Age-related differences in hamstring tendon used as autograft in reconstructive anterior cruciate ligament surgery.

PURPOSE: The hamstring tendon is the most commonly used autograft material in reconstructive surgeries of anterior cruciate ligament (ACL) tears. Younger patients have worse surgical outcomes, with a higher risk of re-rupture. We hypothesized that age-related changes in hamstring tendon properties affect the tendon's propensity to rupture when used as an autograft in ACL reconstructions. The purpose of this study was to compare hamstring tendon samples obtained from people aged 20 years or younger to samples obtained from older people. METHODS: Superfluous hamstring tendon material was collected from 13 young donors (aged 16-20 years) and 17 older donors undergoing ACL reconstructive surgery. Sections of the tendon samples were used for biomechanical testing, structural analysis of collagen fibrils by electron microscopy, and global analysis of gene expression by microarrays. RESULTS: We found that tendon samples from the older group had lower Young's modulus than the younger group (P = 0.015), whereas the stress to failure was similar in the two groups. We found no difference in the average diameter of collagen fibrils between the two groups. Microarray analysis identified 162 differentially expressed genes (fold change ≥ 1.5, P < 0.05), with overrepresentation of several biological processes, including regulation of adhesion, migration, inflammation, and differentiation (fold enrichment > 2.0, false discovery rate P < 0.05). CONCLUSION: The hamstring tendon from younger people has higher stiffness than tendon from older people, and the profile of gene expression in tendon varies with age. These differences may negatively affect the performance of the hamstring tendon in ACL reconstructions in younger people.

Molecular characterisation of osteoblasts from bone obtained from people of Polynesian and European ancestry undergoing joint replacement surgery.

Population studies in Aotearoa New Zealand found higher bone mineral density and lower rate of hip fracture in people of Polynesian ancestry compared to Europeans. We hypothesised that differences in osteoblast proliferation and differentiation contribute to the differences in bone properties between the two groups. Osteoblasts were cultured from bone samples obtained from 30 people of Polynesian ancestry and 25 Europeans who had joint replacement surgeries for osteoarthritis. The fraction of cells in S-phase was determined by flow cytometry, and gene expression was analysed by microarray and real-time PCR. We found no differences in the fraction of osteoblasts in S-phase between the groups. Global gene expression analysis identified 79 differentially expressed genes (fold change > 2, FDR P < 0.1). Analysis of selected genes by real-time PCR found higher expression of COL1A1 and KRT34 in Polynesians, whereas BGLAP, DKK1, NOV, CDH13, EFHD1 and EFNB2 were higher in Europeans (P ≤ 0.01). Osteoblasts from European donors had higher levels of late differentiation markers and genes encoding proteins that inhibit the Wnt signalling pathway. This variability may contribute to the differences in bone properties between people of Polynesian and European ancestry that had been determined in previous studies.

The effect of age on the microarchitecture and profile of gene expression in femoral head and neck bone from patients with osteoarthritis.

Ageing of the skeleton is characterised by decreased bone mineral density, reduced strength, and increased risk of fracture. Although it is known that these changes are determined by the activities of bone cells through the processes of bone modelling and remodelling, details of the molecular mechanisms that underlie age-related changes in bone are still missing. Here, we analysed age-related changes in bone microarchitecture along with global gene expression in samples obtained from patients with osteoarthritis (OA). We hypothesised that changes would be evident in both microarchitecture and gene expression and aimed to identify novel molecular mechanisms that underlie ageing processes in bone. Samples of femoral head and neck were obtained from patients undergoing hip arthroplasty for OA, who were either ≤60 years or ≥70 years of age. Bone microarchitecture was analysed in cores of trabecular bone from the femoral head (17 from the younger group and 18 from the older), and cortical bone from the femoral neck (25 younger/22 older), using a Skyscan 1172 microCT scanner (Bruker). Gene expression was compared between the two age groups in 20 trabecular samples from each group, and 10 cortical samples from each group, using Clariom S Human microarrays (ThermoFisher Scientific). We found no significant changes between the two age groups in indices of trabecular or cortical bone microarchitecture. Gene expression analysis identified seven genes that had higher expression in the older group, including the transcription factor EGR1 and the glucose transporter SLC2A3 (GLUT3), and 21 differentially expressed genes in cortical bone samples (P<0.05, fold change>2). However, none of the comparisons of gene expression had false discovery rate-adjusted P<0.1. In contrast to our working hypothesis, we found only minor differences in gene expression and no differences in bone microarchitecture between the two age-groups. It is possible that pathological processes related to OA provide protection against age-related changes in bone. Our study suggests that in patients with OA, the bone properties measured here in femoral head and neck do not deteriorate significantly from the sixth to the eighth decade of life.

