The performance of artificial intelligence chatbot large language models to address skeletal biology and bone health queries.

Artificial intelligence (AI) chatbots utilizing large language models (LLMs) have recently garnered significant interest due to their ability to generate humanlike responses to user inquiries in an interactive dialog format. While these models are being increasingly utilized to obtain medical information by patients, scientific and medical providers, and trainees to address biomedical questions, their performance may vary from field to field. The opportunities and risks these chatbots pose to the widespread understanding of skeletal health and science are unknown. Here we assess the performance of 3 high-profile LLM chatbots, Chat Generative Pre-Trained Transformer (ChatGPT) 4.0, BingAI, and Bard, to address 30 questions in 3 categories: basic and translational skeletal biology, clinical practitioner management of skeletal disorders, and patient queries to assess the accuracy and quality of the responses. Thirty questions in each of these categories were posed, and responses were independently graded for their degree of accuracy by four reviewers. While each of the chatbots was often able to provide relevant information about skeletal disorders, the quality and relevance of these responses varied widely, and ChatGPT 4.0 had the highest overall median score in each of the categories. Each of these chatbots displayed distinct limitations that included inconsistent, incomplete, or irrelevant responses, inappropriate utilization of lay sources in a professional context, a failure to take patient demographics or clinical context into account when providing recommendations, and an inability to consistently identify areas of uncertainty in the relevant literature. Careful consideration of both the opportunities and risks of current AI chatbots is needed to formulate guidelines for best practices for their use as source of information about skeletal health and biology.

Artificial intelligence chatbots are increasingly used as a source of information in health care and research settings due to their accessibility and ability to summarize complex topics using conversational language. However, it is still unclear whether they can provide accurate information for questions related to the medicine and biology of the skeleton. Here, we tested the performance of three prominent chatbots­ChatGPT, Bard, and BingAI­by tasking them with a series of prompts based on well-established skeletal biology concepts, realistic physician­patient scenarios, and potential patient questions. Despite their similarities in function, differences in the accuracy of responses were observed across the three different chatbot services. While in some contexts, chatbots performed well, and in other cases, strong limitations were observed, including inconsistent consideration of clinical context and patient demographics, occasionally providing incorrect or out-of-date information, and citation of inappropriate sources. With careful consideration of their current weaknesses, artificial intelligence chatbots offer the potential to transform education on skeletal health and science.

Characterization of adult human skeletal cells in different tissues reveals a CD90+CD34+ periosteal stem/progenitor population.

The periosteum plays a crucial role in bone healing and is an important source of skeletal stem and progenitor cells. Recent studies in mice indicate that diverse populations of skeletal progenitors contribute to growth, homeostasis and healing. Information about the in vivo identity and diversity of skeletal stem and progenitor cells in different compartments of the adult human skeleton is limited. In this study, we compared non-hematopoietic populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-dimensional clustering analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+CD146- population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations in vitro. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. CD90+ cells are also highly enriched for CFU-F in bone marrow and endosteum, but not articular cartilage. In conclusion, our study indicates considerable diversity in the non-hematopoietic cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal progenitor cells reside within the CD90+CD34+ population.

Hematopoietic and stromal DMP1-Cre labeled cells form a unique niche in the bone marrow.

Skeletogenesis and hematopoiesis are interdependent. Niches form between cells of both lineages where microenvironmental cues support specific lineage commitment. Because of the complex topography of bone marrow (BM), the identity and function of cells within specialized niches has not been fully elucidated. Dentin Matrix Protein 1 (DMP1)-Cre mice have been utilized in bone studies as mature osteoblasts and osteocytes express DMP1. DMP1 has been identified in CXCL12+ cells and an undefined CD45+ population. We crossed DMP1-Cre with Ai9 reporter mice and analyzed the tdTomato+ (tdT+) population in BM and secondary hematopoietic organs. CD45+tdT+ express myeloid markers including CD11b and are established early in ontogeny. CD45+tdT+ cells phagocytose, respond to LPS and are radioresistant. Depletion of macrophages caused a significant decrease in tdT+CD11b+ myeloid populations. A subset of CD45+tdT+ cells may be erythroid island macrophages (EIM) which are depleted after G-CSF treatment. tdT+CXCL12+ cells are in direct contact with F4/80 macrophages, express RANKL and form a niche with B220+ B cells. A population of resident cells within the thymus are tdT+ and express myeloid markers and RANKL. In conclusion, in addition to targeting osteoblast/osteocytes, DMP1-Cre labels unique cell populations of macrophage and stromal cells within BM and thymus niches and expresses key microenvironmental factors.

