Investigations into the Ulnar Response to Mechanical Stimuli Activating Lamellar and Woven Bone Formation

Woven and lamellar bone formation can be stimulated using mechanical loading. Woven bone forms rapidly in response to damaging loading in a disorganized manner with low mineral density. In contrast, lamellar bone formation can be induced in the absence of damage, and is characterized by its slow, organized deposition and high density. In this dissertation, we first examined the molecular response to woven and lamellar bone formation using damaging and non-damaging dynamic loading protocols, respectively. We observed a significant increase in gene expression related to angiogenesis, cell proliferation and osteogenesis prior to woven bone formation, with significantly lower levels of expression associated with lamellar bone formation. To fully characterize the molecular responses of woven and lamellar bone we used a whole genome microarray. The micorarray results brought to light many inflammatory factors not previously investigated in our model, expanded previous findings about angiogenesis, and strengthened our understanding of the role of osteogenic pathways. Our investigations suggested that angiogenesis is required for successful woven bone formation. We used several angiogenic inhibitors, but were unable to prove the dependence of woven bone formation on angiogenesis. Finally, we sought to separate the effects of static and dynamic strains on bone formation. These findings demonstrate that in the absence dynamic strain, bone damage triggers a woven bone response that leads to a functional repair of whole-bone strength. Overall, the work done in this thesis has enhanced our understanding of bone formation. Future studies will expand on the microarray findings and clarify the role of angiogenesis in woven bone formation

The Small World of Osteocytes: Connectomics of the Lacuno-Canalicular Network in Bone

Osteocytes and their cell processes reside in a large, interconnected network of voids pervading the mineralized bone matrix of most vertebrates. This osteocyte lacuno-canalicular network (OLCN) is believed to play important roles in mechanosensing, mineral homeostasis, and for the mechanical properties of bone. While the extracellular matrix structure of bone is extensively studied on ultrastructural and macroscopic scales, there is a lack of quantitative knowledge on how the cellular network is organized. Using a recently introduced imaging and quantification approach, we analyze the OLCN in different bone types from mouse and sheep that exhibit different degrees of structural organization not only of the cell network but also of the fibrous matrix deposited by the cells. We define a number of robust, quantitative measures that are derived from the theory of complex networks. These measures enable us to gain insights into how efficient the network is organized with regard to intercellular transport and communication. Our analysis shows that the cell network in regularly organized, slow-growing bone tissue from sheep is less connected, but more efficiently organized compared to irregular and fast-growing bone tissue from mice. On the level of statistical topological properties (edges per node, edge length and degree distribution), both network types are indistinguishable, highlighting that despite pronounced differences at the tissue level, the topological architecture of the osteocyte canalicular network at the subcellular level may be independent of species and bone type. Our results suggest a universal mechanism underlying the self-organization of individual cells into a large, interconnected network during bone formation and mineralization

Metaplastic woven bone in bone metastases: A Fourier-transform infrared analysis and imaging of bone quality (FTIR)

Most osteolytic tumors are in fact mixed and contain an osteoblastic component associated with the predominant osteolytic areas. This metaplastic woven bone is always evidenced by histological analysis even in the absence of radiological expression. Metaplastic bone formation reflects the activation of new osteoblasts coming from the stimulation of the dormant lining cells. Twelve patients with secondary metastases of the iliac crest evidenced by hot spots on a Tc-MBP san were diagnosed by histomorphometry on bone biopsies. Fourier Transformed InfraRed analysis and Imaging (FTIRI) was used on 4μm thick sections of undecalcified bone. The mineralization degree, carbonate substitution, crystallinity and the cross-links ratio of collagen (1660/1690cm bands) were determined. The matrix characteristics were analyzed and imaged in the pre-existing residual bone and in the metaplastic woven bone in the vicinity of the tumor cells. FTIRI provided images of the phosphate, amide and combination of peak ratio after having selected the peaks of interest. In addition, the matrix properties can be measured and compared between the old and newly-formed bones. Woven bone appeared poorly calcified with a low phosphate/amide ratio (P=0.03) crystallinity (P<0.0001) and carbonate substitution (P=0.003). Collagen was less mature as evidenced by lower cross-links (P=0.01). Woven bone associated with bone metastasis appears poorly mineralized and rapidly elaborated by osteoblasts. The collagenous phase of the bone matrix has a low level of reticulation. FTIRI is a powerful tool to measure and visualize the various components of the bone matrix in human diseases

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions’ orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young’s modulus E= 1.90 GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young’s modulus in the preferential direction of 9–16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent

Histomorphometric evaluation of bone regeneration induced by biodegradable scaffolds as carriers for dental pulp stem cells in a rat model of calvarial "critical size" defect

