Marrow Space Research Articles

6728 Bilateral Adrenal Hemorrhage: Cause Or Consequence?

Abstract Disclosure: M.N. Rayan: None. R. Horner: None. A. Valenciaga: None. L.E. Ryan: None. Introduction: Before the advent of the CT scan, bilateral adrenal hemorrhage was primarily discovered post-mortem in patients who had succumbed to sepsis, burns, or anticoagulant therapy. Today, non-traumatic hemorrhage is recognized as an infrequent, yet potentially lethal diagnosis causing clinical deterioration in acutely ill patients. Clinical Case: Our patient is a 64-year-old-male with a medical history of Myelodysplastic Syndrome initially presented to an external ED with abdominal pain. CT abdomen revealed hepatomegaly, splenomegaly, and new bilateral adrenal masses—absent in an MRI conducted a year earlier. He was treated conservatively and subsequently discharged. Two weeks later, he returned to the hospital with severe right sided flank pain. His hemoglobin and platelet counts dropped from 11.5 g/dL and 309 K/uL during the previous ED visit to 7.7 g/dL and 80 K/uL, respectively. Additionally, his PT/INR, PTT, fibrinogen, and D-Dimer levels were elevated. A repeat CT scan indicated a significant enlargement of the right adrenal mass, raising suspicion of adrenal hemorrhage. He was appropriately transfused, and a BM biopsy was performed to evaluate for acute transformation of underlying MDS. Cortisol levels were monitored daily for adrenal insufficiency. A complicated hospital course ensued marked by worsening anemia, thrombocytopenia, and coagulopathy, accompanied by high fevers and new-onset atrial fibrillation. Mixing studies showed absence of a factor inhibitor ruling out acquired forms of Hemophilia. Infectious workup including fungal and viral serologies returned negative. A PET scan revealed several small hypermetabolic hypodense liver lesions, and hypermetabolic activity diffusely in the spleen, and adrenal glands leading to suspicion of metastasis. Results of the BM biopsy returned revealing hypercellularity, with aggregates of hemosiderin-laden macrophages replacing the bone marrow space with associated necrosis. Worsening abdominal pain with hematochezia prompted an endoscopy, confirming a diagnosis of metastatic melanoma. Concurrently, seizure activity and altered mentation led to a head CT, revealing new metastatic lesions that confirmed the rapid progression of the disease. Subsequently, the patient was transitioned to comfort care and passed away. Clinical Lesson: It is crucial to review the various causes of adrenal hemorrhage, understand the challenges associated with interpreting adrenal imaging, emphasize the need to monitor adrenal function, and consider the occasional necessity for tissue sampling. In this clinical scenario, metastases with hemorrhage were the most likely etiology. The causes of adrenal hemorrhage are not always apparent; in this presentation, the consequence of the underlying disease state ultimately led to the initial presenting symptom of abdominal pain. Presentation: 6/2/2024

Developing long bones respond to surrounding tissues by trans-pairing of periosteal osteoclasts and endocortical osteoblasts.

Developing long bones alter their shape while maintaining uniform cortical thickness via coordinated activity of bone-forming osteoblasts and bone-resorbing osteoclasts at periosteal and endosteal surfaces, a process we designate trans-pairing. Two types of trans-pairing shift cortical bone in opposite orientations: peri-forming trans-pairing (peri-t-p) increases bone marrow space and endo-forming trans-pairing (endo-t-p) decreases it, via paired activity of bone resorption and formation across the cortex. Here, we focused on endo-t-p in growing bones. Analysis of endo-t-p activity in the cortex of mouse fibulae revealed osteoclasts under the periosteum compressed by muscles, and expression of RANKL in periosteal cells of the cambium layer. Furthermore, mature osteoblasts were localized on the endosteum, while preosteoblasts were at the periosteum and within cortical canals. X-ray tomographic microscopy revealed the presence of cortical canals more closely associated with endo- than with peri-t-p. Sciatic nerve transection followed by muscle atrophy and unloading induced circumferential endo-t-p with concomitant spread of cortical canals. Such canals likely supply the endosteum with preosteoblasts from the periosteum under endo-t-p, allowing bone shape to change in response to mechanical stress or nerve injury.