CD51 labels periosteal injury-responsive osteoprogenitors.

The periosteum is a critical source of skeletal stem and progenitor cells (SSPCs) that form callus tissue in response to injury. There is yet to be a consensus on how to identify SSPCs in the adult periosteum. The aim of this study was to understand how potential murine periosteal SSPC populations behave in vivo and in response to injury. We evaluated the in vivo differentiation potential of Sca1-CD51+ and Sca1+CD51+ cells following transplantation. In vitro, the Sca1+CD51+ population appears to be more primitive multipotent cells, but after transplantation, Sca1-CD51+ cells showed superior engraftment, expansion, and differentiation into chondrocytes and osteoblasts. Despite representing a clear population with flow cytometry, we identified very few Sca1+CD51+ cells histologically. Using a periosteal scratch injury model, we successfully mimicked the endochondral-like healing process seen in unstable fractures, including the expansion and osteochondral differentiation of αSMA+ cells following injury. CD51+ cells were present in the cambium layer of resting periosteum and expanded following injury. Sca1+CD51- cells were mainly localized in the outer periosteal layer. We found that injury increased colony-forming unit fibroblast (CFU-F) formation in the periosteum and led to rapid expansion of CD90+ cells. Several other populations, including Sca1-CD51+ and CD34+ cells, were expanded by day 7. Mice with enhanced fracture healing due to elevated Notch signaling mediated by NICD1 overexpression showed significant expansion of CD51+ and CD34hi cells in the early stages of healing, suggesting these populations contribute to more rapid healing. In conclusion, we demonstrate that periosteal injury leads to the expansion of various SSPC populations, but further studies are required to confirm their lineage hierarchy in the adult skeletal system. Our data indicate that CD51+ skeletal progenitor cells are injury-responsive and show good engraftment and differentiation potential upon transplantation.

Preptin Deficiency Does Not Protect against High-Fat Diet-Induced Metabolic Dysfunction or Bone Loss in Mice.

Preptin is derived from the cleavage of the E-peptide of pro-insulin-like growth factor (IGF)-II and is an insulin secretagogue. Observational studies have linked elevated circulating preptin to metabolic dysfunction in humans; however, a causal role for preptin in metabolic dysfunction has not been established. Additionally, preptin can promote osteoblast proliferation and differentiation, suggesting a link with skeletal health. We previously described a global preptin knockout (KO) model. In this study, we sought to uncover the impact of preptin KO in mice on the response to a moderately high-fat diet (HFD) and low-fat diet (LFD). HFD groups had higher weight and fat mass gain, lower trabecular and cortical bone volume and fracture load, and higher liver triglycerides. In males, preptin deficiency led to lower blood glucose than wild-type (WT) mice under LFD conditions. This was accompanied by differences in bone microarchitecture, including lower trabecular bone volume fraction, trabecular number, and lower cortical thickness. These differences were absent in female mice, although KO females had a HFD-driven increase in fat mass and liver triglycerides that was absent in WT mice. Female WT mice had increased glucose-stimulated insulin secretion under HFD conditions that was absent in female KO mice. Overall, preptin may have a detrimental impact on metabolism and a positive impact on bone health in male mice and may protect against liver fat storage in females while enabling islet compensation under HFD conditions. When we consider that serum preptin levels are elevated in humans of both sexes in pathological states in which insulin levels are elevated, the impact of preptin on comorbidity risk needs to be better understood. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Better post-operative outcomes at 1-year follow-up are associated with lower levels of pre-operative synovitis and higher levels of IL-6 and VEGFA in unicompartmental knee arthroplasty patients.

