The structure of cortical bone as revealed by the application of methods for the calcified matrix study.

Calcination and decalcification are basic procedures useful to a morphological approach of a biological, composite material like cortical bone. The study was carried out on a whole human femur conserved in liquid (from an educational collection). Cortical fracturing and SEM observation of vascular canals surface collagen texture was used to study bone deproteination at scalar temperatures (400-1,200°C) and acid bone decalcification at crescent time intervals. Heating burned and vaporized the organic matrix with shrinkage of the bone specimens as documented by the weight loss and transverse surface morphometry. SEM showed a pattern of aligned spherulites at 400°C which maintained the collagen fibrils layout (like a mineral cast), followed by a spherulites fusion progression with the temperature increments. At 1200°C a crystalline-like structure of tightly-packed trapezohendron units. XRD analysis supported the SEM morphology displaying the complete Debey rings of hydroxyapatite and spotted Debey rings of withlockite. Surface Ca and P elution was documented after 12 hr of exposition to the acid solution by dissolution of spherulites and the whole canal surface decalcified in depth after 15 days by SEM-EDAX analysis. The periodic pattern of collagen fibrils was still evident up to 15 days of decalcification together with fine granular deposits of a not-collagenic proteic material, while after 30 days no period was observed in the decalcified fibrils. Collagen mineral cast at 400°C calcination. Complete crystalline transformation at 1200°C. Up to 15 days of decalcification fibrils period maintained.

Hyperglycemia compromises Rat Cortical Bone by Increasing Osteocyte Lacunar Density and Decreasing Vascular Canal Volume.

Uncontrolled diabetes is associated with increased risk of bony fractures. However, the mechanisms have yet to be understood. Using high-resolution synchrotron micro-CT, we calculated the changes in the microstructure of femoral cortices of streptozotocin-induced hyperglycemic (STZ) Wistar Albino rats and tested the mechanical properties of the mineralized matrix by nanoindentation. Total lacunar volume of femoral cortices increased in STZ group due to a 9% increase in lacunar density. However, total vascular canal volume decreased in STZ group due to a remarkable decrease in vascular canal diameter (7 ± 0.3 vs. 8.5 ± 0.4 µm). Osteocytic territorial matrix volume was less in the STZ group (14,908 ± 689 µm3) compared with healthy controls (16,367 ± 391 µm3). In conclusion, hyperglycemia increased cellularity and lacunar density, decreased osteocyte territorial matrix, and reduced vascular girth, in addition to decreasing matrix mechanical properties in the STZ group when compared with euglycemic controls.

Characterization of a prevascularized biomimetic tissue engineered scaffold for bone regeneration.

Significant bone loss due to disease or severe injury can result in the need for a bone graft, with over 500,000 procedures occurring each year in the United States. However, the current standards for grafting, autografts and allografts, can result in increased patient morbidity or a high rate of failure respectively. An ideal alternative would be a biodegradable tissue engineered graft that fulfills the function of bone while promoting the growth of new bone tissue. We developed a prevascularized tissue engineered scaffold of electrospun biodegradable polymers PLLA and PDLA reinforced with hydroxyapatite, a mineral similar to that found in bone. A composite design was utilized to mimic the structure and function of human trabecular and cortical bone. These scaffolds were characterized mechanically and in vitro to determine osteoinductive and angioinductive properties. It was observed that further reinforcement is necessary for the scaffolds to mechanically match bone, but the scaffolds are successful at inducing the differentiation of mesenchymal stem cells into mature bone cells and vascular endothelial cells. Prevascularization was seen to have a positive effect on angiogenesis and cellular metabolic activity, critical factors for the integration of a graft.

Combined Pedicled Vascularized and Wedge Nonvascularized Bone Graft for Treating Scaphoid Waist Nonunion With Humpback Deformity.