Higher solubility and lower onset temperature of protein denaturation increase the osteoconductive capacity of collagen membranes: A preclinical invivo study.

Collagen membranes are extensively used for guided bone regeneration procedures, primarily for horizontal bone augmentation. More recently, it has been demonstrated that collagen membranes promote bone regeneration. Present study aimed at assessing if structural modifications of collagen membranes may enhance their osteoconductive capacity. Twenty-four adult Wistar rats were used. Bilateral calvaria defects with a diameter of 5 mm were prepared and covered with prototypes of collagen membranes (P1 or P2). The P1 membrane (positive control) presented a lower onset temperature of protein denaturation and a higher solubility than the P2 membrane (test). The contralateral defects were left uncovered (NC). After 1 and 4 weeks, the animals were euthanized. A microcomputed tomography analysis of the harvested samples was performed within and above the bony defect. Undecalcified ground sections were subjected to light microscopy and morphometric analysis. Bone formation was observed starting from the circumferential borders of the defects in all groups at 1-week of healing. The foci of ossification were observed at the periosteal and dura mater sites, with signs of collagen membrane mineralization. However, there was no statistically significant difference between the groups. At 4 weeks, remnants of the collagen fibers were embedded in the newly formed bone. In the P2 group, significantly more bone volume, more new bone, and marrow spaces compared to the NC group were observed. Furthermore, the P2 group showed more bone volume ectocranially then the P1 group. Bone formation subjacent to a P2 membrane was superior than subjacent to the P1 membrane and significantly better compared to the control. Modifications of the physico-chemical properties may enhance the osteoconductive competence of collagen membranes, supporting bone formation outside the bony defects.

Increased nerve growth factor expression and osteoclast density are associated with subchondral bone marrow lesions in osteoarthritic knees

ObjectivesSubchondral bone marrow lesions (BMLs) detected on magnetic resonance imaging in knee osteoarthritis (OA) are associated with knee pain, though the mechanisms remain unknown. Increased nerve growth factor (NGF) expression and osteoclast density in subchondral bone appear to be the key features associated with bone pain in knee OA. Therefore, we aimed to identify associations among NGF, osteoclasts, and BMLs in knee OA. MethodsTwenty tibial plateaus were obtained from patients undergoing total knee arthroplasty for medial knee OA with BMLs at the medial tibial plateau (MTP). Osteochondral tissue samples from the weight-bearing part of the MTP, with and without BML, and from the weight-bearing part of the lateral tibial plateau (LTP), without BML, were collected. NGF expression and density of osteoclasts were compared among the three osteochondral tissue types. ResultsMTP bone with BMLs exhibited significantly higher NGF expression in bone marrow space and osteochondral channel, and higher osteoclast density than MTP bone without BML and LTP bone. The mean differences in NGF-positive area in the bone marrow space and the percentage of NGF-positive channels between MTP bones with and without BML were 9.0% (95% confidence interval [CI]: 5.9–12.1%) and 23.1% (95% CI: 11.3–35.0%), respectively. The difference in osteoclast density between MTP bones with and without BML was 0.6 osteoclasts per mm (95% CI: 0.3–0.9 osteoclasts per mm). ConclusionsIncreased NGF expression and osteoclast density are associated with subchondral BMLs in knee OA, contribute to understanding the mechanisms underlying BML-related bone pain in knee OA.

The Effect of Osteotomy Preparation Technique and Implant Diameter on Primary Stability and Bone-implant Interface of Short Implants (6 mm).

To evaluate the effect of osteotomy preparation technique and implant diameter on primary stability and bone-implant interface of short implants (6mm), when placed in bone with high degree of cancellous content. 90 short (S) implants (6 mm) divided in nine groups based on width (Narrow 4.2 mm, Regular 4.8 mm, Wide 5.4 mm) (N,R,W) and osteotomy preparation (Standard, Osteotome, Osseodensification) (ST, OT, OD) and placed in porcine tibia plateau bone samples: Group SN-ST; Group SN-OT; Group SN-OD; Group SRST; Group SR-OT; Group SR-OD; Group SW-ST; Group SW-OT and Group SW-OD. Insertion torque and Implant Stability Quotient (ISQ) were measured. Four implants from each group SNST, SN-OT, SN-OD were evaluated histomorphometrically. Insertion torque was significantly higher for implants of Group SW-OD compared to Group SW-ST (50.00 ±14.14 Ncm vs 28.00 ±10.85 Ncm, p= 0.005) and Group SW-OT compared to Group SW-ST (46.87 ±17.10 Ncm vs 28.00 ±10.85 Ncm, p=0.026). Insertion torque was significantly higher for implants of Group SW-OD compared to Group SN-OD (50.00 ±14.14 Ncm vs 31.5 ±15.82 Ncm, p=0.04). No significant differences were observed for the percentage of bone, marrow space and connective tissue in contact to the implant surface between studied groups. Osteotomy preparation technique at sites with high degree of cancellous content can influence the implant insertion torque for short and wide implants (5.4x6mm). Implant width can influence the insertion torque of short implants placed with the osseodensification technique.

