Marjolein C. H. van der Meulen

The ability of bone to adapt to external mechanical loads has been extensively studied, with mechanical stimuli increasing cortical and cancellous bone mass. However, the stem cell basis underlying this response is not well understood. To date, most studies focused on the role of differentiated cell populations in the skeletal response to loading. A recently discovered periosteal-specific skeletal stem cell marked by cathepsin K (CTSK) that drives intramembranous bone formation is a promising candidate to mediate load-induced bone formation. In this study, we sought to determine the influence of CTSK-lineage cells on the skeletal response to mechanical loading. We ablated cells expressing CTSK prior to initiating cyclic tibial compression for two weeks beginning at 16 weeks of age. We analyzed cortical and cancellous bone morphology at the tibial metaphysis and cortical bone morphology at the mid-diaphysis. Loading increased cortical, but not cancellous, bone mass. The amount of bone formed in response to loading did not differ when CTSK-expressing cells were ablated. CTSK-lineage cell ablation increased cortical bone mass primarily in regions subjected to tension and loading predominantly affected regions of bone under compression. To analyze the material composition of load-induced bone, we performed Raman spectroscopy along the periosteal surface of the diaphysis. CTSK-lineage cell ablation altered the influence of loading on B-type carbonate substation, a measure of tissue age. Overall, the amount of bone formed in response to loading did not differ in the absence of CTSK-lineage cells, but the material composition of load-induced cortical tissue was altered.

Abstract Applied in vivo loading, such as tibial compression, leads to robust bone formation in young rodents but diminished anabolic responses in adults. To evaluate age-related differences in biological pathways active with loading, we examined the metaphyseal transcriptomes following in vivo tibial compression in young (10-wk-old) and adult (26-wk-old) female C57Bl/6 mice. Animals underwent 1 bout tibial compression and were euthanized at 1, 3, or 24 h post-loading or loaded for 1 wk (n = 4-6/group). Differential gene expression and enriched biological processes were compared between loaded and contralateral control limbs. Few load-induced differentially expressed genes were shared between tissue compartments, across time points, and between young and adult mice. In young animals, the response of cancellous bone to loading was greater than the surrounding metaphyseal cortical shell at all timepoints examined. Following 1 bout of tibial compression, adults also had greater transcriptional responses in cancellous compared with cortical bone. However, load-induced gene expression was increased in the adult metaphyseal cortical shell compared with the cancellous core following 1 wk of loading. Despite previously established age-related reductions in the tissue-level response to loading, adults had 63% more load-induced differentially-expressed genes compared with young animals, 3919 vs 2402 total. Individual bone-associated gene expression in adults did not mirror the anabolic expression measured in young animals; the consistent load-induced upregulation of osteoblast genes in young animals was absent in adults. Most load-induced differentially expressed genes with high magnitude fold changes were novel with unknown roles in bone cells. The skeletally-relevant genes identified were related to osteoblast function and generally upregulated with loading. More bone-specific anabolic pathways were enriched with loading in cancellous compared to cortical bone. In conclusion, in both cancellous and cortical envelopes, adult mice have robust transcriptional responses to physiological loading that do not explain their reduced mechanoresponsiveness with age.

Mechanical loading induces bone formation in young rodents, but mechanoresponsiveness is reduced with age. Glycolytic activity and mitochondrial dysfunction increase with age and may change bone mechanotransduction. To evaluate load-induced changes to bioenergetic activity in young and adult animals, we loaded the tibia of 10-wk and 26-wk female C57BL/6J mice and examined transcriptomic responses at the mid-diaphysis, and metaphyseal cortical shell and cancellous core. Across all biological processes, oxidative phosphorylation and mitochondrial pathways were most often enriched with loading and had contrasting enrichment in young and adult animals. Following loading, young animals had temporally-coordinated differential expression of mitochondrial-associated genes, with greatest expression at the mid-diaphysis. In adults, bioenergetic gene expression was lower compared to young animals. To assess individual contributions of glycolysis and pyruvate-mediated oxidative phosphorylation to load-induced bone formation in vivo, we inhibited each pathway therapeutically and loaded the tibia of young and adult female mice for 2 weeks. In both young and adult mice, loading increased cortical bone mass, but inhibition of oxidative phosphorylation reduced cortical area and moment of inertia in both loaded and control limbs. Conversely, load-induced improvements of adult cancellous bone depended on glycolysis. In summary, mechanical loading transcriptionally activated mitochondrial pathways in an age-specific manner and bioenergetic inhibition revealed unique metabolic programs for cortical and cancellous bone.