PURPOSE: Osteoarthritis (OA) is associated with inflammation, and residual inflammation may influence outcomes following knee arthroplasty. This may be more relevant for patients undergoing unicompartmental knee arthroplasty (UKA) due to larger remaining areas of native tissue. This study aimed to: (1) characterise inflammatory profiles for medial UKA patients and (2) investigate whether inflammation markers are associated with post-operative outcomes. METHODS: This prospective, observational study has national ethics approval. Bloods, synovial fluid, tibial plateaus and synovium were collected from medial UKA patients in between 1 January 2021 and 31 December 2021. Cytokine and chemokine concentrations in serum and synovial fluid (SF) were measured with multiplexed assays. Disease severity of cartilage and synovium was assessed using validated histological scores. Post-operative outcomes were measured with Oxford Knee Score (OKS), Forgotten Joint Score (FJS-12) and pain scores. RESULTS: The study included 35 patients. SF VEGFA was negatively correlated with pre-operative pain at rest (r - 0.5, p = 0.007), and FJS-12 at six-week (r 0.44, p = 0.02), six-month (r 0.61, p < 0.01) and one-year follow-up (r 0.63, p = 0.03). Serum and SF IL-6 were positively correlated with OKS at early follow-up (serum 6 weeks, r 0.39, p = 0.03; 6 months, r 0.48, p < 0.01; SF 6 weeks, r 0.35, p = 0.04). At six weeks, increased synovitis was negatively correlated with improvements in pain at rest (r - 0.41, p = 0.03) and with mobilisation (r - 0.37, p = 0.047). CONCLUSION: Lower levels of synovitis and higher levels of IL-6 and VEGFA were associated with better post-operative outcomes after UKA, which could be helpful for identifying UKA patients in clinical practice. LEVEL OF EVIDENCE: Level IV case series.

Rat model of recalcitrant prosthetic joint infection using biofilm inocula.

Prosthetic joint infection (PJI) is a rare but devastating complication of joint arthroplasty. Biofilm formation around the prosthesis confers tolerance to antibiotics so that treatment is challenging. Most animal models of PJI use planktonic bacteria to establish the infection which fails to reproduce the pathology of chronic infection. We aimed to establish a rat model of Staphylococcus aureus PJI in male Sprague-Dawley rats using biofilm inocula and demonstrate its tolerance to frontline antibiotics. Pilot studies indicated that infection could be introduced to the knee joint by a biofilm-coated pin but that handling the prosthetic without disturbing the biofilm was difficult. We, therefore, developed a pin with a slotted end and used a miniature-biofilm reactor to develop mature biofilm in this niche. These biofilm-laden pins consistently produced infection of the bone and joint space. Treatment with high dose cefazolin, 250 mg/kg, starting the day of surgery reduced or cleared pin-adherent bioburden within 7 days, however when escalation from 25 to 250 mg/kg cefazolin treatment was delayed for 48 h, rats were unable to clear the infection. To track infections, we used bioluminescent bacteria, however, the bioluminescent signal did not accurately track the degree of infection in the bone and joint space as the signal did not penetrate the bone. In conclusion, we demonstrate that using a custom prosthetic pin, we can generate biofilm in a specific niche using a novel bioreactor setup and initiate a rat PJI that rapidly develops tolerance to supra-clinical doses of cefazolin.

Combined Growth Factor Hydrogel Enhances Rotator Cuff Enthesis Healing in Rat But Not Sheep Model.

We hypothesized that a combined growth factor hydrogel would improve chronic rotator cuff tear healing in a rat and sheep model. Insulin-like growth factor 1, transforming growth factor ß1, and parathyroid hormone were combined into a tyraminated poly-vinyl-alcohol (PVA-Tyr) hydrogel and applied directly at the enthesis. In total, 30 Sprague-Dawley rats and 16 Romney ewes underwent unilateral rotator cuff tenotomy and then delayed repairs were performed after 3-4 weeks. The animals were divided into a control group (repair alone) and treatment group. The rotator cuffs were harvested at 12 weeks after surgery for biomechanical and histological analyses of the repair site. In the rat model, the stress at failure and Young's modulus were higher in the treatment group in comparison with the control group (73% improvement, p = 0.010 and 56% improvement, p = 0.028, respectively). Histologically, the repaired entheses in the treatment group demonstrated improved healing with higher semi-quantitative scores (10.1 vs. 6.55 of 15, p = 0.032). In the large animal model, there was no observable treatment effect. This PVA-Tyr bound growth factor system holds promise for improving rotator cuff healing. However, our approach was not scalable from a small to a large animal model. Further tailoring of this growth factor delivery system is still required. Level of Evidence: Basic Science Study; Biomechanics and Histology; Animal Model Impact Statement Previous studies using single-growth factor treatment to improve enthesis healing after rotator cuff repair have reported promising, but inconsistent results. A novel approach is to combine multiple growth factors using controlled-release hydrogels that mimic the normal healing process. In this study, we report that a combined growth factor hydrogel can improve the histological quality and strength of rotator cuff repair in a rat chronic tear model. This novel hydrogel growth factor treatment has the potential to be used in human clinical applications to improve healing after rotator cuff repair.

