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Skeletal stem and progenitor cells (SSPCs) constitute a reservoir of bone-forming cells necessary for bone development, modeling and remodeling, as well as for fracture healing. Recent advances in tools to identify and isolate SSPCs have revealed that cells with multipotent properties are present not only in neonatal bone, but also in adult bone marrow and periosteum. The long bone metaphysis and endosteum have been proposed as an additional SSPC niche, although in vitro approaches to study their cellular and molecular characteristics are still limited. Here, we describe a comprehensive procedure to isolate and culture SSPCs derived from the metaphysis and endosteum of young-adult mice. Based on flow cytometry analysis of known SSPC markers, we found the presence of putative multipotent SSPCs, similar to neonatal bone tissue. In vitro, metaphyseal/endosteal SSPCs possess self-renewing capacity, and their multipotency is underscored by the ability to differentiate into the osteogenic and adipogenic lineage, while chondrogenic potential is limited. Expansion of metaphyseal/endosteal SSPCs under low oxygen conditions increases their proliferation capacity, while progenitor properties are maintained, likely reflecting their hypoxic niche in vivo. Collectively, we propose a validated isolation and culture protocol to study metaphyseal/endosteal SSPC biology in vitro.

Femoral head osteonecrosis, also known as avascular necrosis, is a disease with a multifactorial etiology, characterized by a profound change of bone architecture, which leads to the diminishing of bone resistance and femoral head collapse. The main causes that lead to femoral head necrosis are represented by the decrease of local blood perfusion and increase of intraosseous pressure, because of an excessive development of adipose tissue in the areolas of the trabecular bone tissue in the femoral head. The histopathological and immunohistochemical (IHC) study performed by us showed that most of bone trabeculae were damaged by necrotic-involutive processes, their sizes being reduced, both regarding their length and their diameter; generally, the spans were thin, fragmented, distanced among them, which led to the occurrence of some large areolar cavities, full of conjunctive tissue, rich in adipocytes. Some of the residual bone spans even presented microfractures. In the structure of the trabecular bone tissue, numerous cavities showed lack of content, which indicates the death of osteocytes inside, while the endosteum appeared very thin, with few osteoprogenitor, flattened, difficult to highlight cells. The IHC study showed a low reaction of the bone reparatory processes and a reduced multiplication capacity of bone cells involved in the remodeling and remake of the diseased bone tissue. Nevertheless, there were identified numerous young conjunctive cells (fibroblasts, myofibroblasts), positive to proliferating cell nuclear antigen (PCNA), cells that have a high capacity of multiplication, participating in the formation of a fibrous conjunctive tissue (sclerous) instead of the damaged bone trabeculae. The formation of fibrous conjunctive tissue causes the reduction of mechanical resistance of the femoral head and its collapse. The IHC study of the microvascularization in the femoral head damaged by aseptic osteonecrosis showed the presence of a very low vascular system, both in the residual bone trabeculae and in the sclerous conjunctive tissue. Of the inflammatory cells present in the spongy bone tissue of the femoral head affected by osteonecrosis, the most numerous ones were the macrophages. Both macrophages and T- and B-lymphocytes had a heterogenous distribution.

Up to 70% of patients with late-stage breast cancer have bone metastasis. Current treatment regimens for breast cancer bone metastasis are palliative with no therapeutic cure. Disseminated tumor cells (DTCs) colonize inside the osteogenic niches in the early stage of bone metastasis. Drug delivery into osteogenic niches to inhibit DTC colonization can prevent bone metastasis from entering its late stage and therefore cure bone metastasis. Here, we constructed a 50% DSS6 peptide conjugated nanoparticle to target the osteogenic niche. The osteogenic niche was always located at the endosteum with immature hydroxyapatite. Arsenic-manganese nanocrystals (around 14 nm) were loaded in osteogenic niche-targeted PEG-PLGA nanoparticles with an acidic environment-triggered arsenic release. Arsenic formulations greatly reduced 4T1 cell adhesion to mesenchymal stem cells (MSCs)/preosteoblasts (pre-OBs) and osteogenic differentiation of osteoblastic cells. Arsenic formulations also prevented tumor cell colonization and dormancy via altering the direct interaction between 4T1 cells and MSCs/pre-OBs. The chemotactic migration of 4T1 cells toward osteogenic cells was blocked by arsenic in mimic 3D osteogenic niche. Systemic administration of osteogenic niche-targeted arsenic nanoparticles significantly extended the survival of mice with 4T1 syngeneic bone metastasis. Our findings provide an effective approach for osteogenic niche-specific drug delivery and suggest that bone metastasis can be effectively inhibited by blockage of tumor cell colonization in the bone microenvironment., Graphical abstract Osteogenic niche-targeted nanoparticles selectively delivered arsenic trioxide into the pre-metastatic niche. Locally released arsenic effectively inhibited breast cancer cell colonization and diminished the number of pre-metastatic niches in the bone.Image 1