Parathyroid hormone (PTH) increases bone mass and decreases fracture risk. However, the anabolic effects of PTH are limited to a period of approximately 24 months, motivating the need to maximize bone growth during this timeframe. Concurrent mechanical loading with weight-bearing exercise is synergistic with PTH treatment. We sought to determine if priming with PTH prior to initiating mechanical loading would enhance their synergistic effects. We pre-treated 10-week-old, female C57Bl/6 J mice with either PTH or saline vehicle (VEH) for six weeks. We subsequently initiated cyclic tibial compression for either two or six weeks while continuing PTH or VEH treatment. We analyzed bone morphology in cortical and cancellous compartments of the proximal tibia. To further explore the effects of PTH and loading in cancellous bone, we measured bone cell presence and changes in bone morphology via histology, immunohistochemistry, and dynamic histomorphometry. Concurrent treatment with PTH enhanced load-induced increases in bone mass in cortical bone but blunted the effects of loading in cancellous bone. PTH pre-treatment further increased load-induced changes in cortical bone mass and rescued the load effects in cancellous bone, returning values to those of VEH-treated animals. Osteoclast populations decreased with loading, independent of PTH treatment. Active osteoblast populations increased with PTH pre-treatment but did not change with loading. Bone formation rate increased with PTH pre-treatment in the 2-week group but did not differ between treatment groups after 6-weeks. Collectively, pre-treating with PTH prior to mechanical loading primed the skeletal tissue and enhanced the anabolic response of concurrent treatment and loading.

SUMMARY Objective: While physiological loads maintain cartilage health, both joint overload and abnormal joint mechanical loading contribute to osteoarthritis (OA) development. Here, we examined the role of abnormal mechanical loading on joint health by comparing the severity of OA development following a single overload event and repetitive joint overloads. Method: Cyclic tibial compression was applied to the left limbs of 26-week-old male mice at a peak load of 9N for either a single bout or daily bouts to initiate OA disease. Joint damage severity was morphologically examined using histology and microcomputed tomography at 6 weeks following the start of loading. Early-stage transcriptomic responses to loading were evaluated. Results: Joint damage was more severe at 6 weeks following a single bout of loading than after daily loading bouts. Severe cartilage damage, subchondral plate erosions, and soft tissue calcifications occurred following the single bout of loading. Daily loading bouts resulted in less severe cartilage damage and preserved subchondral plate integrity. A diverging transcriptomic response was identified in cartilage at 1 week with increased expression of fibrosis- and inflammation-related genes following a single bout of loading compared to daily loading. Conclusions: Even applied at hyperphysiological load magnitudes known to initiate cartilage damage, repetitive loading may induce protective effects in the joint and attenuate OA progression over time relative to a single bout of loading. Our findings suggest the potential of mechanotherapies that use repetitive loading as disease-modifying treatments for OA disease.

Osteoarthritis (OA) treatment is limited by the lack of effective nonsurgical interventions to slow disease progression. Here, we examined the contributions of the subchondral bone properties to OA development. We used parathyroid hormone (PTH) to modulate bone mass before OA initiation and alendronate (ALN) to inhibit bone remodeling during OA progression. We examined the spatiotemporal progression of joint damage by combining histopathological and transcriptomic analyses across joint tissues. The additive effect of PTH pretreatment before OA initiation and ALN treatment during OA progression most effectively attenuated load-induced OA pathology. Individually, PTH directly improved cartilage health and slowed the development of cartilage damage, whereas ALN primarily attenuated subchondral bone changes associated with OA progression. Joint damage reflected early transcriptomic changes. With both treatments, the structural changes were associated with early modulation of immunoregulation and immunoresponse pathways that may contribute to disease mechanisms. Overall, our results demonstrate the potential of subchondral bone-modifying therapies to slow the progression of OA.