Disease progression, aseptic loosening and bearing dislocations are the main revision indications after lateral unicompartmental knee arthroplasty: a systematic review.

IMPORTANCE: Lateral unicompartmental knee arthroplasty (UKA) is a surgical option for patients with isolated lateral osteoarthritis however, the procedure has higher revision rates than medial UKA. The reason for this remains unclear; therefore, a better understanding of the indications for lateral UKA revision is needed. AIM: The primary aim of this systematic review was to identify revision indications for lateral UKA. Secondary aims were to further investigate if revision indications were influenced by implant design and time from surgery. EVIDENCE REVIEW: A systematic literature review was performed according to the PRISMA 2020 guidelines. Search was performed in January 2022 in MedLine, EMBASE, CINAHL and the Cochrane Library using the keywords "knee arthroplasty", "unicompartmental", "reoperation", synonyms and abbreviations. Articles published in 2000-2021 that were at least level III retrospective cohort studies with at least 10 lateral UKAs and reported all failure modes were included. Risk of bias was assessed using the ROBINS-I tool. Revision indications, patient characteristics, study design, implant types and time to failure were extracted from the selected studies. Collated data were tabulated and differences were tested using Chi-square or Fisher's exact test. FINDINGS: A total of 29 cohort and 4 registry studies that included 7,668 UKAs met the inclusion criteria. Studies were judged as having moderate or severe risk of bias; this was associated with the retrospective nature of studies required to investigate long-term outcomes of knee arthroplasty. The main indications for lateral UKA revision were OA progression (35%), aseptic loosening (17%) and bearing dislocation (14%). The incidence of revision was similar for mobile-bearing implants (7.6%) and fixed-bearing (6.4%). For mobile-bearing implants, there was introduction of bearing dislocations as an additional mode of failure (24% cf. 0%, p < 0.001). For fixed-bearing implants, the incidence of revision was higher for all-poly-ethylene (13.9%) than metal-backed (1.8%) tibial components. Early lateral UKA failures were associated with bearing dislocations (sequential decrease from 69% under 6 months to 0% 10+ years, p < 0.001), whereas late failures were associated with OA progression (sequential increase from 0% under 6 months to 100% > 10+ years, p < 0.01). Compared with medial UKA, OA progression (41% cf. 30%, p = 0.004), malalignment (2.7% cf. 0.8%, p = 0.02), instability (4% cf. 1%, p = 0.02) and bearing dislocations (20% cf. 10%, p < 0.001) were more common for lateral UKA. CONCLUSIONS AND RELEVANCE: OA progression, aseptic loosening and bearing dislocation were the three main revision indications for lateral UKA. Compared to medial UKA, OA progression, malalignment, instability and bearing dislocations were more common revision indications for lateral UKA. Higher survivorship of metal-backed fixed-bearing implants was found. The findings suggest that the outcomes of lateral UKA may be improved with more optimal alignment, gap balancing and patient selection. LEVEL OF EVIDENCE: Level III systematic review.

Cystinosin-deficient rats recapitulate the phenotype of nephropathic cystinosis.

