Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass.

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.

Use of computed tomography to compare two femoral head and neck excision ostectomy techniques as performed by two novice veterinarians.

OBJECTIVES: To compare the results of femoral head and neck excision (FHNE) ostectomy performed by two novice veterinarians using an osteotome and mallet or microsagittal saw. METHODS: In this ex vivo cadaveric study, hindlimbs of eight canine cadavers were randomized to FHNE with osteotome or micro sagittal saw as performed by two recently graduated veterinarians. The hindimbs were imaged by computed tomography (CT) before and after the osteotomy. Post FHNE CT images were evaluated by a board certified radiologist blinded to the ostectomy technique for assessment of the number of bone fragments, fissures, smoothness of osteotomy margination, and volume of residual femoral neck. RESULTS: Femoral head and neck excision performed with the osteotome produced more peri-ostectomy bone fragments, cortical fissures, irregular margins, and residual femoral neck volume, compared with osteotomy using a saw. CLINICAL RELEVANCE: Compared to FHNE performed with a sagittal saw, osteotome FHNE resulted in a greater bone trauma and residual neck bone volume, which would require post-ostectomy modification in a clinical setting.

Real time neutron diffraction and NMR of the Empress II glass-ceramic system.

OBJECTIVE: This study reports real time neutron diffraction on the Empress II glass-ceramic system. METHODS: The commercial glass-ceramics was characterized by real time neutron diffraction, ³¹P and ²9Si solid-state MAS-NMR, DSC and XRD. RESULTS: On heating, the as-received glass ceramic contained lithium disilicate (Li2Si2O5), which melted with increasing temperature. This was revealed by neutron diffraction which showed the Bragg peaks for this phase had disappeared by 958°C in agreement with thermal analysis. On cooling lithium metasilicate (Li2SiO3) started to form at around 916°C and a minor phase of cristobalite at around 852°C. The unit cell volume of both Li-silicate phases increased linearly with temperature at a rate of +17×10⁻³ ų.°C⁻¹. Room temperature powder X-ray diffraction (XRD) of the material after cooling confirms presence of the lithium metasilicate and cristobalite as the main phases and shows, in addition, small amount of lithium disilicate and orthophosphate. ³¹P MAS-NMR reveals presence of the lithiorthophosphate (Li3PO4) before and after heat treatment. The melting of lithium disilicate on heating and crystallisation of lithium metasilicate on cooling agree with endothermic and exotermic features respectively observed by DSC. ²9Si MAS-NMR shows presence of lithium disilicate phase in the as-received glass-ceramic, though not in the major proportion, and lithium metasilicate in the material after heat treatment. Both phases have significantly long T1 relaxation time, especially the lithium metasilicate, therefore, a quantitative analysis of the ²9Si MAS-NMR spectra was not attempted. Significance. The findings of the present work demonstrate importance of the commercially designed processing parameters in order to preserve desired characteristics of the material. Processing the Empress II at a rate slower than recommended 60°C min⁻¹ or long isothermal hold at the maximal processing temperature 920°C can cause crystallization of lithium metasilicate and cristobalite instead of lithium disilicate as major phase.

Influence of strontium and the importance of glass chemistry and structure when designing bioactive glasses for bone regeneration.

The purpose of this article is to highlight some recent in vitro and in vivo studies of bioactive glasses containing strontium and to review selected literature on the in vitro and in vivo behaviour of bioactive glasses to relate this to the structure of the glass. The strontium-glass studies were performed well scientifically, but the results and conclusions could be misleading in terms of the effect of strontium, or more broadly glass chemistry, on the bioactivity and in vivo behaviour of bioactive glasses due to substitutions made on a weight basis. When strontium is substituted by weight for a lighter element such as calcium this will have a significant effect on structure and properties in particular biological response.

The effect of phosphate content on the bioactivity of soda-lime-phosphosilicate glasses.