Over the past two decades Biomedical Engineering has emerged as a major discipline that bridges societal needs of human health care with the development of novel technologies. Every medical institution is now equipped at varying degrees of sophistication with the ability to monitor human health in both non-invasive and invasive modes. The multiple scales at which human physiology can be interrogated provide a profound perspective on health and disease. We are at the nexus of creating "avatars" (herein defined as an extension of "digital twins") of human patho/physiology to serve as paradigms for interrogation and potential intervention. Motivated by the emergence of these new capabilities, the IEEE Engineering in Medicine and Biology Society, the Departments of Biomedical Engineering at Johns Hopkins University and Bioengineering at University of California at San Diego sponsored an interdisciplinary workshop to define the grand challenges that face biomedical engineering and the mechanisms to address these challenges. The Workshop identified five grand challenges with cross-cutting themes and provided a roadmap for new technologies, identified new training needs, and defined the types of interdisciplinary teams needed for addressing these challenges. The themes presented in this paper include: 1) accumedicine through creation of avatars of cells, tissues, organs and whole human; 2) development of smart and responsive devices for human function augmentation; 3) exocortical technologies to understand brain function and treat neuropathologies; 4) the development of approaches to harness the human immune system for health and wellness; and 5) new strategies to engineer genomes and cells.

Objective: The crosstalk of joint pathology with local lymph nodes in osteoarthritis (OA) is poorly understood. We characterized the change in T cells in lymph nodes following load-induced OA and established the association of the presence and migration of T cells to the onset and progression of OA. Methods: We used an in vivo model of OA to induce mechanical load-induced joint damage. After cyclic tibial compression of mice, we analyzed lymph nodes for T cells using flow cytometry and joint pathology using histology and microcomputed tomography. The role of T-cell migration and presence of T-cell type was examined using TCRα−/− and an immunomodulatory drug, Sphingosine-1-phosphate (S1P) receptor inhibitor-treated mice, respectively. Results: We demonstrate a significant increase in T-cell populations in local lymph nodes in response to joint injury in 10, 16, and 26-week-old mice, and as a function of load duration, 1, 2, and 6 weeks. T-cell expression of inflammatory cytokine markers increased in the local lymph nodes and was associated with load-induced OA progression in the mouse knee. Joint loading in TCRα−/− mice reduced both cartilage degeneration (OARSI: TCRα 0.568, 0.981–0.329 CI; WT 1.328, 2.353–0.749 CI) and osteophyte formation. Inhibition of T-cell egress from lymph nodes attenuated load-induced cartilage degradation (OARSI: Fingolimod: 0.509, 1.821–0.142 CI; Saline 1.210, 1.932–0.758 CI) and decreased localization of T cells in the synovium. Conclusions: These results establish the association of lymph node-resident T cells in joint damage and suggest that the S1P receptor modulators and T-cell immunotherapies could be used to treat OA.

Bone morphogenetic protein (BMP) gene delivery to Lewis rat lumbar intervertebral discs (IVDs) drives bone formation anterior and external to the IVD, suggesting the IVD is inhospitable to osteogenesis. This study was designed to determine if IVD destruction with a proteoglycanase, and/or generating an IVD blood supply by gene delivery of an angiogenic growth factor, could render the IVD permissive to intra-discal BMP-driven osteogenesis and fusion. Surgical intra-discal delivery of naïve or gene-programmed cells (BMP2/BMP7 co-expressing or VEGF165 expressing) +/- purified chondroitinase-ABC (chABC) in all permutations was performed between lumbar 4/5 and L5/6 vertebrae, and radiographic, histology, and biomechanics endpoints were collected. Follow-up anti-sFlt Western blotting was performed. BMP and VEGF/BMP treatments had the highest stiffness, bone production and fusion. Bone was induced anterior to the IVD, and was not intra-discal from any treatment. chABC impaired BMP-driven osteogenesis, decreased histological staining for IVD proteoglycans, and made the IVD permissive to angiogenesis. A soluble fragment of VEGF Receptor-1 (sFlt) was liberated from the IVD matrix by incubation with chABC, suggesting dysregulation of the sFlt matrix attachment is a possible mechanism for the chABC-mediated IVD angiogenesis we observed. Based on these results, the IVD can be manipulated to foster vascular invasion, and by extension, possibly osteogenesis.

Posttraumatic osteoarthritis (PTOA) is a subset of osteoarthritis that occurs after joint injury and is associated with degradation of articular cartilage and subchondral bone. As compared with primary osteoarthritis, PTOA occurs in a time window initiated by a traumatic event resulting in damage to layers of joint structure and alterations in joint shape. As techniques in open reduction and internal fixation continue to mature, our success in preventing posttraumatic osteoarthritis has not kept pace. Advances in research in the subchondral bone, inflammatory response, and joint mechanics continue to open our understanding of this posttraumatic process. In addition, there are possibilities emerging as biological agents to therapeutically alter the progression of PTOA.