The lysosomal storage disease cystinosis is caused by mutations in CTNS, encoding the cystine transporter cystinosin, and in its severest form leads to proximal tubule dysfunction followed by kidney failure. Patients receive the drug-based therapy cysteamine from diagnosis. However, despite long-term treatment, cysteamine only slows the progression of end-stage renal disease. Preclinical testing in cystinotic rodents is required to evaluate new therapies; however, the current models are suboptimal. To solve this problem, we generated a new cystinotic rat model using CRISPR/Cas9-mediated gene editing to disrupt exon 3 of Ctns and measured various parameters over a 12-mo time course. Ctns-/- rats display hallmarks of cystinosis by 3-6 mo of age, as demonstrated by a failure to thrive, excessive thirst and urination, cystine accumulation in tissues, corneal cystine crystals, loss of LDL receptor-related protein 2 in proximal tubules, and immune cell infiltration. High levels of glucose, calcium, albumin, and protein were excreted at 6 mo of age, consistent with the onset of Fanconi syndrome, with a progressive diminution of urine urea and creatinine from 9 mo of age, indicative of chronic kidney disease. Kidney histology and immunohistochemistry showed proximal tubule atrophy and glomerular damage as well as classic "swan neck" lesions. Overall, Ctns-/- rats show a disease progression that more faithfully recapitulates nephropathic cystinosis than existing rodent models. The Ctns-/- rat provides an excellent new rodent model of nephropathic cystinosis that is ideally suited for conducting preclinical drug testing and is a powerful tool to advance cystinosis research.NEW & NOTEWORTHY Animal models of disease are essential to perform preclinical testing of new therapies before they can progress to clinical trials. The cystinosis field has been hampered by a lack of suitable animal models that fully recapitulate the disease. Here, we generated a rat model of cystinosis that closely models the human condition in a timeframe that makes them an excellent model for preclinical drug testing as well as being a powerful tool to advance research.

Improve Integration of In Vitro Biofilm Body of Knowledge to Support Clinical Breakthroughs in Surgical Site Infection.

Prosthetics increase the risk of deep surgical site infections in procedures intended to restore function. In orthopaedics, prosthetic joint infections can lead to repetitive surgeries, amputation, or worse. Biofilm formation both in vitro and in vivo involves stages of attachment, accumulation, and maturation. The level of maturation affects susceptibility to antibiotics, the immune system, and the success of surgical interventions. A review of the literature indicates that orthopedic publications are less likely to mention biofilm. We have reviewed animal models of infection to assess in vivo models of prosthetic infection. Although most prosthetic infections seem to originate from local skin microbiota, clinically representative biofilm inocula are unusual. Biofilm-related end points are more widely adopted, but studies rarely include both quantification of adherent microbial burden and imaging of the in vivo biofilm. Failure to differentiate between planktonic and biofilm infections can skew research away from needed chronic disease models. In this review, we address prosthetic joint infections as an important model for chronic biofilm infection research, identify critical requirements for in vivo models of chronic infection, and propose that resistance to the terminology of biofilm research exists within both research and regulation, which could limit progress toward important orthopaedic targets.

The Impact of Maternal High-Fat Diet on Bone Microarchitecture in Offspring.

The incidence of obesity in women of reproductive age has significantly increased over the past 100 years. There is a well-established connection between maternal obesity during pregnancy and an increased risk of developing non-communicable cardiometabolic diseases in her offspring. This mini-review focuses on evidence examining the effect of maternal high-fat diet (HFD) on skeletal development and bone health in later life in offspring. The majority of rodent studies indicate that maternal HFD generally negatively affects both embryonic bone development and bone volume in adult animals. Details surrounding the mechanisms of action that drive changes in the skeleton in offspring remain unclear, although numerous studies suggest that some effects are sex-specific. Human studies in this area are limited but also suggest that HFD during pregnancy may impair bone formation and increase fracture risk during childhood. Given the consequences of low bone mass and deranged bone microarchitecture for offspring, advances in our understanding of the developmental origins of bone health is critical in the battle against osteoporosis.

Heterogeneity of murine periosteum progenitors involved in fracture healing.

The periosteum is the major source of cells involved in fracture healing. We sought to characterize progenitor cells and their contribution to bone fracture healing. The periosteum is highly enriched with progenitor cells, including Sca1+ cells, fibroblast colony-forming units, and label-retaining cells compared to the endosteum and bone marrow. Using lineage tracing, we demonstrate that alpha smooth muscle actin (αSMA) identifies long-term, slow-cycling, self-renewing osteochondroprogenitors in the adult periosteum that are functionally important for bone formation during fracture healing. In addition, Col2.3CreER-labeled osteoblast cells contribute around 10% of osteoblasts but no chondrocytes in fracture calluses. Most periosteal osteochondroprogenitors following fracture can be targeted by αSMACreER. Previously identified skeletal stem cell populations were common in periosteum but contained high proportions of mature osteoblasts. We have demonstrated that the periosteum is highly enriched with skeletal progenitor cells, and there is heterogeneity in the populations of cells that contribute to mature lineages during periosteal fracture healing.