Pedicled vascularized bone graft (VBG) is a useful method in treating the scaphoid fracture nonunion, especially when the avascular necrosis exists. Humpback deformity is an important issue that we have to correct it during the treatment. We describe a method by using combined wedge non-VBG to correct the nonunion deformity when treating scaphoid nonunion with pedicled VBG. The wedge bone graft was harvested just proximal to the 2,3 intercompartmental supraretinacular artery VBG and was used as an inlay at the volar site to correct the humpback deformity, whereas the VBG was set at the dorsal site for bone bridging and blood supply. We also present our results of 10 patients with scaphoid fracture nonunion and humpback deformity treated with this method. Bone healing was achieved and the lateral intrascaphoid angles could be improved in all the 10 patients. Functional outcomes, including the Visual Analog Pain Scale for pain during activity, grip strength, the shortened Disabilities of the Arm, Shoulder, and Hand questionnaire (QuickDASH), and the modified Mayo Wrist Scores, were significantly improved.

PYY is a negative regulator of bone mass and strength.

OBJECTIVE: Bone loss in anorexia nervosa and following bariatric surgery is associated with an elevated circulating concentration of the gastrointestinal, anorexigenic hormone, peptide YY (PYY). Selective deletion of the PYY receptor Y1R in osteoblasts or Y2R in the hypothalamus results in high bone mass, but deletion of PYY in mice has resulted in conflicting skeletal phenotypes leading to uncertainty regarding its role in the regulation of bone mass. As PYY analogs are under development for treatment of obesity, we aimed to clarify the relationship between PYY and bone mass. METHODS: The skeletal phenotype of Pyy knockout (KO) mice was investigated during growth (postnatal day P14) and adulthood (P70 and P186) using X-ray microradiography, micro-CT, back-scattered electron scanning electron microscopy (BSE-SEM), histomorphometry and biomechanical testing. RESULTS: Bones from juvenile and Pyy KO mice were longer (P < 0.001), with decreased bone mineral content (P < 0.001). Whereas, bones from adult Pyy KO mice had increased bone mineral content (P < 0.05) with increased mineralisation of both cortical (P < 0.001) and trabecular (P < 0.001) compartments. Long bones from adult Pyy KO mice were stronger (maximum load P < 0.001), with increased stiffness (P < 0.01) and toughness (P < 0.05) compared to wild-type (WT) control mice despite increased cortical vascularity and porosity (P < 0.001). The increased bone mass and strength in Pyy KO mice resulted from increases in trabecular (P < 0.01) and cortical bone formation (P < 0.05). CONCLUSIONS: These findings demonstrate that PYY acts as a negative regulator of osteoblastic bone formation, implicating increased PYY levels in the pathogenesis of bone loss during anorexia or following bariatric surgery.

Microanatomical Record of Cortical Bone Remodeling and High Vascularity in a Fossil Giant Rat Midshaft Femur.

Rat cortical bone does not typically undergo secondary (Haversian) remodeling. Haversian organization of rat bone has been mainly observed in experimental settings following biomechanical or dietary manipulation. Here, we report an observation of cortical secondary osteons within a histological femur cross-section from an extinct (late Quaternary) form of Timorese giant rat (Murinae gen. et sp. indet). The medio-lateral midshaft diameter of its femur, used as a measure of bone size, is 6.15 mm and indicates a heavier than normal skeletal frame. We compare this sample to bone histology in a small rat's midshaft femur of 2.33 mm diameter. A complete lack of Haversian bone remodeling characteristics is noted for the smaller sample, which is dominated by radial vascular canals. The giant rat shows clear secondary osteons and diffuse vascularity mainly composed of tightly packed longitudinal canals across its cortex. It appears that rat cortical bone can undergo bone remodeling, and is organized in a highly vascularized manner, in insular giant cases. Our findings from Timor align with results reported in experimental rat model skeletal biology literature and other insular fossil rat material. Where macroanatomical examination is limited, histological observations on fossil rat limb bones have the potential to aid reconstructions of life history and skeletal growth aspects in these rodents. Anat Rec, 302:1934-1940, 2019. © 2019 American Association for Anatomy.