Emergence of the brain-border immune niches and their contribution to the development of neurodegenerative diseases.

Historically, the central nervous system (CNS) was regarded as 'immune-privileged', possessing its own distinct immune cell population. This immune privilege was thought to be established by a tight blood-brain barrier (BBB) and blood-cerebrospinal-fluid barrier (BCSFB), which prevented the crossing of peripheral immune cells and their secreted factors into the CNS parenchyma. However, recent studies have revealed the presence of peripheral immune cells in proximity to various brain-border niches such as the choroid plexus, cranial bone marrow (CBM), meninges, and perivascular spaces. Furthermore, emerging evidence suggests that peripheral immune cells may be able to infiltrate the brain through these sites and play significant roles in driving neuronal cell death and pathology progression in neurodegenerative disease. Thus, in this review, we explore how the brain-border immune niches may contribute to the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). We then discuss several emerging options for harnessing the neuroimmune potential of these niches to improve the prognosis and treatment of these debilitative disorders using novel insights from recent studies.

Clinical Application of Unidirectional Porous Hydroxyapatite to Bone Tumor Surgery and Other Orthopedic Surgery.

Unidirectional porous hydroxyapatite (UDPHAp) was developed as a remarkable scaffold characterized by a distinct structure with unidirectional pores oriented in the horizontal direction and connected through interposes. We evaluated the radiographic changes, clinical outcomes, and complications following UDPHAp implantation for the treatment of bone tumors. Excellent bone formation within and around the implant was observed in all patients treated with intralesional resection and UDPHAp implantation for benign bone tumors. The absorption of UDPHAp and remodeling of the bone marrow space was observed in 45% of the patients at a mean of 17 months postoperatively and was significantly more common in younger patients. Preoperative cortical thinning was completely regenerated in 84% of patients at a mean of 10 months postoperatively. No complications related to the implanted UDPHAp were observed. In a pediatric patient with bone sarcoma, when the defect after fibular resection was filled with UDPHAp implants, radiography showed complete resorption of the implant and clear formation of cortex and marrow in the resected part of the fibula. The patient could walk well without crutches and participate in sports activities. UDPHAp is a useful bone graft substitute for the treatment of benign bone tumors, and the use of this material has a low complication rate. We also review and discuss the potential of UDPHAp as a bone graft substitute in the clinical setting of orthopedic surgery.

Preliminary study of proton magnetic resonance spectroscopy to assess bone marrow adiposity in the third metacarpus or metatarsus in Thoroughbred racehorses.

Magnetic resonance spectroscopy (MRS) has been used to investigate metabolic changes within human bone. It may be possible to use MRS to investigate bone metabolism and fracture risk in the distal third metacarpal/tarsal bone (MC/MTIII) in racehorses. To determine the feasibility of using MRS as a quantitative imaging technique in equine bone by using the 1H spectra for the MC/MTIII to calculate fat content (FC). Observational cross-sectional study. Limbs from Thoroughbred racehorses were collected from horses that died or were subjected to euthanasia on racecourses. Each limb underwent magnetic resonance imaging (MRI) at 3 T followed by single-voxel MRS at three regions of interest (ROI) within MC/MTIII (lateral condyle, medial condyle, proximal bone marrow [PBM]). Percentage FC was calculated at each ROI. Each limb underwent computed tomography (CT) and bone mineral density (BMD) was calculated for the same ROIs. All MR and CT images were graded for sclerosis. Histology slides were graded for sclerosis and proximal marrow space was calculated. Pearson or Spearman correlations were used to assess the relationship between BMD, FC and marrow space. Kruskal-Wallis tests were used to check for differences between sclerosis groups for BMD or FC. Eighteen limbs from 10 horses were included. A negative correlation was identified for mean BMD and FC for the lateral condyle (correlation coefficient = -0.60, p = 0.01) and PBM (correlation coefficient = -0.5, p = 0.04). There was a significant difference between median BMD for different sclerosis grades in the condyles on both MRI and CT. A significant difference in FC was identified between sclerosis groups in the lateral condyle on MRI and CT. Small sample size. 1H Proton MRS is feasible in the equine MC/MTIII. Further work is required to evaluate the use of this technique to predict fracture risk in racehorses.