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.

Markers for Identification of Postnatal Skeletal Stem Cells In Vivo.

PURPOSE OF REVIEW: The adult skeleton contains stem cells involved in growth, homeostasis, and healing. Mesenchymal or skeletal stem cells are proposed to provide precursors to osteoblasts, chondrocytes, marrow adipocytes, and stromal cells. We review the evidence for existence and functionality of different skeletal stem cell pools, and the tools available for identifying or targeting these populations in mouse and human tissues. RECENT FINDINGS: Lineage tracing and single cell-based techniques in mouse models indicate that multiple pools of stem cells exist in postnatal bone. These include growth plate stem cells, stem and progenitor cells in the diaphysis, reticular cells that only form bone in response to injury, and injury-responsive periosteal stem cells. New staining protocols have also been described for prospective isolation of human skeletal stem cells. Several populations of postnatal skeletal stem and progenitor cells have been identified in mice, and we have an increasing array of tools to target these cells. Most Cre models lack a high degree of specificity to define single populations. Human studies are less advanced and require further efforts to refine methods for identifying stem and progenitor cells in adult bone.

Modulation of Notch1 signaling regulates bone fracture healing.

Fracture healing involves interactions of different cell types, driven by various growth factors, and signaling cascades. Periosteal mesenchymal progenitor cells give rise to the majority of osteoblasts and chondrocytes in a fracture callus. Notch signaling has emerged as an important regulator of skeletal cell proliferation and differentiation. We investigated the effects of Notch signaling during the fracture healing process. Increased Notch signaling in osteochondroprogenitor cells driven by overexpression of Notch1 intracellular domain (NICD1) (αSMACreERT2 mice crossed with Rosa-NICD1) during fracture resulted in less cartilage, more mineralized callus tissue, and stronger and stiffer bones after 3 weeks. Periosteal cells overexpressing NICD1 showed increased proliferation and migration in vitro. In vivo data confirmed that increased Notch1 signaling caused expansion of alpha-smooth muscle actin (αSMA)-positive cells and their progeny including αSMA-derived osteoblasts in the callus without affecting osteoclast numbers. In contrast, anti-NRR1 antibody treatment to inhibit Notch1 signaling resulted in increased callus cartilage area, reduced callus bone mass, and reduced biomechanical strength. Our study shows a positive effect of induced Notch1 signaling on the fracture healing process, suggesting that stimulating the Notch pathway could be beneficial for fracture repair.

Perivascular osteoprogenitors are associated with transcortical channels of long bones.

Bone remodeling and regeneration are dependent on resident stem/progenitor cells with the ability to replenish mature osteoblasts and repair the skeleton. Using lineage tracing approaches, we identified a population of Dmp1+ cells that reside within cortical bone and are distinct from osteocytes. Our aims were to characterize this stromal population of transcortical perivascular cells (TPCs) in their resident niche and evaluate their osteogenic potential. To distinguish this population from osteoblasts/osteocytes, we crossed mice containing inducible DMP1CreERT2/Ai9 Tomato reporter (iDMP/T) with Col2.3GFP reporter (ColGFP), a marker of osteoblasts and osteocytes. We observed iDMP/T+;ColGFP- TPCs within cortical bone following tamoxifen injection. These cells were perivascular and located within transcortical channels. Ex vivo bone outgrowth cultures showed TPCs migrated out of the channels onto the plate and expressed stem cell markers such as Sca1, platelet derived growth factor receptor beta (PDGFRß), and leptin receptor. In a cortical bone transplantation model, TPCs migrate from their vascular niche within cortical bone and contribute to new osteoblast formation and bone tube closure. Treatment with intermittent parathyroid hormone increased TPC number and differentiation. TPCs were unable to differentiate into adipocytes in the presence of rosiglitazone in vitro or in vivo. Altogether, we have identified and characterized a novel stromal lineage-restricted osteoprogenitor that is associated with transcortical vessels of long bones. Functionally, we have demonstrated that this population can migrate out of cortical bone channels, expand, and differentiate into osteoblasts, therefore serving as a source of progenitors contributing to new bone formation.