Volar Radius Vascularized Bone Flaps for the Treatment of Scaphoid Nonunion.

Vascularized bone flaps (VBFs) improve union rates for scaphoid nonunions compared with nonvascularized grafts. Volar VBFs are indicated in cases of scaphoid nonunion with avascular necrosis and/or humpback deformity. Four volar VBFs are described in this article. The volar carpal artery and pronator quadratus VBFs are most commonly used. The pisiform VBF can be used for replacement of the proximal pole of the scaphoid; it is covered by articular cartilage. The ulna VBF has greater donor morbidity; the ulnar artery is harvested and a palpable donor site deformity results.

Parathyroid suppression therapy normalizes chronic kidney disease-induced elevations in cortical bone vascular perfusion: a pilot study.

Interventions that alter PTH levels in an animal model of chronic kidney disease have effects on the perfusion of bone and bone marrow. INTRODUCTION: Patients with chronic kidney disease (CKD) have accelerated bone loss, vascular calcification, and abnormal biochemistries, together contributing to an increased risk of cardiovascular disease and fracture-associated mortality. Despite evidence of vascular pathologies and dysfunction in CKD, our group has shown that cortical bone tissue perfusion is higher in a rat model of high-turnover CKD. The goal of the present study was to test the hypothesis that parathyroid hormone (PTH) suppressive interventions would normalize cortical bone vascular perfusion in the setting of CKD. METHODS: In two separate experiments, 35-week-old CKD animals and their normal littermates underwent intra-cardiac fluorescent microsphere injection to assess the effect of 10 weeks of PTH suppression (Experiment 1: calcium supplementation, Experiment 2: calcimimetic treatment) on alterations in bone tissue perfusion. RESULTS: In Experiment 1, CKD animals had serum blood urea nitrogen (BUN) and PTH levels significantly higher than NL (+ 182% and + 958%; p < 0.05). CKD+Ca animals had BUN levels that were similar to CKD, while PTH levels were significantly lower and comparable to NL. Both femoral cortex (+ 220%, p = 0.003) and tibial cortex (+ 336, p = 0.005) tissue perfusion were significantly higher in CKD animals when compared to NL; perfusion was normalized to those of NL in CKD+Ca animals. MicroCT analysis of the proximal tibia cortical porosity showed a trend toward higher values in CKD (+ 401%; p = 0.017) but not CKD+Ca (+ 111%; p = 0.38) compared to NL. Experiment 2, using an alternative method of PTH suppression, showed similar results as those of Experiment 1. CONCLUSIONS: These data demonstrate that PTH suppression-based interventions normalize cortical bone perfusion in the setting of CKD.

Microcomputed tomography of the femur of diabetic rats: alterations of trabecular and cortical bone microarchitecture and vasculature-a feasibility study.

BACKGROUND: To better understand bone fragility in type 2 diabetes mellitus and define the contribution of microcomputed tomography (micro-CT) to the evaluation of bone microarchitecture and vascularisation, we conducted an in vitro preliminary study on the femur of Zucker diabetic fatty (ZDF) rats and Zucker lean (ZL) rats. We first analysed bone microarchitecture, then determined whether micro-CT allowed to explore bone vascularisation, and finally looked for a link between these parameters. METHODS: Eight ZDF and six ZL rats were examined for bone microarchitecture (group 1), and six ZDF and six ZL rats were studied for bone vascularisation after Microfil® perfusion which is a radiopaque casting agent (group 2). In group 1, we used micro-CT to examine the trabecular and cortical bone microarchitecture of the femoral head, neck, shaft, and distal metaphysis. In group 2, micro-CT was used to study the blood vessels in the head, neck, and distal metaphysis. RESULTS: Compared to ZL rats, the ZDF rats exhibited significantly lower trabecular bone volume and number and higher trabecular separation in the three locations (p = 0.02, p = 0.02, p = 0.003). Cortical porosity was significantly higher in the ZDF rats at the neck and shaft (p = 0.001 and p = 0.005). We observed a dramatically poorer bone vascularisation in the femur of ZDF rats, especially in distal metaphysis (p < 0.047). CONCLUSIONS: Micro-CT demonstrated not only significant alterations in the bone microarchitecture of the femurs of ZDF rats, but also significant alterations in bone vascularisation. Further studies are required to demonstrate the causal link between poor vascularisation and impaired bone architecture.

