Tendons are characterised by an inferior healing capacity when compared to other tissues, ultimately resulting in the formation of a pathologically altered extracellular matrix structure. Although our understanding of the underlying causes for the development and progression of tendinopathies remains incomplete, mounting evidence indicates a coordinated interplay between tendon-resident cells and the ECM is critical. Our recent results demonstrate that the matricellular protein SPARC (Secreted protein acidic and rich in cysteine) is essential for regulating tendon tissue homeostasis and maturation by modulating the tissue mechanical properties and aiding in collagen fibrillogenesis [1,2]. Consequently, we speculate that SPARC may also be relevant for tendon healing.

In a rat patellar tendon window defect model, we investigated whether the administration of recombinant SPARC protein can modulate tendon healing. Besides the increased mRNA expression of collagen type 1 and the downregulation of collagen type 3, a robust increase in the expression of pro-regenerative fibroblast markers in the repair tissue after a single treatment with rSPARC protein was observed. Additionally, pro-fibrotic markers were significantly decreased by the administration of rSPARC. Determination of structural characteristics was also assessed, indicating that the ECM structure can be improved by the application of rSPARC protein. Therefore, we believe that SPARC plays an important role for tendon healing and the application of recombinant SPARC to tendon defects has great potential to improve functional tendon repair.

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network.

hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting.

Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics.

We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases.

Osteoporosis (OP) and osteoarthritis (OA) are leading causes of musculoskeletal dysfunction in elderly, with chondrocyte senescence, inflammation, oxidative stress, subcellular organelle dysfunction, and genomic instability as prominent features. Age-related intestinal disorders and gut dysbiosis contribute to host tissue inflammation and oxidative stress by affecting host immune responses and cell metabolism. Not surprisingly, the development of OP and OA correlate with dysregulations of the gut microflora in rodents and humans. Intestinal microorganisms produce metabolites, including short-chain fatty acids, bile acids, trimethylamine N-oxide, and liposaccharides, affecting mitochondrial function, metabolism, biogenesis, autophagy, and redox reactions in chondrocytes to regulate joint homeostasis. Modulating the abundance of specific gut bacteria, like Lactobacillus and Bifidobacterium, by probiotics or fecal microbiota transplantation appears to suppress age-induced chronic inflammation and oxidative damage in musculoskeletal tissue and holds potential to slow down OP development. The talk will highlight treatment options with probiotics or metabolites for modulating the progression of OA and OP.

Calcium is an important element for a wide range of physiological functions including muscle contraction, neuronal activity, exocytosis, blood coagulation and cell communication. In the musculoskeletal system calcium is crucial for the structural integrity of bones, teeth, intervertebral disc and articular cartilage. At the cellular level calcium acts as a second messenger. Calcium signalling uses intracellular calcium ions to drive intracellular communication and signal transduction processes. When calcium enters the cell it exerts allosteric regulatory effects on many enzymes and proteins. Examining the role of calcium in chondrocyte biology is important for understanding the role for this divalent ion in the metabolic modulation of chondrocyte function in health and disease. This includes the study of calcium transport systems such as channels, transporters and pumps involved in calcium homeostasis in chondrocytes and how existing pharmacological drugs act on these transport systems. L-type calcium channel blockers are drugs used as cardiac antiarrhythmics or antihypertensives, depending on whether the drugs have higher affinity for the heart (the phenylalkylamines, like verapamil), or for the blood vessels (the dihydropyridines, like nifedipine). L-type calcium channels are present in many musculoskeletal tissues including skeletal muscle, smooth muscle, bone and cartilage. L-type calcium channel inhibitors like nifedipine used for the treatment of some forms of hypertension modulate calcium-mediated events in chondrocytes under dynamic loading, thus affecting metabolism, osmotic responses and extracellular matrix turnover in cartilage. The aim of our work is to understand the impact of L-type calcium channel inhibitors used for the treatment of hypertension on chondrocytes and on the chondrogenic differentiation of bone marrow derived mesenchymal stem cells (MSCs). This knowledge will enhance our understanding of the development of osteoarthritis (OA) and may lead to new opportunities for chondroprotection and regenerative medicine for OA. We have used electrophysiology to demonstrate L-type calcium currents in chondrocytes immediately after pharmacological activation with the calcium channel opener Bay-K8644. We have also used immunohistochemistry to demonstrate expression of the a1C subunit Ca_v1.2 (CACNA1C) in human chondrocytes and MSCs. Inhibitors of L-type calcium channels such as nifedipine downregulate mitochondrial respiration and ATP production in MSCs but not in chondrocytes. Nifedipine inhibits proliferation of chondrocytes and enhances glycolytic capacity in chondrocytes, promoting glycolytic reserve in both MSCs and chondrocytes. Nifedipine can also stimulate chondrogenic differentiation in MSCs (with or without growth factors). Metabolic responses to nifedipine differs in mesenchymal stem cells and chondrocytes highlighting important metabolic differences between these cells. In summary, antihypertensive drugs such as nifedipine can affect the biological function of chondrocytes and MSCs and may modulate the course of OA progression and impact on cartilage repair.