Blood vessels serve as a versatile transport system and play crucial roles in organ development, regeneration, and stem cell behavior. In the skeletal system, certain capillaries support perivascular stem cells or osteoprogenitor cells and thereby regulate bone formation. Recent studies reported that a specialized capillary subtype, termed type H vessels, is shown to couple angiogenesis and osteogenesis in rodents and humans. They can be distinguished by specific cell surface markers, as the endothelial cells in the metaphysis and endosteum highly express the junctional protein CD31 and the sialoglycoprotein endomucin. Here, we provide an overview of the role of type H vessels in bone homeostasis and summarize their linkage with various cytokines to control bone cell behavior and bone formation. We also discuss the potential clinical application for bone disorders by targeting these specific vessels according to their physiological and pathobiological settings.

The biology of hematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. Particularly challenging has been the study of dynamic HSC behaviors given that live animal HSC visualization in the native niche still represents an elusive goal in the field. Here, we describe a dual genetic strategy in mice that restricts reporter labeling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow (BM)3–5. We find that this subset of LT-HSCs resides in close proximity to both sinusoidal blood vessels and the endosteal surface. In contrast, multipotent progenitor cells (MPPs) display a broader distance distribution from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in BM niches with the deepest hypoxia and instead are found in similar hypoxic environments as MPPs. In vivo time-lapse imaging reveals that LT-HSCs display limited motility at steady-state. Following activation, LT-HSCs display heterogenous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of BM cavities exhibiting bone-remodeling activities. In contrast, cavities with low bone-resorbing activities do not harbor expanding HSCs. These findings point to a new degree of heterogeneity within the BM microenvironment, imposed by the stages of bone turnover. Overall, our approach enables direct visualization of HSC behaviors and dissection of heterogeneity in HSC niches.

Hematopoietic aging is marked by a loss of regenerative capacity and skewed differentiation from hematopoietic stem cells (HSC) leading to dysfunctional blood production. Signals from the bone marrow (BM) niche dynamically tailor hematopoiesis, but the effect of aging on the niche microenvironment and the contribution of the aging niche to blood aging still remains unclear. Here, we characterize the inflammatory milieu in the aged marrow cavity that drives both stromal and hematopoietic remodeling. We find decreased numbers and functionality of osteogenic mesenchymal stromal cells (MSC) at the endosteum and expansion of pro-inflammatory perisinusoidal MSCs with deterioration of sinusoidal endothelium in the central marrow, which together create a degraded and inflamed old niche. Molecular mapping at single cell resolution confirms disruption of cell identities and enrichment of inflammatory response genes in niche populations. Niche inflammation, in turn, drives chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors (MPP), which promotes myeloid differentiation at the expense of lymphoid and erythroid commitment and hinders hematopoietic regeneration. Remarkably, niche deterioration, HSC dysfunction and defective hematopoietic regeneration, can be improved by blocking inflammatory IL-1 signaling. Our results demonstrate that targeting niche inflammation is a tractable strategy to restore blood production during aging.