Human Cartilage Homogenates Influence the Crystallization of Monosodium Urate and Inflammatory Response to Monosodium Urate Crystals: A Potential Link Between Osteoarthritis and Gout.

OBJECTIVE: Monosodium urate (MSU) crystal deposition and gout flares frequently affect osteoarthritic joints. This study was undertaken to examine the effects of human cartilage homogenates on MSU crystallization and MSU crystal-induced inflammation. METHODS: Human cartilage homogenates were prepared from macroscopically healthy and macroscopically diseased knee joint samples. Crystallization assays were used to test the effects of cartilage homogenates or individual cartilage factors on MSU crystallization. Changes in urate solubility, crystal nucleation, crystal growth, and total crystal mass were determined. THP-1 cell assays were used to assess cytokine release following culture with MSU crystals grown in the presence or absence of cartilage homogenates or individual proteins. RESULTS: Addition of either 5% or 10% healthy cartilage homogenate increased the total mass of MSU crystals formed and resulted in formation of shorter MSU crystals compared to controls without cartilage homogenate. MSU crystal bows were observed in both the presence and absence of cartilage homogenate; however, bows formed in the presence of cartilage homogenates were significantly shorter than bows formed in their absence. There were no effect differences between macroscopically healthy and macroscopically diseased cartilage homogenates in all assessments. Addition of either type II collagen or albumin also led to the formation of shorter MSU crystals. In THP-1 cell assays, MSU crystals grown with healthy cartilage homogenate increased the release of interleukin-8, whereas MSU crystals grown with type II collagen or albumin had no effect on inflammatory cytokine release. CONCLUSION: In the presence of elevated urate levels, human cartilage homogenates increase MSU crystal formation and promote the formation of smaller crystals, which have greater inflammatory potential. These processes may contribute to the predilection of osteoarthritic joints to develop gout.

Reduced Bone Density and Cortical Bone Indices in Female Adiponectin-Knockout Mice.

A positive association between fat and bone mass is maintained through a network of signaling molecules. Clinical studies found that the circulating levels of adiponectin, a peptide secreted from adipocytes, are inversely related to visceral fat mass and bone mineral density, and it has been suggested that adiponectin contributes to the coupling between fat and bone. Our study tested the hypothesis that adiponectin affects bone tissue by comparing the bone phenotype of wild-type and adiponectin-knockout (APN-KO) female mice between the ages of 8-37 weeks. Using a longitudinal study design, we determined body composition and bone density using dual energy x-ray absorptiometry. In parallel, groups of animals were killed at different ages and bone properties were analyzed by microcomputed tomography, dynamic histomorphometry, 3-point bending test, nanoindentation, and computational modelling. APN-KO mice had reduced body fat and decreased whole-skeleton bone mineral density. Microcomputed tomography analysis identified reduced cortical area fraction and average cortical thickness in APN-KO mice in all the age groups and reduced trabecular bone volume fraction only in young APN-KO mice. There were no major differences in bone strength and material properties between the 2 groups. Taken together, our results demonstrate a positive effect of adiponectin on bone geometry and density in our mouse model. Assuming adiponectin has similar effects in humans, the low circulating levels of adiponectin associated with increased fat mass are unlikely to contribute to the parallel increase in bone mass. Therefore, adiponectin does not appear to play a role in the coupling between fat and bone tissue.