Aim

Staphylococcus aureus (SA) chronic bone and joint infections (BJI) are characterized by a progressive destruction of bone tissue associated to SA persistence which results in a large number of relapses (10–20%). The main factors proposed for these failures are: i) a weak diffusion of antibiotics in bone tissue, ii) formation of biofilm, iii) the bacterial internalization by the cells responsible for bone mineralization, namely the osteoblasts (OB).

Our in vitro and in vivo work aimed at providing new information on the impact of SA, more specifically of internalized SA, on bone homeostasis.

Mechanical loading plays an essential role in both tendon development and degradation. However, the underlying mechanism of how tendons sense and response to mechanical loading remains largely unknown. SPARC, a multifunctional extracellular matrix glycoprotein, modulates cell extracellular matrix contact, cell-cell interaction, ECM deposition and cell migration. Adult mice with SPARC deficiency exhibited hypoplastic tendons in load-bearing zone. By investigating tendon maturation in different stages, we found that hypoplastic tendons developed at around postnatal 3 weeks when the mice became actively mobile. The in vitro experiments on primary tendon derived stem cells demonstrated that mechanical loading induced SPARC production and AKT/S6K signalling activation, which was disrupted by deleting SPARC causing reduced collagen type I production, suggesting that mechanical loading was harmful to tendon homeostasis without SPARC. In vivo treadmill training further confirmed that increased loading led to reduced Achilles tendon size and eventually caused tendon rupture in SPARC-/− mice, whereas no abnormality was seen in WT mice after training. We then investigate whether paralysing the hindlimb of SPARC-/− mice using BOTOX from postnatal 2 weeks to 5 weeks would delay the hypoplastic tendon development. Increased patellar tendon thickness was shown in SPARC-/− mice by reducing mechanical loading, whereas opposite effect was seen in WT mice. Finally, we identified a higher prevalence of a missense SNP in the SPARC gene in patients who suffered from a rotator cuff tear. In conclusion, SPARC is a mechano-sensor that regulates tendon development and homeostasis.

Osteoporosis is a major healthcare burden, responsible for significant morbidity and mortality. Manipulating bone homeostasis would be invaluable in treating osteoporosis and optimising implant osseointegration. Strontium increases bone density through increased osteoblastogenesis, increased bone mineralisation, and reduced osteoclast activity. However, oral treatment may have significant side effects, precluding widespread use. We have recently shown that controlled disorder nanopatterned surfaces can control osteoblast differentiation and bone formation. We aimed to combine the osteogenic synergy of nanopatterning with local strontium delivery to avoid systemic side effects.

