for hyaluronan synthase-1 and -2 and/or the increased expression of the message for hyaluronidase-2 may be reflected in the reduced concentration and decreased average molecular weight of hyaluronan in the joint fluids of patients with osteoarthritis and rheumatoid arthritis.

Bone Quality in Diabetes

Diabetes is associated with increased risk of fracture, although type 2 diabetes is characterized by normal bone mineral density (BMD). The fracture risk of type 1 diabetes increases beyond an explained by a decrease of BMD. Thus, diabetes may be associated with a reduction of bone strength that is not reflected in the measurement of BMD. Based on the present definition, both bone density and quality, which encompass the structural and material properties of bone, are important factors in the determination of bone strength. Diabetes reduces bone quality rather than BMD. Collagen cross-linking plays an important role in bone strength. Collagen cross-links can be divided into lysyl hydroxylase and lysyl oxidase-mediated enzymatic immature divalent cross-links, mature trivalent cross-links, and glycation- or oxidation-induced non-enzymatic cross-links (Advanced Glycation End-products: AGEs) such as pentosidine. These types of cross-links differ in the mechanism of formation and in function. Not only hyperglycemia, but also oxidative stress induces the reduction in enzymatic beneficial cross-links and the accumulation of disadvantageous AGEs in bone. In this review, we describe the mechanism of low bone quality in diabetes.

Comparison of effects of alfacalcidol and alendronate on mechanical properties and bone collagen cross-links of callus in the fracture repair rat model.

Both bone density and quality are important determinants of bone strength. Bone quality is prescribed by matrix characteristic including collagen cross-linking and bone structural characteristics and is important in reinforcement of bone strength. We investigated the effects of alfacalcidol (ALF), a prodrug of calcitriol, and alendronate (ALN), a bisphosphanate, on the mechanical properties and content of enzymatic cross-links in femoral bone using a fracture repair rat model. Forty 3-month-old female Wistar-Imamichi rats were randomized into 4 groups: SHAM (sham-operated+vehicle), OVX (ovariectomy+vehicle), ALF (ovariectomy+ALF, 0.1 microg/kg/d, p.o.) and ALN (ovariectomy+ALN, 10 microg/kg/d, s.c.). Treatment began immediately after SHAM or OVX surgery. Three weeks later, all animals underwent transverse osteotomies at the midshaft of the left femur. Treatment was continued and rats were sacrificed at 12 weeks post-fracture for evaluation by X-ray radiography, micro-CT, pQCT, biomechanical testing and bone histomorphometry. In the ALN group, no new cortical shell appeared and the callus diameter was significantly larger than in the OVX group (p<0.05). Stiffness of fractured callus in the ALF group, but not in the ALN group, was significantly higher than in the OVX group. Youngs modulus in the ALN group was significantly decreased compared to the OVX group. Moreover, micro-CT analysis showed that ALN treatment increased the lowly mineralized bone in the callus by, resulting in the highest content of woven bone area and lowest content of lamellar bone. The total amount of enzymatic cross-links in both the ALF and ALN groups was significantly higher than in the OVX control group. Of particular interest, the Pyr-to-Dpyr ratio was significantly decreased by ALF administration, suggesting that ALF but not ALN normalized the enzymatic cross-link patterns in fractured bone to the control level. In conclusion, ALN and ALF treatment increased bone strength via the distinctive effect on bone mass and quality. ALN formed larger calluses and increased enzymatic cross-links despite delayed woven bone remodeling into lamellar bone, whereas ALF treatment induced lamellar bone formation coincided with increasing in the enzymatic cross-linking and normalizing the cross-link pattern in callus to native bone pattern.

Effects of Collagen Crosslinking on Bone Material Properties in Health and Disease

Data have accumulated to show that various types of collagen crosslinking are implicated in the health of individuals, as well as in a number of disease states, such as osteoporosis, diabetes mellitus, chronic kidney disease, inflammatory bowel disease, or in conditions of mild hyperhomocysteinemia, or when glucocorticoid use is indicated. Collagen crosslinking is a posttranslational modification of collagen molecules and plays important roles in tissue differentiation and in the mechanical properties of collagenous tissue. The crosslinking of collagen in the body can form via two mechanisms: one is enzymatic crosslinking and the other is nonenzymatic crosslinking. Lysyl hydroxylases and lysyl oxidases regulate tissue-specific crosslinking patterns and quantities. Enzymatic crosslinks initially form via immature divalent crosslinking, and a portion of them convert into mature trivalent forms such as pyridinoline and pyrrole crosslinks. Nonenzymatic crosslinks form as a result of reactions which create advanced glycation end products (AGEs), such as pentosidine and glucosepane. These types of crosslinks differ in terms of their mechanisms of formation and function. Impaired enzymatic crosslinking and/or an increase of AGEs have been proposed as a major cause of bone fragility associated with aging and numerous disease states. This review focuses on the effects of collagen crosslinking on bone material properties in health and disease.