Implant Survival in Renal-Transplanted Patients: A Prospective Long-Term Study.

The present study aims to assess the feasibility of implant rehabilitation in kidney-transplanted patients. Patients with kidney transplantation included in periodontal supportive care and at least one year of dialysis with mono- or partial edentulism were eligible for this study. Histomorphometric evaluation of the harvested bone was matched with radiological bone assessment. Implant stability was also monitored with resonance frequency analysis and insertion torque value. Fixed cemented prostheses have been delivered after conventional loading protocol. Supportive periodontal therapy has been administered. Eleven patients (9 males and 2 females) were included. The mean age was 58.1 ± 9.9 years. A total of 17 implants were inserted and analyzed. Mean ITV was 39.3 ± 23.8 Ncm. The mean primary stability (implant stability quotient) at T0 was 71.7 ± 10.5, whereas the mean secondary stability at T1 was 73.0 ± 7.3. The minimum follow-up was 62 months, with a maximum of 84 months (7 years) reached by 4 patients. Fourteen out of 15 implants were in function at a 5-year follow-up (survival rate: 93.3%). Two implants showed peri-implantitis. Seventeen bone samples were collected (13 in the mandible and 4 in the maxilla). The mean percentage of marrow spaces and lamellar bone was 41.6% and 58.4%, respectively. Class 3, according to Misch classification, was found as the mean value of radiological bone density. It can be concluded that implant-supported rehabilitation in kidney-transplanted patients is possible. Adequate periodontal maintenance allows implant rehabilitation in kidney-transplanted patients with long-term sufficient survival rates.

Jaw Fibro-Osseous Lesions: Use of a Predictive Index in Grading Probable Malignant Changes and a Review of Cases.

Fibro-osseous lesions (FLs), may rarely exhibit malignant features likewise undergo malignant transformation. Awareness of these features can assist in screening for potentially malignant cases and identifying low-grade central osteogenic sarcoma (LGCOS) that may mimic FLs. The objective of this study was to determine the usability of an index in predicting malignant changes in jaw FLs. This was a retrospective study where hematoxylin and eosin (H&E) slides and archival records of fibrous dysplasia (FD) and ossifying fibroma (OF) cases were reviewed. The sections were assessed for permeation of marrow spaces, stromal growth pattern, cytologic atypia, mitotic activity, and pattern of bone growth, which are parameters for diagnosing LGCOS. The predictive histologic index of malignancy (PHIM) was determined by a sum of the scores and graded as 0=nil, 1=low, 2 & 3=moderate, and 4 & 5=high. Data were presented using descriptive analysis. Ninety-three cases of FLs met the inclusion criteria, consisting of 40(43%) cases of FD and 53(57%) cases of OF. The peak age of presentation for FD and OF was 2nd and 3rd decade. There was a female preponderance of 1:1.6. The maxilla was the most common site affected by FD, while the mandible was most commonly affected by OF. For FD cases, the PHIM was moderate in 10(25%) cases and low in 21(52.5%) cases. Similarly, for OF cases, 30(56.6%) cases had low grade PHIM while 10(17%) cases had moderate grade PHIM. The PHIM depicted low to moderate malignancy grade in some of the cases studied. Follow up studies would be necessary to assess the PHIM.

Fosl2 Deficiency Predisposes Mice to Osteopetrosis, Leading to Bone Marrow Failure.