Using a sol-gel technique we developed strontium doped and/or nanopatterned titanium surfaces, with flat titanium controls including osteogenic and strontium doped media controls. These were characterised using atomic force microscopy and ICP-mass spectroscopy. Cellular response assessed using human osteoblast/osteoclast co-cultures including scanning electron microscopy, quantitative immunofluorescence, histochemical staining, ELISA and PCR techniques. We further performed RNAseq gene pathway combined with metabolomic pathway analysis to build gene/metabolite networks.

The surfaces eluted 800ng/cm2 strontium over 35 days with good surface fidelity. Osteoblast differentiation and bone formation increased significantly compared to controls and equivalently to oral treatment, suggesting improved osseointegration. Osteoclast pre-cursor survival and differentiation reduced via increased production of osteoprotegrin. We further delineated the complex cellular signalling and metabolic pathways involved including unique targets involved in osteoporosis.

We have developed unique nanopatterned strontium eluting surfaces that significantly increase bone formation and reduce osteoclastogenesis. This synergistic combination of topography and chemistry has great potential merit in fusion surgery and arthroplasty, as well as providing potential targets to treat osteoporosis.

Proliferation of synovial Mesenchymal Stromal/Stem Cells (MSCs) leads to synovial hyperplasia (SH) following Joint Surface Injury (JSI). Uncontrolled Yap activity causes tissue overgrowth due to modulation of MSC proliferation. We hypothesised that YAP plays a role in SH following JSI.

A spatiotemporal analysis of Yap expression was performed using the JSI model in C57Bl/6 mice. Synovial samples from patients were similarly analysed. Gdf5-Cre;Yap1fl/fl;Tom mice were created to determine the effect YAP1 knockout in Gdf5 lineage cells on SH after JSI.

In patients, Yap expression was upregulated in activated synovium, including a subset of CD55 positive fibroblast-like synoviocytes in the synovial lining (SL). Cells staining positive for the proliferation marker Ki67 expressed active YAP.

In mice, Yap was highly expressed in injured knee joint synovium compared to controls. Yap mRNA levels at 2 (p<0.05) and 8 days (p<0.001) after injury were increased.

Conditional Yap1 knockout in Gdf5 progeny cells prevented hyperplasia of synovial lining (SL) after JSI. Cellularity was significantly decreased in the SL but not in the sub-lining of injured Yap1 knockout- compared to control mice. The percentage of cells in synovium that were Tom+ increased in response to JSI in control and haplo-insufficient but not in YAP1 knockout mice (p<0.05).

Modulation of YAP and proliferation of MSCs in the synovium after JSI provides a system to study the role of SH after trauma in re-establishing joint homeostasis and is a potential novel therapeutic target for the treatment of post traumatic OA.

Industrialized countries experience a population aging. Elderly patients, due to the experienced immunity, have a constant pro-inflammatory milieu. Little is known on how adaptive immunity impacts the tissue homeostasis and regeneration. The standardized housing of lab animals is specific pathogen free (SPF). However, this housing condition hinders antigen exposure and thus an aging of the adaptive immune system. We hypothesized that exposure to antigens and a developing adaptive immunity will impact tissue homeostasis and regeneration in mice. Mice kept under SPF housing or non-SPF were examined towards their immune status via flow cytometry, bone structure via microCT and bone competence via biomechanical torsional testing. MSCs from these mice were analyzed regarding their differentiation potential and ECM production under various immune cell signaling. Bone regeneration was analyzed in vivo in a mouse osteotomy model. The memory and effector compartment of the adaptive immunity was significantly increased in mice under non-SPF housing. This housing led to an increased femoral cortical thickness and torsional stiffness (p<0,05), whereas the tissue mineral density was not affected. The differentiation potential of stem cells under the influence of an aged immune milieu was significantly reduced. Bone formation was highly affected by the immune status and availed of a naïve immune cell milieu. Adaptive immunity directly impacts bone tissue formation, by exhibiting a constant stress, leading to structural differences in bone tissue organization as well as mechanical competence. For experimental settings, it appears highly relevant if mouse models have had the chance to develop an experienced immune system.