The aim of this study was to describe the pathologic changes in temporal bones with cochlear otosclerosis that may have implications for cochlear implantation. Fifteen human temporal bones with otosclerosis were used for this study. In each temporal bone was studied: (1) medial wall of the middle ear and in particular the horizontal segment of the facial nerve canal; (2) the round window niche and the corresponding part of the scala tympani; (3) the area of the labyrinthine segment and first genu of the facial nerve. Six temporal bones revealed otosclerotic pathology spanning the otic capsule between the cochlear endosteum and the facial nerve canal. The labyrinthine facial nerve was enveloped by otosclerosis in two cases, while the horizontal segment was involved in four temporal bones. Complete obstruction of the round window niche was observed in three cases, while round window niche obstruction was observed in four temporal bones. Otosclerosis presents difficulties for implant candidates. These otosclerotic changes with obstruction are usually present at the round window or at the scala tympani. The histology described underpins the need to counsel patients regarding the possibility of facial nerve stimulation and difficult implantation. Key words: Cochlear otosclerosis, histopathology, round window, obstruction.

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.

Osteoma is a benign, slow growing lesion that consists of compact or cancellous bone. Three types of osteomas could be classified: the central osteoma arising from the endosteum, the peripheral osteoma from the periosteum, and the extraskeletal soft tissue osteoma. In the craniofacial region, peripheral osteomas of the zygoma are quite rare. A literature review identified 7 cases of zygomatic arch and 3 cases of zygomatic body. This is the first report of zygomatic osteoma that was endoscopically removed. This report presents a rare case of osteoma of the zygoma and its endoscopic approach. The authors were able to confirm that endoscopic approach of this zygomatic osteoma was safe and effective surgical choice.

Labyrinthitis most commonly results from an infectious and less commonly from an inflammatory process of the inner ear, but it can be associated with temporal bone trauma, hemorrhage, or tumor. This inflammation (regardless of the etiology) disrupts the transmission of sensory information from the ear to the brain. Labyrinthitis ossificans is the pathological ossification of the membranous labyrinthine spaces in response to an insult to the inner ear involving membranous labyrinth or the endosteum of the otic capsule. Herein, we present a case of a 67-year-old female with progressive bilateral sensorineural hearing loss and positional vertigo.

In situ hybridization of decorin and biglycan mRNA, principal members of small leucine-rich proteoglycan, was performed using [35S]-labeled RNA probes, in the context of the hypothesis that they show different expression patterns associated with osteoblast differentiation in mice. We adopted two ossifying sites that can clearly follow the developmental process of bone formation: ossifying tympanic ring and developing bone collar of mandibular condylar cartilage. Decorin mRNA was expressed in osteoblasts of developing tympanic ring at E14.0, as well as of developing bone collar at E15.0, but biglycan mRNA was not, indicating decorin mRNA was expressed earlier in newly differentiating osteoblasts than biglycan. With maturation of osteoblasts, biglycan mRNA became expressed and maintained its expression both in the outer region (periosteum) and in the interior region (endosteum) of bone. By contrast, decorin mRNA expression was maintained in the outer region but diminished in the interior region. These results indicate that decorin and biglycan show differential expression patterns in differentiating osteoblasts and play specific roles in bone formation.

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 for progenitor cells, including Sca1+ cells, CFU-F and label-retaining cells compared to the endosteum and bone marrow. Using lineage tracing, we demonstrate that 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 for skeletal progenitor cells and there is heterogeneity in the populations of cells that contribute to mature lineages during periosteal fracture healing.

Hematopoietic stem cells (HSCs) with superior reconstitution potential are reported to be enriched in the endosteal compared to central bone marrow (BM) region. To investigate whether specific factors at the endosteum may contribute to HSC potency, we screened for candidate HSC niche factors enriched in the endosteal compared to central BM regions. Together with key known HSC supporting factors Kitl and Cxcl12, we report that prostacyclin/prostaglandin I2 (PGI2 ) synthase (Ptgis) was one of the most highly enriched mRNAs (>10-fold) in endosteal compared to central BM. As PGI2 signals through receptors distinct from prostaglandin E2 (PGE2 ), we investigated functional roles for PGI2 at the endosteal niche using therapeutic PGI2 analogs, iloprost and cicaprost. We found PGI2 analogs strongly reduced HSC differentiation in vitro. Ex vivo iloprost pulse treatment also significantly boosted long-term competitive repopulation (LT-CR) potential of HSCs upon transplantation. This was associated with increased tyrosine-phosphorylation of transducer and activator of transcription-3 (STAT3) signaling in HSCs but not altered cell cycling. In vivo, iloprost administration protected BM HSC potential from radiation or granulocyte colony-stimulating factor (G-CSF)-induced exhaustion, and restored HSC homing potential with increased Kitl and Cxcl12 transcription in the BM. In conclusion, we propose that PGI2 is a novel HSC regulator enriched in the endosteum that promotes HSC regenerative potential following stress. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: We discovered prostacyclin as a novel HSC regulator enriched in endosteal bone marrow niches. Ex vivo and in vivo treatment with clinical prostacyclin analogs enhanced HSC reconstitution potential and protect HSC from stress-mediated exhaustion. These findings open new therapeutic avenues to improve HSC engraftment after autologous transplantations by short ex vivo pulse treatment with prostacyclin analogs; and limit loss of HSC and acquired bone marrow failure by protecting HSC in patients undergoing radiation or chemotherapy treatment for non-hematologic malignancies. Prostacyclin analogs are safe, used clinically for vascular diseases, and thus have potential for swift repurposing for the therapies mentioned above.