Arthritis causes Fos-like 2 (Fosl2) inactivation, and various immune cells contribute to its pathogenesis. However, little is known about the role of Fosl2 in hematopoiesis and the possible pathological role of Fosl2 inactivation in the hematopoietic system in arthritis. In this study, we show that Fosl2 maintains hematopoietic stem cell (HSC) quiescence and differentiation while controlling the inflammatory response via macrophages. Fosl2-specific deletion in the hematopoietic system caused the expansion of HSCs and myeloid cell growth while affecting erythroid and B cell differentiation. Fosl2 inactivation enhanced macrophage M1 polarization and stimulated proinflammatory cytokines and myeloid growth factors, skewing HSCs toward myeloid cell differentiation, similar to hematopoietic alterations in arthritic mice. Loss of Fosl2 mediated by Vav-iCre also displays an unexpected deletion in embryonic erythro-myeloid progenitor-derived osteoclasts, leading to osteopetrosis and anemia. The reduced bone marrow cellularity in Vav-iCreFosl2f/f mice is a consequence of the reduced bone marrow space in osteopetrotic mice rather than a direct role of Fosl2 in hematopoiesis. Thus, Fosl2 is indispensable for erythro-myeloid progenitor-derived osteoclasts to maintain the medullary cavity to ensure normal hematopoiesis. These findings improve our understanding of the pathogenesis of bone-destructive diseases and provide important implications for developing therapeutic approaches for these diseases.

IMPLANT SURVIVAL IN RENAL-TRANSPLANTED PATIENTS: A PROSPECTIVE LONG-TERM STUDY.

The present study aims to assess the feasibility of implant rehabilitation in kidney-transplanted patients. Patients with kidney transplantation included in periodontal supportive care and at least one year of dialysis with mono- or partial edentulism were eligible for this study. Histomorphometric evaluation of the harvested bone was matched with radiological bone assessment. Implant stability was also monitored with RFA and ITV. Fixed cemented prostheses have been delivered after conventional loading protocol. Supportive periodontal therapy has been administered. Eleven patients (9 males and 2 females) were included. The mean age was 58.1 ± 9.9 years. A total of 17 implants were inserted and analyzed. Mean ITV was 39.3 ± 23.8 Ncm. The mean primary stability (ISQ) at T0 was 71.7 ± 10.5, whereas the mean secondary stability at T1 was 73.0 ± 7.3. The minimum follow-up was 62 months, with a maximum of 84 months (7 years) reached by four patients. Fourteen out of 15 implants were in function at a 5-year follow-up (survival rate 93.3%). Two implants showed peri-implantitis. Seventeen bone samples were collected (13 in the mandible and 4 in the maxilla). The mean percentage of marrow spaces and lamellar bone was 41.6% and 58.4%, respectively. Class 3, according to Misch classification, was found as the mean value of radiological bone density. It can be concluded that implant-supported rehabilitation in kidney-transplanted patients is possible. Adequate periodontal maintenance allows implant rehabilitation in kidney transplanted patients with long-term sufficient survival rates.

Age and Gender Estimation Using the Osseous Microanatomy: Original Research

ABSTRACT Aim: This study was performed with the idea of assessing age and gender utilizing differences in osseous microanatomy in human jawbones. Materials and Methods: The study was conducted retrospectively among human jawbone samples. Various morphometric assessments such as trabecular width, marrow space, and their corelation were studied. In the samples, variations among osteon numbers, differences in the shapes of the osteocytes of jawbones, and amount of inflammation in the bony areas were recorded. Results: It was noted in this study that mean values of the diameter of the Haversian canal and vessel density had a noteworthy increase in female jawbone samples. The amount of osteocytes in both female and male bone samples was also statistically significant in terms of the correlation coefficient. Conclusion: We concluded that more sensitive identification of human remains, that is, age and gender analysis, can be performed by histomorphometric evaluation of bone remains.

Hedgehog activation promotes osteogenic fates of growth plate resting zone chondrocytes through transient clonal competency.

The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP-Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1-floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone ("patched roses") and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.

Alveolar Ridge Preservation Procedures Performed with a Freeze-Dried Bone Allograft: Clinical and Histologic Outcomes in a Case Series: Part II.