Bone is a connective tissue that undergoes constant remodeling. Any disturbances during this process may result in undesired pathological conditions. A single nucleotide substitution (596T-A) in exon eight which leads to a M199K mutation in human RANKL was found to cause osteoclast-poor autosomal recessive osteopetrosis (ARO). Patients with ARO cannot be cured by hematopoietic stem cell transplantation and, without proper treatments, will die in their early age. To date, how this mutation alters RANKL function has not been characterized. We thus hypothesized that hRANKL M199 residue is a structural determinant for normal RANKL-RANK interaction and osteoclast differentiation. By sharing our findings, we aim to achieve an improved clinical outcome in treating bone-related diseases such as osteoporosis, ARO and osteoarthritis.

Site-directed mutagenesis was employed to create three rat RANKL mutants, replacing the methionine 200 (human M199 equivalent residue) with either lysine (M200K), alanine (M200A) or glutamic acid (M200E). Recombinant proteins were subsequently purified through affinity chromatography and visualized by Coomassie blue staining and western blot. MTS was carried out before osteoclastogenesis assay in vitro to measure the cellular toxicity. Bone resorption pit assay, immuno-fluorescent staining, luciferase reporter assay, RT-PCR, western blot and calcium oscillation detection were also conducted to explore the biological effect of rRANKL mutants. Computational modeling, thermal Shift Assay, western blot and protein binding affinity experiments were later carried out for structural analyses.

rRANKL mutants M200K/A/E showed a drastically reduced ability to induce osteoclast formation and did not demonstrate features of competitive inhibition against wild-type rRANKL. These mutants are all incapable of supporting osteoclastic polarization and bone resorption or activating RANKL-induced osteoclast marker gene transcription. Consistently, they were unable to induce calcium flux, and also showed a diminished induction of IκBa degradation and activation of NF-kB and NFATc1 transcriptional activity. Furthermore, the transcriptional activation of the antioxidant response element (ARE) crucial in modulating oxidative stress and providing cytoprotection was also unresponsive to stimulation with rM200s. Structural analyses showed that rM200 is located in a hydrophobic pocket critical for protein folding. Thermal shift and western blot assays suggested that rM200 mutants formed unstructured proteins, with disturbed trimerisation and the loss of affinity to its intrinsic receptors RANK and OPG.

Taken together, we first demonstrates the underlying cause of M199-meidated ARO in a cellular and molecular level by establishing a phenotype in BMMs similar to observed in human samples. Further investigation hints the structural significance of a hydrophobic pocket within the TNF-like region. Combined with pharmaceutical studies on small-molecule drugs, this finding may represent a therapeutic target motif for future development of anti-resorptive treatments.

Background/Aims

Bisphosphonates play an important role in the treatment of catabolic bone diseases such as osteoporosis. In addition to their anti-resorptive activity exerted by their proapoptotic effect on osteoclasts, recent data suggest that nitrogen-containing bisphosphonates (N-BP) may also promote osteogenic differentiation by an unknown mechanism. Similar bone-anabolic effects have been attributed to cholesterol-lowering statins, which represent another class of mevalonate pathway inhibitors besides N-BP, suggesting a common mode of action. In vascular endothelial cells statins were recently shown to activate the Mek5/Erk5 mitogen-activated protein kinase cascade, which plays an important role in cellular differentiation, apoptosis or inflammatory processes. Here we evaluated whether N-BPs may also target the Mek5/Erk5 pathway and analysed the consequence of Erk5 activation on bone-relevant gene expression, calcification and osteoblast differentiation.

At present bone scan is the only objective indicator of homeostasis in the bone and adjacent joint. This prospective study of 19 consecutive osteotomies in 17 patients was undertaken to see whether homeostasis is achieved around the knee after high tibial osteotomy for medial compartment osteoarthritis.