We report on the bioactivity of two series of glasses in the SiO(2)-Na(2)O-CaO-P(2)O(5) system after immersion in simulated body fluid (SBF) after 21 days. The effect of P(2)O(5) content was examined for compositions containing 0-9.25 mol.% phosphate. Both series of glasses degraded to basic pH, but the solutions tended towards to neutrality with increasing phosphate content; a result of the acidic phosphate buffering the effect of the alkali metal and alkaline earth ions on degradation. Bioactivity was assessed by the appearance of features in the X-ray diffraction (XRD) traces and Fourier transform infrared (FTIR) spectra consistent with crystalline hydroxyl-carbonate-apatite (HCAp): such as the appearance of the (002) Bragg reflection in XRD and splitting of the P-O stretching vibration around 550 cm(-1) in the FTIR respectively. All glasses formed HCAp in SBF over the time periods studied and the time for formation of this crystalline phase occurred more rapidly in both series as the phosphate contents were increased. For P(2)O(5) content >3 mol.% both series exhibited highly crystalline apatite by 16 h immersion in SBF. This indicates that in the compositions studied, phosphate content is more important for bioactivity than network connectivity (NC) of the silicate phase and compositions showing rapid apatite formation are presented, superior to 45S5 Bioglass which was tested under identical conditions for comparison.

Structural analysis of a series of strontium-substituted apatites.

A series of Sr-substituted hydroxyapatites, (Sr(x)Ca(1-)(x))(5)(PO(4))(3)OH, where x=0.00, 0.25, 0.50, 0.75 and 1.00, were made by a standard wet chemical route and investigated using X-ray diffraction (XRD), Rietveld refinement and Raman spectroscopy. We report apatites manufactured by two synthesis routes under 90 degrees C, and only the fully Sr-substituted sample had a small amount of an impurity phase, which is believed to be strontium pyrophosphate. Lattice parameters (a and c), unit cell volume and density were shown to increase linearly with strontium addition and were consistent with the addition of a slightly larger and heavier ion (Sr) in place of Ca. XRD Lorentzian peak widths increased to a maximum at x=0.50, then decreased with increasing Sr content. This indicated an increase in crystallite size when moving away from the x=0.50 composition (d approximately 9.4nm). There was a slight preference for strontium to enter the Ca(II) site in the mixed apatites (6 to 12% depending on composition). The position of the Raman band attributed to v(1)PO(4)(3-) at around 963cm(-1) in hydroxyapatite decreased linearly to 949cm(-1) at full Sr-substitution. The full width at half maximum of this peak also correlated well and increased linearly with increasing crystallite size calculated from XRD.

Ultrastructure of lung elastin and collagen in mouse models of spontaneous emphysema.

The tight-skin (Tsk) and beige (bg) mutants of the C57B1/6J strain of mouse spontaneously develop air-space enlargement reminiscent of human emphysema. To determine if this enlargement is accompanied by matrix destruction, as in the human disease, we examined the elastin and collagen matrices of the lungs of both mutants. The ultrastructure of these matrix components was separately visualized by scanning electron microscopy following controlled alkali digestion, which preserves collagen, and formic acid digestion, which enables visualization of elastin. Significant elastin destruction suggestive of an elastolytic process was observed in the lungs of Tsk mice. Thickening of elastin lamellae was observed in the lungs of bg mice, suggesting that congenital matrix remodeling may underlie air-space enlargement in this strain.

Elastin and collagen remodeling in emphysema. A scanning electron microscopy study.

The relationship between elastin degradation and emphysema is well known. Recent evidence suggests that a complex process of pulmonary remodeling occurs within the emphysematous lung. The aim of this study was to assess the extent of extracellular matrix remodeling in emphysema by ultrastructural examination of elastin and collagen templates in an animal model of emphysema and in human emphysematous lungs. Emphysema was induced in rats by the intratracheal administration of porcine pancreatic elastase. Human lung samples were obtained at surgical resection for lung carcinoma. Emphysema was confirmed morphometrically and quantitated using the mean linear intercept. Matching sections were treated with sodium hydroxide and formic acid to expose collagen and elastin templates, respectively. Scanning electron microscopy with stereo-pair imaging allowed three-dimensional visualization of the exposed templates. In emphysematous lungs from both sources, sheets of elastin were disrupted and perforated with multiple fenestrations. In elastase-induced emphysema, this disintegration was accompanied by a marked increase in thickness of collagen fibrils, which contrasted with the fine fibrillar network of control lungs. Similarly, a pattern of thickened fibrils and disorganized deposition of collagen was observed in human lungs. In conclusion, these findings support the novel concept of increased collagen deposition and aberrant collagen remodeling in the pathogenesis of emphysema.