Decellularized Cortical Bone Scaffold Promotes Organized Neovascularization In Vivo.

IMPACT STATEMENT: Bone loss and skeletal deficiencies due to musculoskeletal diseases, traumatic injury, abnormal development, and cancer are major problems worldwide, frequently requiring surgical intervention. There has been a shift in paradigm to utilize tissue engineering applications. This novel bone technology has the potential to promote bone regeneration for large bone defects without the addition of growth factors and offers a unique architecture for cell attachment, proliferation, and differentiation. This scaffold serves as a tailored therapeutic for bone injuries and defects, leading to an increased quality of life by decreasing the risk of reoccurring surgeries and complications.

Volar Vascularized Strut Graft for Avascular Scaphoid Nonunion Using the 1,2 Intercompartmental Supraretinacular Artery.

In this retrospective study, we report the preliminary results of a novel technique for volar vascularized strut grafting to treat avascular scaphoid nonunion by using the 1,2 intercompartmental supraretinacular artery through a single incision. Forty-three of 45 patients with avascular scaphoid nonunion healed at a mean of 13 weeks (range, 3 to 10 mo). Complications consisted of 1 pin tract infection that resolved with oral antibiotics and 4 cases of transient dysesthesia of the radial sensory nerve. In 4 patients with equivocal radiographs, computed tomography scans confirmed bony union. The 2 patients who remained unhealed subsequently underwent proximal row carpectomy. Two other patients had persistent pain with the progression of radiocarpal arthritis. Our technique provides good results for the treatment of avascular scaphoid fracture nonunion. Notable advantages include performance through a single incision, use of an already established vascularized bone graft, volar graft placement, and no requirement for microvascular free graft reconstruction. It also provides the surgeon with the ability to adjust the procedure intraoperatively in the event of unexpected avascularity, without requiring substantially longer operative time or additional equipment.

Regional diversity in the murine cortical vascular network is revealed by synchrotron X-ray tomography and is amplified with age.

Cortical bone is permeated by a system of pores, occupied by the blood supply and osteocytes. With ageing, bone mass reduction and disruption of the microstructure are associated with reduced vascular supply. Insight into the regulation of the blood supply to the bone could enhance the understanding of bone strength determinants and fracture healing. Using synchrotron radiation-based computed tomography, the distribution of vascular canals and osteocyte lacunae was assessed in murine cortical bone and the influence of age on these parameters was investigated. The tibiofibular junction from 15-week- and 10-month-old female C57BL/6J mice were imaged post-mortem. Vascular canals and three-dimensional spatial relationships between osteocyte lacunae and bone surfaces were computed for both age groups. At 15 weeks, the posterior region of the tibiofibular junction had a higher vascular canal volume density than the anterior, lateral and medial regions. Intracortical vascular networks in anterior and posterior regions were also different, with connectedness in the posterior higher than the anterior at 15 weeks. By 10 months, cortices were thinner, with cortical area fraction and vascular density reduced, but only in the posterior cortex. This provided the first evidence of age-related effects on murine bone porosity due to the location of the intracortical vasculature. Targeting the vasculature to modulate bone porosity could provide an effective way to treat degenerative bone diseases, such as osteoporosis.

Regeneration of Vascularized Corticocancellous Bone and Diploic Space Using Muscle-Derived Stem Cells: A Translational Biologic Alternative for Healing Critical Bone Defects.