The lacunar-canaliculi system is a network of channels that is created and maintained by osteocytes as they are embedded throughout cortical bone. As osteocytes modify their lacuna space, the local tissue composition and tissue strength are subject to change. Although continual exposure to parathyroid hormone (PTH) can induce adaptation at the lacunar wall, the impact of intermittent PTH treatment on perilacunar adaptation remains unclear. Therefore, the primary objective of this study was to establish how intermittent PTH(1-34) treatment influences perilacunar adaptation with respect to changes in tissue composition. We hypothesized that local changes in tissue composition following PTH(1-34) are associated with corresponding gains in tissue strength and resistance to microdamage at the whole bone level. Adult male C57BL/6J mice were treated daily with PTH(1-34) or vehicle for 3 weeks. In response to PTH(1-34), Raman spectroscopy revealed a significant decrease in the carbonate-to-phosphate ratio and crystallinity across the entire tissue, while the mineral-to-matrix ratio demonstrated a significant decrease in just the perilacunar region. The shift in perilacunar composition largely explained the corresponding increase in tissue strength, while the degree of new tissue added at the endosteum and periosteum did not produce any significant changes in cortical area or moment of inertia that would explain the increase in tissue strength. Furthermore, fatigue testing revealed a greater resistance to crack formation within the existing tissue following PTH(1-34) treatment. As a result, the shift in perilacunar composition presents a unique mechanism by which PTH(1-34) produces localized differences in tissue quality that allow more energy to be dissipated under loading, thereby increasing tissue strength and resistance to microdamage. In addition, our findings demonstrate the potential for PTH(1-34) to amplify osteocytes' mechanotransduction by producing a more compliant tissue. Overall, the present study demonstrates that changes in tissue composition localized at the lacuna wall contribute to the strength and fatigue resistance of cortical bone gained in response to intermittent PTH(1-34) treatment.

Hematopoietic stem cells (HSCs) reside in specialized microenvironments within the marrow designated as stem cell niches, which function to support HSCs at homeostasis and promote HSC engraftment after radioablation. We previously identified marrow space remodeling after hematopoietic ablation, including osteoblast thickening, osteoblast proliferation, and megakaryocyte migration to the endosteum, which is critical for effective engraftment of donor HSCs. To further evaluate the impact of hematopoietic cells on marrow remodeling, we used a transgenic mouse model (CD45Cre/iDTR) to selectively deplete hematopoietic cells in situ. Depletion of hematopoietic cells immediately before radioablation and hematopoietic stem cell transplantation abrogated donor HSC engraftment and was associated with strikingly flattened endosteal osteoblasts with preserved osteoblast proliferation and megakaryocyte migration. Depletion of monocytes, macrophages, or megakaryocytes (the predominant hematopoietic cell populations that survive short-term after irradiation) did not lead to an alteration of osteoblast morphology, suggesting that a hematopoietic-derived cell outside these lineages regulates osteoblast morphologic adaptation after irradiation. Using 2 lineage-tracing strategies, we identified a novel CD45-F4/80lo HSC-derived cell that resides among osteoblasts along the endosteal marrow surface and, at least transiently, survives radioablation. This newly identified marrow cell may be an important regulator of HSC engraftment, possibly by influencing the shape and function of endosteal osteoblasts.