Tooth extractions can result in alveolar bone dimensional changes, necessitating additional bone grafting for implant placement. Alveolar Ridge Preservation (ARP) aims to counteract post-extraction changes. This study evaluates the bone regenerative properties of a freeze-dried bone allograft (FDBA) and the clinical outcomes of implants in grafted extraction sites. This case series enrolled 33 patients undergoing single/multiple tooth extractions followed by ARP. Biopsies were harvested during implant placement for histologic and histomorphometric analysis. Clinical outcomes included marginal bone loss and Pink Esthetic Score (PES). 25 patients completed the study. FDBA augmented sockets exhibited new bone formation adjacent to graft particles. Implants (n&#61;25) showed 100% survival and success rates at 1 and 2 years. PES improved significantly over time (p<.001), while marginal bone loss did not significantly differ at 1 and 2 years (p&#61;.096). Specimens showed trabecular bone, residual FDBA particles, and marrow spaces. High magnification revealed immature bone and woven bone bridges around graft particles. No inflammatory cells were observed. The case series provides valuable insights into ARP performed with FDBA; implants were placed after 3 months of healing without any additional bone augmentation, the histologic outcomes were favorable, and implants were successful after a 2-year period of follow-up.

The Clinical, Histological, and Immunohistochemical Features of Bone Metastases - A Review

Malignant tumors can spread differently, and bone metastasis is a complex process starting years before detection. Cancerous cells adhere in bone marrow spaces, growing undetected. Immunohistochemistry helps diagnose, but up to 30% have unidentified bone metastases despite tests and imaging. This review study introduces a deep learning model (DL-BM) to trace bone metastasis genesis, aiding pathologists when biopsy alone is insufficient. Clinicopathological analysis identifies the main carcinoma site in metastases with unclear origins. Thorough histological examinations of bone biopsies are crucial for suspecting the primary cancer. This review emphasizes using bone degradation indicators to identify bone metastases, tracking disease development, and monitoring treatment response.

Histological Evaluation of Arthritis in Mice

Arthritis is one the almost commonly prevalent painful diseases which cause limit the movement of joints. This study conducts to investigate histological alterations resulted in rheumatoid arthritis (RA)-induced in mice and estimating the levels of some serum biomarkers including serum IL-6, CRP and RF. Totally, 30 were purchased, acclimated, and divided into two groups; control that injected distilled water, and arthritis that injected adjuvant (CFA). Post the 25th day of experiment, all study mice of both groups were subjected for collection of blood and right ankle joints. Serologically, the findings of IL-6, CRP and RF of arthritis group were elevated significantly when compare to control mice. Histologically, the findings observed in mice of control group were included the proliferation of chondrocytes of articular cartilage with moderate cellular and subchondral marrow space, infiltration few inflammatory cells in dermis, proliferation of chondrocytes in articular cartilage with absence of inflammatory reaction in synovial cavity, moderate infiltration of inflammatory cells in subcutaneous tissue, nearly normal articular cartilage with regular tidemark and absence of sclerosis in marrow spaces, regenerative articular cartilage and regular tidemark line with presence of very narrow distance between articular cartilage and marrow bone, and existence of normal bone trabeculae. In arthritis mice, there were an infiltration of inflammatory cells in subcutaneous tissues and dermis, marked increases in numbers of osteoclasts, sclerosis, regular tidemark and proliferation of chondrocytes, low amonunt of abscesses in subcutaneous tissue, thickness of bone trabeculae, fragments of bone, subchondral sclerosis, moderate distance between bone and articular cartilage. In conclussion, RA causes severe impacts on tissues of affected joints. Studying the mechanism of inhibition during different inflammatory process appear needed.

Ras Activity Is Significantly Elevated in Patients with Primary Myelofibrosis and Is Agnostic to the Presence of RAS and JAK pathwaymutations