Three-dimensional structure of lung elastin demonstrated by scanning electron microscopy/stereo-pair images.

The aim of this study was to expose the inflated 3-D structure of lung elastin. Formic acid digestion followed by freeze-drying unveiled the lamellar framework. The 3-D structure of elastin was well preserved within the alveolar septa and ducts, as demonstrated by scanning electron microscopy/stereo-pair photography. Elastin fibers are seen in the alveolar septa, which are continuous with the lamellae. The removal of collagen fibers and cells by formic acid was visualised as a function of time: The optimum was 48 hours. Transverse sections still retained some collagen fibrils and partially digested cells in addition to elastin as shown by transmission electron microscopy (TEM). Formic acid digestion followed by critical point drying caused damage to the lamellar structures and they appeared to collapse. Sodium hydroxide digestion combined with freeze-drying did not preserve the 3-D lamellar structure of elastin, but converted it into flat ribbonlike bands. The main structures remaining following alkali treatment were identified by TEM as collagen fibrils well preserved in their original locations.

An alkali digestion method to expose connective tissue fibers: a scanning electron microscopy study of rat lung.

Alkali digestion has been used to remove cellular elements of tissues thus exposing the underlying connective tissue framework. We studied the action of this severe alkali treatment on the delicate tissues of rat lung. The lungs of male Sprague-Dawley rats were perfused with saline to remove blood and then inflated by fixation through the airways at 20 cm pressure. Sections of lung 2 x 5 x 5 mm were immersed in 2.5 M NaOH at 25 degrees C for 6 h, 16 h, 24 h, 48 h, and 72 h. The alkali was changed daily. Tissues were washed to neutral with water (24 h), treated with tannic acid (1%, 3h), post-fixed with osmic acid (1%, 3 h) and processed for SEM. At 6 h, epithelial cells started to peel off the alveolar surface. At 16 h the digestion process was well advanced. At 48 h the cells were completely removed revealing the lattice network of connective tissue fibers within the alveolar surface. The method allows the complete removal of cellular elements of the lung while retaining the very fine 3D structure of the connective tissue matrix.

The skeletal framework of human kidney and renal cell carcinoma. A scanning electron microscopic study.

The three dimensional architecture of the connective tissue framework of normal human kidney and three renal cell carcinomas was studied. A sodium hydroxide maceration technique was used to remove the cellular elements thus exposing the underlying connective tissue structures. The collagen fibrillar network was visualized using the scanning electron microscope. In normal kidney the fibres were fine, and smooth, and corresponded to the shapes of the original parenchymal constituents. The fibres of the kidney tumours were coarse in nature and irregularly distributed. The technique provides a rapid method for studying connective tissue fibres in normal and diseased tissue. The three dimensional architecture thus exposed enhances our knowledge of tumour stroma.

Suitability of control materials in the differential inhibition assay for human pancreatic and salivary amylase.

We investigated the behavior of 26 quality-control sera with the inhibitor method for differential amylase (EC 3.2.1.1) assay. We also studied the sensitivity to the wheat-derived inhibitor of pancreatic amylases from 10 different animals in comparison with human pancreatic and salivary amylase. The results indicate that only control materials containing human amylases can be measured accurately. The animal amylases (bovine, equine, porcine) used in many quality control sera are relatively insensitive to the inhibitor as compared with human pancreatic and salivary amylase.

Problems associated with the radioimmunoassay of serum trypsin.