Engraftment of skeletal progenitor cells by bone-directed transplantation improves osteogenesis imperfecta murine bone phenotype.

Osteogenesis imperfecta (OI) is a genetic disorder most commonly caused by mutations associated with type I collagen, resulting in a defective collagen bone matrix. Current treatments for OI focus on pharmaceutical strategies to increase the amount of defective bone matrix, but do not address the underlying collagen defect. Introducing healthy donor stem cells that differentiate into osteoblasts producing normal collagen in OI patients has the potential to increase bone mass and correct the mutant collagen matrix. In this study, donor bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) expressing both αSMACreERT2/Ai9 progenitor reporter and osteoblast reporter Col2.3GFP were locally transplanted into the femur of OI murine (OIM) mice. One month post-transplantation, 18% of the endosteal surface was lined by donor Col2.3GFP expressing osteoblasts indicating robust engraftment. Long-term engraftment in the marrow was observed 3 and 6 months post-transplantation. The presence of Col1a2-expressing donor cell-derived cortical bone matrix was detected in transplanted OIM femurs. Local transplantation of BMSCs increased cortical thickness (+12%), the polar moment of inertia (+14%), bone strength (+30%), and stiffness (+30%) 3 months post-transplantation. Engrafted cells expressed progenitor markers CD51 and Sca-1 up to 3 months post-transplantation. Most importantly, 3 months post-transplantation donor cells maintained the ability to differentiate into Col2.3GFP+ osteoblasts in vitro, and in vivo following secondary transplantation into OIM animals. Locally transplanted BMSCs can improve cortical structure and strength, and persist as continued source of osteoblast progenitors in the OIM mouse for at least 6 months.

PDGF Modulates BMP2-Induced Osteogenesis in Periosteal Progenitor Cells.

BMPs are used in various clinical applications to promote bone formation. The limited success of the BMPs in clinical settings and supraphysiological doses required for their effects prompted us to evaluate the influence of other signaling molecules, specifically platelet-derived growth factor (PDGF) on BMP2-induced osteogenesis. Periosteal cells make a major contribution to fracture healing. We detected broad expression of PDGF receptor beta (PDGFRß) within the intact periosteum and healing callus during fracture repair. In vitro, periosteum-derived progenitor cells were highly responsive to PDGF as demonstrated by increased proliferation and decreased apoptosis. However, PDGF blocked BMP2-induced osteogenesis by inhibiting the canonical BMP2/Smad pathway and downstream target gene expression. This effect is mediated via PDGFRß and involves ERK1/2 MAPK and PI3K/AKT signaling pathways. Therapeutic targeting of the PDGFRß pathway in periosteum-mediated bone repair might have profound implications in the treatment of bone disease in the future.

Skeletal phenotype of the neuropeptide Y knockout mouse.

Neuropeptide Y (NPY) is involved in multiple processes such as behavior, energy and bone metabolism. Previous studies have relied on global NPY depletion to examine its effects on bone. However, this approach is unable to distinguish the central or local source of NPY influencing bone. Our aim was to identify which cells within the skeleton express Npy and establish a model that will enable us to differentiate effects of NPY derived from different cell types. We have generated the NPY floxed (NPYflox) mice using CRISPR technology. By crossing the NPYflox mice with Hypoxanthine Phosphoribosyltransferase 1 (Hprt)-cre to generate a global knockout, we were able to validate and confirm loss of Npy transcript and protein in our global NPYKO. Global deletion of NPY results in a smaller femoral cortical cross-sectional area (-12%) and reduced bone strength (-18%) in male mice. In vitro, NPY-deficient bone marrow stromal cells (BMSCs) showed increase in osteogenic differentiation detected by increases in alkaline phosphatase staining and bone sialoprotein and osteocalcin expression. Despite both sexes presenting with increased adiposity, female mice had no alterations in bone mass, suggesting that NPY may have sex-specific effects on bone. In this study we identified Npy expression in the skeleton and examined the effect of global NPY depletion to bone mass. The differential impact of NPY deletion in cortical and cancellous compartments along with differences in phenotypes between in vitro and in vivo, highlights the complex nature of NPY signaling, indicative of distinct sources that can be dissected in the future using this NPYflox model.