BACKGROUND: Regeneration of functional bone substrate remains a priority in reconstructive surgery especially for patients suffering from complex skeletal defects. Efforts to develop implantable osteoinductive constructs and novel osteoconductive materials remain at the forefront of industry forces and product line development. Despite advancement in clinical practice and bone biology, cancellous autograft remains the gold standard for procedures requiring osteogenic mechanisms of healing. This study investigates the utility of muscle-derived stem cells as a cellular therapy for definitive bone regeneration through a form of neo-osteogenesis. METHODS: Adipose-derived stem cell, bone marrow-derived mesenchymal stem cell, and muscle-derived stem cell populations were isolated separately from C57BL/6 murine tissues and supplemented with collagen scaffolding with or without bone morphogenetic protein-2 to compare relative osteogenic potency and ultrastructure organization in both two- and three-dimensional systems. Parallel populations were bound to a deployable collagen implant within a syngeneic murine cranial defect model. RESULTS: Although all populations provided and maintained mesenchymal stem cell multilineage capacity, adipose-derived stem cell- and bone marrow-derived mesenchymal stem cell-enriched constructs were capable of forming small bone aggregates. Defects receiving muscle-derived stem cells self-assembled a form of organized corticocancellous structures within two- and three-dimensional in vitro systems and within the in vivo model. Muscle-derived stem cells also augmented healing, implant angiogenesis, and diploic space formation. CONCLUSION: Muscle-derived stem cell-enriched implants appear to provide an autologous response to current industry-derived products and an attractive alternative to mesenchymal stem cells for the regeneration of corticocancellous bone and a vascularized diploic space.

A method for measuring the three-dimensional orientation of cortical canals with implications for comparative analysis of bone microstructure in vertebrates.

The orientation of vascular canals in cortical bone can reveal information about the growth rate and loading environment of a bone. For example, in birds it has been proposed that a high proportion of circumferential canals (a laminar cortex) is related to fast growth or torsional loading related to active flight. In this paper we present a method to measure the three dimensional (3D) orientation of vascular canals. Image data are obtained from micro-CT and two angles are measured: phi, determining how longitudinal a canal is; and theta, determining whether a canal is radial or circumferential. This method can measure the orientation of each canal contained in the scanned images. Here we demonstrate the approach on two samples - a rat tibia and a hawk humerus. This method offers a direct (3D) method for quantifying features of canal orientation, such as the degree of laminarity, and can be applied easily and non-destructively to multiple species and bones. The growth and development of the cortical canal network and its impact on factors such as bone strength and bone quality remains relatively unexplored. Our method provides a new tool to examine the impact of the orientation of cortical bone canals on bone and explore the origins of cortical canals formed during modelling and remodeling. This method has applications in comparative bone biology, small animal models, and human bone studies.

The influence of cortical bone perforation on guided bone regeneration in humans.

The purpose of this study was to evaluate the effect of cortical bone perforation on angiogenesis and osteogenesis of the augmented ridge in guided bone regeneration. Eighteen patients who had osseous defects in the mandible were selected. In the test group (n=9), alveolar cortical bone in the area of regeneration was perforated. No decortication was performed in the control group (n=9). Subsequently, defects were augmented by guided bone regeneration using resorbable membrane and bovine bone. After a healing period of 7 months, trephine cores were harvested for histological and histomorphometric analysis of the grafted areas. Histomorphometry demonstrated that the amount of newly formed bone in the test group (27.8%) was greater than that in the control group (25.3%), but the difference was not statistically significant (P=0.13). However, the mean number of microvessels in the test group was significantly higher than that in the control group (P=0.01). This study found that cortical bone perforation favourably affects the amount of new bone formation in the grafted sites after 7 months of healing. Cortical bone perforation significantly increase number of new vessels (angiogenesis) of the regenerated bone. Further randomized clinical trials are required to confirm these results.