A commercial trypsin radioimmunoassay (RIA) kit was used for its ability to measure trypsin bound to the serum protease inhibitors, alpha 2 macroglobulin (alpha 2 M) and alpha 1 anti-trypsin (alpha 1 AT). Only 20% of trypsin bound to alpha 2 M and 70% bound to alpha 1 AT was detected by the assay system. Recovery of trypsin added to human serum varied from 0 to 20%. Standard curves prepared from purified human cationic trypsin did not exhibit parallelism with the kit standard curves. Inclusion of horse serum in the standard solutions improved the parallelism observed. Immunoreactive trypsin (IRT) levels obtained for serum samples were found to vary considerably depending on the standard curve used to calculate the assay results. Lower IRT levels were observed when trypsin standards prepared in the absence of horse serum were used as reference.

Plasma pancreatic and salivary-type amylase and immunoreactive trypsin concentrations: variations with age and reference ranges for children.

The differential alpha-amylase (EC 3.2.1.1) assay was applied to 166 control children in the age range 0.1--13 years. Circulating levels of both pancreatic and salivary-type amylase were very low (mean 20 U/l) in the first four months of life. Pancreatic levels increased gradually with age, reaching adult levels (mean 74 U/l) by the age of eight years. Salivary amylase levels showed a sharp rise in the 0.9--1.9 year period reaching maximum levels (mean 99 U/l) by age 5--6 years. While linear regression analysis showed significant correlation between age of subject and pancreatic and salivary amylase levels, no such correlation was evident between age and plasma immunoreactive trypsin levels over the age range studied. Plasma trypsin levels in children were lower than reported adult values. Reference ranges for pancreatic and salivary-type amylase and immunoreactive trypsin in children are presented. The importance of age-matching, when pancreatic amylase and plasma trypsin are being investigated in children, is emphasised.

Renal clearance of pancreatic and salivary amylase relative to creatinine in patients with chronic renal insufficiency.

Pancreatic and salivary amylase/creatinine clearance ratios in patients with various degrees of renal impairment were compared with those obtained for control subjects. In chronic renal insufficiency (mean GFR 30 ml/min +/- 15 SD; n = 13) the clearance ratios for pancreatic (mean 3.5 +/- 1.85 SD) and salivary (mean 2.3 +/- 1.3 SD) amylase were significantly higher (P less than 0.05) than those in controls. Corresponding control values (n = 26) were 2.64 +/- 0.86 (pancreatic) and 1.64 +/- 0.95 (salivary). Three patients showed values above the normal limit. In the diabetic group (mean GFR 41 ml/min +/- 22 SD; n = 10) salivary amylase/creatinine clearance ratios (mean 2.36 +/- 1.55 SD) were significantly higher than in controls (P less than 0.05). Three patients showed raised values. Pancreatic amylase clearance was raised in only one of these patients. Three patients with terminal disease (mean GFR 10 ml/min) showed markedly raised (two- to threefold) clearance ratios for both salivary and pancreatic amylase. Of a total of 26 patients, eight had increased total amylase/creatinine clearance ratios. Pancreatic amylase/creatinine clearance was increased in seven patients, while nine patients showed raised salivary amylase/creatinine ratios. Patients with raised clearance ratios did not have clinical evidence of pancreatitis. We suggest that, in the presence of impaired renal function, a high amylase/creatinine clearance ratio need not be indicative of pancreatic disease.

Pancreatic and salivary amylase/creatinine clearance ratios in normal subjects and in patients with chronic pancreatitis.

The clearance of pancreatic and salivary amylase relative to creatinine was measured in 26 control subjects and 22 patients with chronic pancreatitis. Control values for pancreatic amylase clearance (+/- SD) were 2.64 +/- 0.86% compared with 1.54 +/- 0.95% for salivary amylase. In chronic pancreatitis, pancreatic amylase clearance ratios were significantly higher than controls (P less than 0.0005, mean 4.09 +/- 1.63 SD). The difference in clearance rate of salivary amylase did not reach a level of significance when compared with the control group. Twelve of the 22 patients showed pancreatic amylase clearance values above the normal limit of 4.4, while only five were abnormal when the clearance of total amylase was measured. The patients also showed statistically higher (P less than 0.0005) levels of serum salivary amylase when compared with 69 control sera. No such difference was found for the pancreatic component of serum amylase. Comparison of beta2-microglobulin clearance values showed no statistical difference between patients and controls.