Therapeutics reducing bone turnover, such as denosumab (Dmab), an anti-RANKL antibody, can provide treatments for patients with bone destruction. However, some patients with osteoporosis or localized primary bone tumors and many patients with various types of bone-metastatic cancer display unsatisfactory responses to Dmab. For achieving greater efficiency of RANKL neutralization in the bone microenvironment by enhancing the distribution of Dmab to the bone, we reengineered Dmab by fusing with single-chain variable fragments of an antibody specific for osteonectin (On), which is abundantly expressed in osseous tissues. The bispecific antibody, Dmab-FvOn, showed a similar activity as Dmab in inhibiting RANKL as examined in an osteoclast differentiation assay. When administered to mice, Dmab-FvOn was found to localize in increased proportions at the endosteum of the bone where osteonectin is abundant. Our study suggests that by linking anti-RANKL with an osteonectin-targeting moiety, a greater proportion of the therapeutic effector can be distributed in the bone. Future studies are needed to investigate whether the bispecific antibody can achieve higher therapeutic efficacy and lower toxicity.

CD31hiEmcnhi vessels were a subtype of vessels in the murine skeletal system, with high levels of platelet and endothelial cell adhesion molecule-1 (PECAM-1/CD31) and endomucin (Emcn). They were reported coupling angiogenesis and osteogenesis during bone development. We investigated the distribution of these vessels in rat tibiae and their temporal and spatial distribution during the bone defect repair process to improve our understanding of the importance of these vessels. We confirmed that CD31hiEmcnhi vessels were specially distributed around the trabecular bones near metaphysis and endosteum in rat tibiae. At 3 days post bone injury, CD31hiEmcnhi vessels proliferated and were extensively distributed across the entire repair area. At 7 and 14 days post-injury, these vessels decreased but were specially distributed around the growing trabecular bones near the frontier growth area, suggesting that these vessels support new bone formation. The distribution of CD31hiEmcnhi vessels and the transcriptions of Hif-1α and VEGFA, as well as BMP2 and Osterix decreased at 7 and 14 days post-injury under osteoporotic conditions, in combination with insufficient osteogenesis. Our research is of great significance to help understand the important role of CD31hiEmcnhi vessels in supporting new trabecular bones formation during bone defect repair process.

Tissue engineering technology platforms constitute a unique opportunity to integrate cells and extracellular matrix (ECM) proteins into scaffolds and matrices that mimic the natural microenvironment in vitro. The development of tissue-engineered 3D models that mimic the endosteal microenvironment enables researchers to discover the causes and improve treatments for blood and immune-related diseases. The aim of this study was to establish a physiologically relevant in vitro model using 3D printed scaffolds to assess the contribution of human cells to the formation of a construct that mimics human endosteum. Melt electrospun written scaffolds were used to compare the suitability of primary human osteoblasts (hOBs) and placenta-derived mesenchymal stem cells (plMSCs) in (non-)osteogenic conditions and with different surface treatments. Using osteogenic conditions, hOBs secreted a dense ECM with enhanced deposition of endosteal proteins, such as fibronectin and vitronectin, and osteogenic markers, such as osteopontin and alkaline phosphatase, compared to plMSCs. The expression patterns of these proteins were reproducibly identified in hOBs derived from three individual donors. Calcium phosphate-coated scaffolds induced the expression of osteocalcin by hOBs when maintained in osteogenic conditions. The tissue-engineered endosteal microenvironment supported the growth and migration of primary human haematopoietic stem cells (HSCs) when compared to HSCs maintained using tissue culture plastic. This 3D testing platform represents an endosteal bone-like tissue and warrants future investigation for the maintenance and expansion of human HSCs. Statement of Significance This work is motivated by the recent interest in melt electrospinning writing, a 3D printing technique used to produce porous scaffolds for biomedical applications in regenerative medicine. Our team has been among the pioneers in building a new class of melt electrospinning devices for scaffold-based tissue engineering. These scaffolds allow structural support for various cell types to invade and deposit their own ECM, mimicking a characteristic 3D microenvironment for experimental studies. We used melt electrospun written polycaprolactone scaffolds to develop an endosteal bone-like tissue that promotes the growth of HSCs. We combine tissue engineering concepts with cell biology and stem cell research to design a physiologically relevant niche that is of prime interest to the scientific community.