Objective: The aim of this study was to test specific stem cells that could enhance bone formation in combination with specific scaffolds. Methods: Dental Pulp Stem Cells (DPSCs) were seeded with Granular Deproteinized Bovine Bone (GDPB) or Beta-Tricalcium Phosphate (ß-TCP) in a rat model of calvarial "critical size" defect. DPSCs were isolated from permanent human teeth, obtained and characterized using specific stem cells markers (Nanog and Oct-4) by real time-PCR and immunofluorescence. Cells were differentiated for 10-15 days towards the osteoblastic phenotype with 100μM L-ascorbic acid, added every day in culture medium and 20 vol. percentage of FBS in α-MEM medium. Osteogenic commitment was evaluated with real time-PCR by measuring the expression of specific markers (osteonectin and runx2). When a sufficient cell number was obtained, DPSCs were trypsinized, washed in culture medium and seeded onto the GDPB and ß-TCP scaffold sat a density of 0.5-1×106 cells/scaffold. Two bilateral critical-size circular defects (5 mm diameter; 1 mm thickness) were created from the parietal bone of the 8 athymic T-cell deficient nude rats. One cranial defect for each rat was filled with the scaffold alone and the other defect with the scaffold seeded with stem cells. After 12 weeks post-surgery animals were euthanized and histomorphometric analysis was performed. Differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's Protected Least Significant Difference (PLSD) post-hoc test. A p-value <0.05 was considered statistically significant. Results: GDPB group presented higher percentage of lamellar bone than that of GDPB/DPSC, ß-TCP alone had lower levels as compared to ß-TCP/DPSC. The addition of stem cells significantly increased woven bone formation in both scaffold-based implants, although still higher in GDPB based implants. Conclusion: Our findings indicate that GDPB and ß-TCP used as scaffold to induce bone regeneration may benefit from adding DPSC to tissue-engineered constructs

Mechanical Competence and Bone Quality Develop During Skeletal Growth.

Bone fracture risk is influenced by bone quality, which encompasses bone's composition as well as its multiscale organization and architecture. Aging and disease deteriorate bone quality, leading to reduced mechanical properties and higher fracture incidence. Largely unexplored is how bone quality and mechanical competence progress during longitudinal bone growth. Human femoral cortical bone was acquired from fetal (n = 1), infantile (n = 3), and 2- to 14-year-old cases (n = 4) at the mid-diaphysis. Bone quality was assessed in terms of bone structure, osteocyte characteristics, mineralization, and collagen orientation. The mechanical properties were investigated by measuring tensile deformation at multiple length scales via synchrotron X-ray diffraction. We find dramatic differences in mechanical resistance with age. Specifically, cortical bone in 2- to 14-year-old cases exhibits a 160% greater stiffness and 83% higher strength than fetal/infantile cases. The higher mechanical resistance of the 2- to 14-year-old cases is associated with advantageous bone quality, specifically higher bone volume fraction, better micronscale organization (woven versus lamellar), and higher mean mineralization compared with fetal/infantile cases. Our study reveals that bone quality is superior after remodeling/modeling processes convert the primary woven bone structure to lamellar bone. In this cohort of female children, the microstructural differences at the femoral diaphysis were apparent between the 1- to 2-year-old cases. Indeed, the lamellar bone in 2- to 14-year-old cases had a superior structural organization (collagen and osteocyte characteristics) and composition for resisting deformation and fracture than fetal/infantile bone. Mechanistically, the changes in bone quality during longitudinal bone growth lead to higher fracture resistance because collagen fibrils are better aligned to resist tensile forces, while elevated mean mineralization reinforces the collagen scaffold. Thus, our results reveal inherent weaknesses of the fetal/infantile skeleton signifying its inferior bone quality. These results have implications for pediatric fracture risk, as bone produced at ossification centers during children's longitudinal bone growth could display similarly weak points. © 2019 American Society for Bone and Mineral Research

Bone Regeneration Induced by Bone Porcine Block with Bone Marrow Stromal Stem Cells in a Minipig Model of Mandibular “Critical Size” Defect