Myelofibrosis (MF) is a myeloproliferative stem cell disorder arising from various clonal mutations in early myeloid-committed hematopoietic progenitor cells. The subsequent acquisition of additional mutations and cytokine signaling then heralds the recruitment of fibroblasts to the marrow space, leading to collagen deposition. MF disease behavior varies widely and hematopoietic stem cell transplantation remains the only curative therapeutic option. In non-transplant candidates, managing cytopenic disease is uniquely challenging as patients develop resistance to commonly-utilized Jak-inhibitors. Expanding the molecular landscape of the disease beyond Jak/Stat signaling with the goal of identifying new therapeutic targets has, accordingly, been an area of growing interest in MF research. Recently-published studies, including results from our own institution, have identified mutations that potentially activate Ras signaling as being associated with features of advanced MF (higher somatic mutation burden) as well as with shorter overall survival and progression to AML. The goals of our study is to expand on this prior data, characterize the activity of Ras signaling and determine its role in patients with MF. Peripheral blood mononuclear cells (PBMCs) from 27 patients with myelofibrosis (MF) at a single institution underwent integrated genomic-transcriptomic sequencing analyses using the University of Michigan MiOncoSeq platform. PBMCs were separated by Ficoll® followed by cell lysis and protein quantification. Ras activity was measured by the binding of effector Ras binding domain (RBD) via ELISA. Patients with MF were compared to healthy donors and to patients with either polycythemia vera (PV) or essential thrombocythemia (ET). Ras activity in MF was examined across different mutations and with respect to various clinical parameters. Multivariate analyses were performed to assess the impact of each individual mutation on Ras activity. Accompanying statistical analyses for all assessments were conducted using two-way unpaired t-tests. Overall, Ras activity in 21 assessable patients with MF was 1.64 fold higher than in PV/ET (12 patients, p=0.0462) and 1.74 fold higher than healthy donors (13 volunteers, p=0.0304). While the majority of patients in the MF cohort had driver mutations, including in JAK2 (73%) and CALR (15.4%), the individual types of driver mutations did not impact Ras activity. Within the Ras/MAP Kinase signaling pathway, mutations were present at a range of variant allele frequencies (VAF), including 2 patients with NRAS (one with VAF 42%, one &amp;lt;5%), 2 KRAS (both &amp;lt;5%), 2 NF1, 1 CBL and 1 RAF1 (VAFs not assessed). The individual impact of various driver mutations, epigenetic regulators and splicing factors as assessed by multivariate analysis identified a trend toward enhanced Ras activity with mutations in RAS, CALR, ASXL1, TET2 and TP53, suggesting that in addition to RAS, other mutations also activate Ras signaling in MF. With regards to clinical parameters, Ras activity was weakly correlated with spleen size and high molecular risk mutation (HMR) presence, although this did not reach statistical significance in this cohort due to a relatively small sample size. Next, we performed a differential enrichment of myeloid and lymphoid cells from PBMCs using EasySep™ and measured Ras activity in lymphoid and myeloid enriched cells. We observed that the ratio of myeloid:lymphoid activity in patients with MF with RAS/MAP Kinase pathway mutations is 3.8 times higher than in patients without such mutations (p=0.0012), suggesting that RAS pathway mutations have a predominant role in myeloid cells. Our results show that the majority of patients with MF have hyperactive Ras signaling compared to PV/ET or healthy donors, regardless of the presence of alterations in the Ras signaling pathway. This underscores the importance of functional assessment of Ras activity in patients with MF and provides evidence that targeting Ras signaling in MF may lead to therapeutic benefit. We are currently expanding our analysis to a larger cohort of MF patients and performing preclinical testing of Ras/MAP Kinase pathway inhibitors as a means of overcoming Jak-inhibitor resistance.

An Intravital Imaging Protocol to Visualize and Phenotype the Hematopoietic Niche of Clonal Hematopoiesis