In vitro evaluation of a novel non-mulberry silk scaffold for use in tendon regeneration.

Tearing of the rotator cuff tendon in the shoulder is a significant clinical problem, with large/full-thickness tears present in ∼22% of the general population and recurrent tear rates postarthroscopic repair being quoted as high as 94%. Tissue-engineered biomaterials are increasingly being investigated as a means to augment rotator cuff repairs, with the aim of inducing host cell responses to increase tendon tissue regeneration. Silk-derived materials are of particular interest due to the high availability, mechanical strength, and biocompatibility of silks. In this study, Spidrex(®), a novel knitted, non-mulberry silk fibroin scaffold was evaluated in vitro for its potential to improve tendon regeneration. Spidrex was compared with a knitted Bombyx mori silk scaffold, a 3D collagen gel and Fiberwire(®) suture material. Primary human and rat tenocytes successfully adhered to Spidrex and significantly increased in number over a 14 day period (p<0.05), as demonstrated by fluorescent calcein-AM staining and alamarBlue(®) assays. A similar growth pattern was observed with human tenocytes cultured on the B. mori scaffold. Morphologically, human tenocytes elongated along the silk fibers of Spidrex, assuming a tenocytic cell shape, and were less circular with a higher aspect ratio compared with human tenocytes cultured on the B. mori silk scaffold and within the collagen gel (p<0.05). Gene expression analysis by real-time PCR showed that rat tenocytes cultured on Spidrex had increased expression of tenocyte-related genes such as fibromodullin, scleraxis, and tenomodulin (p<0.05). Expression of genes that indicate transdifferentiation toward a chondrocytic or osteoblastic lineage were significantly lower in tenocytes cultured on Spidrex in comparison to the collagen gel (p<0.05). Immunogenicity assessment by the maturation of and cytokine release from primary human dendritic cells demonstrated that Spidrex enhanced dendritic cell maturation in a similar manner to the clinically used suture material Fiberwire, and significantly upregulated the release of proinflammatory cytokines (p<0.05). This suggests that Spidrex may induce an early immune response postimplantation. While further work is required to determine what effect this immune response has on the tendon healing process, our in vitro data suggests that Spidrex may have the cytocompatibility and bioactivity required to support tendon regeneration in vivo.

Novel homozygous mutations in the osteoprotegerin gene TNFRSF11B in two unrelated patients with juvenile Paget's disease.

Most patients with juvenile Paget's disease (JPD) are homozygous for mutations in the gene TNFRSF11B that result in deficiency of osteoprotegerin (OPG) - a key regulator of bone turnover. So far, about 10 different OPG mutations have been described. The current study presents two novel OPG mutations in JPD patients. Patient 1 was diagnosed at the age of 9months when he presented with inability to sit up, slow growth, marked bone pain and very high levels of serum alkaline phosphatase. Patient 2 presented a milder phenotype. He was initially diagnosed with osteogenesis imperfecta, and although he had numerous fractures and bone deformity, he was still independently mobile at the age of 19years, when a diagnosis of JPD was confirmed. Sequence analysis of DNA samples from the patients determined two novel homozygous mutations in TNFSRF11B. Patient 1 (severe phenotype) had a large (245-251kbp) homozygous deletion beginning in intron 1 that resulted in loss of 4 of the 5 exons of TNFSRF11B, including the whole ligand-binding domain. Patient 2 had a homozygous missense mutation resulting in a Thr>Pro change in exon 2 of TNFSRF11B that is predicted to disrupt the OPG ligand-binding domain. Taken in conjunction with other published cases, these results are consistent with the hypothesis that the most severe phenotypes in JPD are seen in patients with major gene deletions or mutations affecting cysteine residues in the ligand-binding domain.