Apart from its nutritive functions, the periosteum critically affects bone regeneration via its stem/osteoprogenitor cell content. Normal healing after bone fractures, trauma–orthopedic interventions and invasive dental procedures is critically linked to the reestablishment of the periosteal microcirculation, but the reconstruction, replacement or repair of lost tissues may also be performed with autologous periosteum. Besides the initiation of cell differentiation during bone repair and remodeling processes, the periosteum together with the endosteum plays significant roles in the pathogenesis of both hormone-related and trauma-induced osteoporotic alterations in the bone metabolism. Nevertheless, the axial bones, and in particular the jawbones, and the appendicular bones display differences not only in their blood supply and fracture healing characteristics, but also in respect of the development of osteoporosis and their reactions to treatment modalities (i.e. bisphosphonates). These reactions may also be linked to the differences in periosteal microcirculatory reactions. The present overview summarizes the relevant data of microcirculatory studies focusing on the periosteal reactions in different anatomical locations together with the optimal background methodologies, study models and the most significant observations.

Bone healing, biocompatibility, and safety employing the IlluminOss System (IS), comprised of an inflatable balloon filled with photopolymerizable liquid monomer, was evaluated in New Zealand white rabbits. Successful bone healing and callus remodeling over 6 months was demonstrated radiologically and histologically with IS implants in fenestrated femoral cortices. Biocompatibility was demonstrated with IS implants in brushed, flushed femoral intramedullary spaces, eliciting no adverse, local, or systemic responses and with similar biocompatibility to K-wires in contralateral femurs up to 1 year post-implant. Lastly simulated clinical failures demonstrated the safety of IS implants up to 1 year in the presence of liquid or polymerized polymer within the intramedullary space. Polymerized material displayed cortical bone and vasculature effects comparable to mechanical disruption of the endosteum. In the clinically unlikely scenario with no remediation or polymerization, a high dose monomer injection resulted in marked necrosis of cortical bone, as well as associated vasculature, endosteum, and bone marrow. Overall, when polymerized and hardened within bone intramedullary spaces, this light curable monomer system may provide a safe and effective method for fracture stabilization. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2181-2190, 2017.

Purpose: The purpose of this experimental study was to evaluate the osteointegration of a bioactive 3D-cylindrical titanium-alloy implant (bone-graft substitute) for tibial shaft defect reconstruction. Methods: An experimental study was done in 7 mongrel dogs. Tibial shaft defect was repaired using an original titanium-alloy (Ti6Al4V) cellular cylindrical implant. with a bioactive layer of hydroxyapatite by anode microarc oxidation. Histological study (hematoxylin-eosin stain and immunohistological reaction using ostepontin polyclonal antibodies) and scanning electron microscopy (electron probe X-ray microanalysis for calcium and phosphorus saturation in the tissue matrix) were applied to assess bone tissue regeneration. Results: Experimental study revealed osteoconduction starting from the endosteum of bone fragments adjacent to the bone defect and developed to the central part of the implant. In 4 weeks, graft osteointegration was achieved in all animals. Implant cells were filled with spongy bone tissue and the graft external surface was covered with a connective tissue structures similar to the periosteum ones. Conclusions: Cellular titanium bone-graft substitute with bioactive coatings placed into bone defect stimulates reparative osteogenesis and graft osteointegration.