Clonal Hematopoiesis of indeterminate potential (CHIP) is a condition where blood cells are produced from a few clones of hematopoietic stem cells (HSCs) carrying leukemia-associated driver mutations 1. Although selective pressures from low-grade inflammation of the bone marrow microenvironment have been shown to promote mutant cell expansion 2-7, we focus on the disease-initiating niche, which is defined as a subset of marrow cells surrounding rare HSCs. Indeed, as we have recently described, clonal expansion of acute myeloid leukemia and activated HSCs 8,9 was not uniform, and only occurred in a subset of marrow spaces harboring bone remodeling activities. To identify the disease-initiating niche for therapeutic targeting, we developed an intravital imaging protocol to visualize the rare pre-malignant clones in the minimally perturbed microenvironment and phenotype their niche. The standard practice to generate CHIP models relies on transplanting syngeneic or allogeneic cells into a sub-lethally irradiated recipient. However, concerns arise as irradiation compromises the microenvironment 10. Successful engraftment in non-conditioned mice can be achieved by injecting a high number of cells (1.5 x 10 7 cells) 11,12, but modeling early clonal expansion events from single cells remain challenging, as a crowd of cells is often observed in proximity. Here, we established a low dose (0.5-1.5 Gy), whole body or local irradiation regimen that preserves the integrity of the bone marrow microenvironment. We performed high-resolution in-vivo imaging to capture dynamics of single cells and cells undergoing early expansion. Specifically, as little as 0.5Gy irradiation enabled survival of the transplanted cells (2x10 6 GFP + healthy whole bone marrow) in non-irradiated side, and allowed direct visualization of early clonal expansion in vivo through 16 weeks . Engraftment was negligible in non-irradiated mice.Through in-vivo, video-rate tracking of Rhodamine dextran dye (70kDa) leakage from vessels into the extravascular space showed no increase in permeability 13 or signs of vessel dilation, suggesting minimal inflammation after the radiation insult (N= 3 mice, data points are individual vessel segments. Mann-Whitney U test). Preliminary findings with in-vitro cultures of mesenchymal stromal cells (MSCs) have also suggested that the MSCs retain their ability for tri-lineage differentiation after the low-dose irradiation. With this imaging protocol, we showed that hot spots of cell expansion exist in the Tet2 +/- murine model. To capture the highly localized niche factors responsible for these hot spots, we implemented image-guided live-cell labeling adapted from the previously published Image-seq protocols 9. As illustrated in Figure 1, a small channel is etched in the bone with plasma-mediated laser ablation. The micropipette was then inserted to the region of interest for delivery of fluorescent antibodies under image guidance. By administering FITC-conjugated anti-CD45 antibodies into the marrow cavities, we can label and recover ~10,000 live cells with 98% viability from two regions of interest, suitable for next generation single cell sequencing. The technique can be expanded to employ antibody cocktails to visualize and enrich particular cell lineages of interest during cell isolation, while preserving spatial information. Its compatibility with standard cell isolation protocols for FACS may help collect cellular targets that are geometrically inaccessible for cell aspiration. In conclusion, we established a working model to visualize expansion of healthy and Tet2 +/- hematopoietic cells in vivo. This can be followed by image-assisted live-cell tagging to isolate local microenvironment cells and study cell-niche coordination at high spatial precision. The imaging protocol may also be broadly applied to study microenvironment regulations in non-malignant clonal disorders.

Alveolar Ridge Preservation Procedures Performed with A Freeze-Dried Bone Allograft: Histologic Outcomes in a Cohort Study: Part I.

The primary aim of this study was to assess the histomorphometric outcomes of extraction sockets grafted with freeze-dried bone allograft (FDBA) and sealed with a collagen membrane after 3 months of healing in the ROI (region of interest) areas. The secondary aims were to analyze the biomaterial resorption rate, the bone to biomaterial contact and the area/perimeter of grafted particles compared with commercially available FDBA particles. Fifteen patients underwent tooth extractions and ridge preservation procedures performed with FDBA and a collagen membrane. Bone biopsies were harvested after 3 months at the time of implant insertion for histologic and histomorphometric analysis. Two areas of concern ROI1 and ROI2 with different histological features were identified within the biopsy samples; the ROI1, ROI2 and commercially available particles were analyzed and compared . The following parameters were analyzed: newly formed bone (NFB), marrow space (MS), residual graft particles (RGP), perimeter/area of FDBA particles and bone to biomaterial contact. The histomorphometric analysis showed 35.22 ± 10.79 % of newly formed bone, 52.55 ± 16.06 % of marrow spaces and 12.41 ± 7.87 % of residual graft. Moreover, the histologic data from the regions of interest (ROI) 1 and 2 showed that: a) the mean percentage of bone-to-biomaterial contact (BBC) was 64.61 ± 27.14 %; b) the Newly Formed Bone was significantly higher in ROI1 than in ROI2; c) the Marrow Space was significantly lower in ROI1 than ROI2 and d) the FDBA particles in ROI1 areas showed significant lower area and perimeter when compared to commercially available FDBA particles. This latter data lead to the hypothesis that FBA particles embedded in newly formed bone undergo a resorption/remodeling process.