Introduction. Adding stem cells to biodegradable scaffolds to enhance bone regeneration is a valuable option. Different kinds of stem cells with osteoblastic activity were tested, such as bone marrow stromal stem cells (BMSSCs). Aim. To assess a correct protocol for osteogenic stem cell differentiation, so BMSSCs were seeded on a bone porcine block (BPB). Materials and Methods. Bone marrow from six minipigs was extracted from tibiae and humeri and treated to isolate BMSSCs. After seeding on BPB, critical-size defects were created on each mandible of the minipigs and implanted with BPB and BPB/BMSSCs. After three months, histomorphometric analysis was performed. Results. Histomorphometric analysis provided percentages of the three groups. Tissues present in control defects were 23 ± 2% lamellar bone, 28 ± 1% woven bone, and 56 ± 4% marrow spaces; in BPB defects were 20 ± 5% BPB, 32 ± 2% lamellar bone, 24 ± 1% woven bone, and 28 ± 2% marrow spaces; in BPB/BMSSCs defects were 17 ± 4% BPB/BMSSCs, 42 ± 2% lamellar bone, 12 ± 1% woven bone, and 22 ± 3% marrow spaces. Conclusion. BPB used as a scaffold to induce bone regeneration may benefit from the addition of BDPSCs in the tissue-engineered constructs

An Organoid for Woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell‐based organoids are promising as functional, self‐organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self‐organizing co‐culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration)

Opioids Delay Healing of Spinal Fusion: A Rabbit Posterolateral Lumbar Fusion Model

Background Context Opioid use is prevalent in the management of pre- and postoperative pain in patients undergoing spinal fusion. There is evidence that opioids downregulate osteoblasts in vitro, and a previous study found that morphine delays the maturation and remodeling of callus in a rat femur fracture model. However, the effect of opioids on healing of spinal fusion has not been investigated before. Isolating the effect of opioid exposure in humans would be limited by the numerous confounding factors that affect fusion healing. Therefore, we have used a well-established rabbit model to study the process of spinal fusion healing that closely mimics humans. Purpose The objective of this work was to study the effect of systemic opioids on the process of healing of spinal fusion in a rabbit posterolateral spinal fusion model. Study Design/Setting This is a preclinical animal study. Materials and Methods Twenty-four adult New Zealand white rabbits were studied in two groups after approval from the Institutional Animal Care and Use Committee (IACUC). The opioid group (n=12) received 4 weeks\u27 preoperative and 6 weeks\u27 postoperative transdermal fentanyl. Serum fentanyl levels were measured just before surgery and 4 weeks postoperatively to ensure adequate levels. The control group (n=12) received only perioperative pain control as necessary. All animals underwent a bilateral L5–L6 posterolateral spinal fusion using iliac crest autograft. Animals were euthanized at the 6-week postoperative time point, and assessment of fusion was done by manual palpation, plain radiographs, microcomputed tomography (microCT), and histology. Results Twelve animals in the control group and 11 animals in the opioid group were available for analysis at the end of 6 weeks. The fusion scores on manual palpation, radiographs, and microCT were not statistically different. Three-dimensional microCT morphometry found that the fusion mass in the opioid group had a lower bone volume (p=.09), a lower trabecular number (p=.02), and a higher trabecular separation (p=.02) compared with the control group. Histologic analysis found areas of incorporation of autograft and unincorporated graft fragments in both groups. In the control group, there was remodeling of de novo woven bone to lamellar organization with incorporation of osteocytes, formation of mature marrow, and relative paucity of hypertrophied osteoblasts lining new bone. Sections from the opioid group showed formation of de novo woven bone, and hypertrophied osteoblasts were seen lining the new bone. There were no sections showing lamellar organization and development of mature marrow elements in the opioid group. Less dense trabeculae on microCT correlated with histologic findings of relatively immature fusion mass in the opioid group. Conclusions Systemic opioids led to an inferior quality fusion mass with delay in maturation and remodeling at 6 weeks in this rabbit spinal fusion model. These preliminary results lay the foundation for further research to investigate underlying cellular mechanisms, the temporal fusion process, and the dose-duration relationship of opioids responsible for our findings

Cutibacterium (formerly Propionibacterium) acnes clavicular infection.

Cutibacterium (formerly Propionibacterium) acnes13, 16 is a slow growing, gram-positive bacteria that is naturally found in higher concentrations as skin flora on the chest and back, as well as in other areas with greater numbers of hair follicles.25, 37 Most of the reported cases of C. acnes shoulder girdle infection follow arthroplasty surgery,18, 20, 26, 27, 32, 35 which then often requires debridement, administration of intravenous antibiotics, and surgical revision of the implanted device.12, 15, 21, 28-30 In a recent study, 56% of 193 shoulder revisions had a positive culture, 70% of which grew C. acnes.30 Despite the relatively common presumed association of C. acnes humeral osteomyelitis with prosthetic infection, infection of the scapula or clavicle secondary to C. acnes is rare.4, 23, 36 Osteomyelitis of the clavicle involving any organism is also an uncommon event that can arise spontaneously via presumed hematogenous spread, or secondary to open fractures or internal fixation.6, 33 The most commonly found organism in clavicular osteomyelitis is Staphylococcus aureus.9 We here report two cases of clavicular infection secondary to C. acnes that were not associated with implants