During the recent years, we published some articles on gamma correction medical imaging, gamma correction 99mTc-HDP pinhole bone scan (GCPBS) in particular, for the specific diagnosis of callused trabecular microfracture (CTMF), bone contusion, and bone marrow edema and hemorrhage (Bahk et al. 2010, 2016, 2017, 2019). Those studies showed that the high 99mTc-HDP uptake in contused trabeculae is not suppressed by gamma correction, but the low uptake in intact bone trabeculae and edema and the moderate uptake in hemorrhage was cleanly suppressed. Originally, Francis et al. (1980) noted in their Sprague-Dawley rat experiment that 99mTc-HDP uptake was significantly increased in injured trabeculae because bone tracer was actively adsorbed onto amorphous calcium phosphate, which is actively engaged with osteoneogenesis in osteoblastic rimming of contused trabeculae. It was also shown that the nominal tracer uptake in intact bone and mild to moderate tracer uptake in bone edema and hemorrhage is due to loose adhesion of tracer onto crystalline hydroxyapatite. Recently, we also performed young rat experiment and confirmed the fact that the nominal tracer uptake in the normal trabeculae and mild to moderate tracer uptake (Fig. 4.1) in edema and hemorrhage are all cleanly suppressed by gamma correction, but the high uptake in CTMF is not suppressed at all (Fig. 4.2) (Bahk et al. 2016). To be exact the normal control study showed that the intact femur uniformly accumulates mild tracer, which is neatly suppressed by gamma correction barely showing endosteum on H&E stain (Fig. 4.1). Contrary, however, when trabeculae are well defined after gamma correction it may well denote that endosteal rimming is formed (Fig. 4.2). Unsuppressed high tracer uptake in epiphysis indicates that the physis in young rats is actively engaged with physiologically growing osteoneogenesis. The same biophysical phenomenon can be seen in CTMF, which is similarly engaged with active repair osteoneogenesis. It can be said that distinctly defined trabeculae may well be interpreted to designate endosteal rimming (Fig. 4.2).

Hematopoiesis is hosted, supported and regulated by a special bone marrow (BM) microenvironment known as “niche.” BM niches have been classified based on micro-anatomic distance from the bone surface into “endosteal” and “central” niches. Whilst different blood vessels have been found in both BM niches in mice, our knowledge of the human BM architecture is much more limited. Here, we have used a combination of markers including NESTIN, CD146, and αSMA labeling different blood vessels in benign human BM. Applying immunohistochemical/immunofluorescence techniques on BM trephines and performing image analysis on almost 300 microphotographs, we detected high NESTIN expression in BM endothelial cells (BMECs) of small arteries (A) and endosteal arterioles (EA), and also in very small vessels we named NESTIN+ capillary-like tubes (NCLTs), not surrounded by sub-endothelial perivascular cells that occasionally reported low levels of NESTIN expression. Statistically, NCLTs were detected within 40 μm from bone trabecula, frequently found in direct contact to the bone line and spatially correlated with hematopoietic stem/progenitor cells. Our results support the expression of NESTIN in human BMECs of EA and A in accordance with the updated classification of murine BM micro-vessels. NCLTs for their peculiar characteristics and micro-anatomical localization have been here proposed as transitional vessels possibly involved in regulating human hematopoiesis.

In the bone marrow, preB cells are found adjacent to the bone endosteum where bone synthesizing osteoblast and bone resorbing osteoclasts reside. Although there is evidence of interactions between preB and bone cells, the factors that contribute to such interactions are poorly understood. A critical checkpoint for preB cell development assesses the integrity of the nascent immunoglobulin μ heavy chain (HC) by testing whether it can participate in the formation of a preB cell receptor (preBCR), composed of the μ HC and surrogate light chain (LC). In this work, we tested whether loss of preBCR components can affect bone synthesis. A panel of gene targeted mice with sequential blocks in preBCR formation or function [surrogate light chain component lambda 5 deleted (λ5-/-), transmembrane domain of μHC deleted (IgM-mem-/-), and CD19 preBCR co-receptor deleted (CD19-/-)] were evaluated for effects on postnatal bone synthesis. Postnatal bone mass was analyzed in 6 month old mice using μ-CT, histomorphometry and double calcein labeling. Both cortical and trabecular bone mass were significantly decreased in the femurs of the λ5 and IgM-mem deficient mice. Histomorphometric analysis showed a decrease in the numbers of osteoblasts and osteoclasts in all three mutant strains. Double calcein labeling revealed a significant decrease in dynamic synthesis and mineralization of bone in λ5-/- mice. Our data strongly suggest that interference with preBCR formation or function affects bone homeostasis independent of the presence or absence of mature B cells, and that components of the preBCR play important, and potentially distinct, roles in regulating adult bone mass.