All cases underwent preoperative clinical, radiological and bone scan evaluation. Clinical evaluation included an SF12 score. Radiographs included standard anteroposterior, lateral, intercondylar and skyline patellar views, as well as standing views for measuring the mechanical axis and calculating the degree of correction. The three-phase technecium-99 m scan included blood-flow, blood pool uptake and delayed static imaging. As part of the surgical procedure an arthroscopic examination was performed and the degree of degeneration in all three compartments of the knee were noted. An excision wedge osteotomy was performed, aiming to achieve a 3° mechanical valgus alignment. Tension wire fixation allowed immediate mobilisation. One year postoperatively the clinical examination, standing radiographs and bone scans were repeated and the results statistically analysed.

One year postoperatively the mean SF12 score had improved from a preoperative 13.6 to 21.2 and the mean mechanical alignment from 6.3° of varus (3° to 12°) to 2° of valgus (0° to 4°). The significant decrease in isotope uptake in the medial compartment correlated with the clinical improvement and improved alignment. There was a significant reciprocal change from high uptake in the medial compartment preoperatively to high uptake in the lateral compartment one year postoperatively.

Medial compartment homeostasis was achieved one year after tibial osteotomy, but homeostasis was not achieved in the whole joint. Although correction was so conservative that it resulted in a mean of only 2° of mechanical valgus alignment, the area of increased uptake shifted from medial to lateral compartment. It is probable that the preoperative varus alignment led to reduced load-bearing and disuse osteoporosis in the lateral compartment, and that the increased uptake represents metabolic response to greater load. Possibly lateral compartment homeostasis will be restored over time.

This study shows that even a mild realignment improves homeostasis in the medial compartment. With the development of biological resurfacing, the importance of osteotomy may increase.

Osteoarthritis (OA) is a highly prevalent degenerative joint disorder characterized by joint pain and physical disability. Aberrant subchondral bone induces pathological changes and is a major source of pain in OA. In the subchondral bone, which is highly innervated, nerves have dual roles in pain sensation and bone homeostasis regulation. The interaction between peripheral nerves and target cells in the subchondral bone, and the interplay between the sensory and sympathetic nervous systems, allow peripheral nerves to regulate subchondral bone homeostasis. Alterations in peripheral innervation and local transmitters are closely related to changes in nociception and subchondral bone homeostasis, and affect the progression of OA. Recent literature has substantially expanded our understanding of the physiological and pathological distribution and function of specific subtypes of neurones in bone. This review summarizes the types and distribution of nerves detected in the tibial subchondral bone, their cellular and molecular interactions with bone cells that regulate subchondral bone homeostasis, and their role in OA pain. A comprehensive understanding and further investigation of the functions of peripheral innervation in the subchondral bone will help to develop novel therapeutic approaches to effectively prevent OA, and alleviate OA pain.

Cite this article: Bone Joint Res 2022;11(7):439–452.

This article presents a unified clinical theory that links established facts about the physiology of bone and homeostasis, with those involved in the healing of fractures and the development of nonunion. The key to this theory is the concept that the tissue that forms in and around a fracture should be considered a specific functional entity. This ‘bone-healing unit’ produces a physiological response to its biological and mechanical environment, which leads to the normal healing of bone. This tissue responds to mechanical forces and functions according to Wolff’s law, Perren’s strain theory and Frost’s concept of the “mechanostat”. In response to the local mechanical environment, the bone-healing unit normally changes with time, producing different tissues that can tolerate various levels of strain. The normal result is the formation of bone that bridges the fracture – healing by callus. Nonunion occurs when the bone-healing unit fails either due to mechanical or biological problems or a combination of both. In clinical practice, the majority of nonunions are due to mechanical problems with instability, resulting in too much strain at the fracture site. In most nonunions, there is an intact bone-healing unit. We suggest that this maintains its biological potential to heal, but fails to function due to the mechanical conditions. The theory predicts the healing pattern of multifragmentary fractures and the observed morphological characteristics of different nonunions. It suggests that the majority of nonunions will heal if the correct mechanical environment is produced by surgery, without the need for biological adjuncts such as autologous bone graft.

Cite this article: Bone Joint J 2016;98-B:884–91.