Quantitative Magnetic Resonance Imaging of Cortical and Trabecular Bone.

Bone is a composite material consisting of mineral, organic matrix, and water. Water in bone can be categorized as bound water (BW), which is bound to bone mineral and organic matrix, or as pore water (PW), which resides in Haversian canals as well as in lacunae and canaliculi. Bone is generally classified into two types: cortical bone and trabecular bone. Cortical bone is much denser than trabecular bone that is surrounded by marrow and fat. Magnetic resonance (MR) imaging has been increasingly used for noninvasive assessment of both cortical bone and trabecular bone. Bone typically appears as a signal void with conventional MR sequences because of its short T2*. Ultrashort echo time (UTE) sequences with echo times 100 to 1,000 times shorter than those of conventional sequences allow direct imaging of BW and PW in bone. This article summarizes several quantitative MR techniques recently developed for bone evaluation. Specifically, we discuss the use of UTE and adiabatic inversion recovery prepared UTE sequences to quantify BW and PW, UTE magnetization transfer sequences to quantify collagen backbone protons, UTE quantitative susceptibility mapping sequences to assess bone mineral, and conventional sequences for high-resolution imaging of PW as well as the evaluation of trabecular bone architecture.

Which type of bone releases the most vancomycin? Comparison of spongious bone, cortical powder and cortico-spongious bone.

Bone infections can be challenging to treat and can lead to several surgeries and relapses. When a graft is needed, cavitary bone loss can be grafted with cancellous or cortical bone. Both can be used for grafting. However, the antibiotic releasing capacity of these grafts has not been compared. Which type of bone is best at releasing the most antibiotic has not been well established. The aim of this study was to determine which type of bone is best for antibiotic release when the bone is suffused with antibiotics by the surgeon. The hypothesis is that there would be a difference between the type of bone tested due to different release capacities of cortical and cancellous bone. This was an experimental study. Cortical spongy bone in chips, Spongy bone in chips and demineralized cortical bone powder were compared. For each type of bone, 5 samples were tested. Processed and decontaminated grafts were freeze-dried to be kept at room temperature. The primary endpoint was the amount of vancomycin released by the graft as it affects the concentration of antibiotic around the graft in clinical practice. The procedure for the study consisted of full graft immersion in a vancomycin solution. Then, the liquid was removed with aspiration. In order to measure the quantity of antibiotic released, the bone was put into distilled water in agitation in a heated rocker at 37 °C. After 30 min of soaking, 1 mL of the liquid was removed. The same extraction process was also carried out after 60 min soaking, 2 h, 3 h, 24 h, and 48 h. No differences were found between each type of bone relative to the concentration of vancomycin released at each time of the assessment. There was a significant difference in the weight of the bone with a higher weight for the cortical powder (1.793 g) versus cortical spongy bone and spongy bone (1.154 g and 1.013 g) with a p value < 0.0001. A significant difference was seen in the weight of the bone with vancomycin after the aspiration of the liquid with 3.026 g for cortical powder, 2.140 g and 2.049 g for the cortical spongy bone and the spongy bone with a p value < 0.0001. In daily clinical practice, one can use cancellous bone, cortico-cancellous bone or cortical powder in order to add vancomycin to a bone graft. Our results show the release kinetics of the soaked allografts. With a maximum of 14 mg/mL in the first minutes and a rapid decrease it shows a pattern comparable to antibiotic loaded bone cement. The method used appears favourable for prophylactic use, protecting the graft against contamination at implantation, but is not sufficient for treating chronic bone infection. LEVEL OF EVIDENCE: V.

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition.

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.

Evaluation of RNA from human trabecular bone and identification of stable reference genes.

The isolation of good quality RNA from tissues is an essential prerequisite for gene expression analysis to study pathophysiological processes. This study evaluated the RNA isolated from human trabecular bone and defined a set of stable reference genes. After pulverization, RNA was extracted with a phenol/chloroform method and then purified using silica columns. The A260/280 ratio, A260/230 ratio, RIN, and ribosomal ratio were measured to evaluate RNA quality and integrity. Moreover, the expression of six candidates was analyzed by qPCR and different algorithms were applied to assess reference gene stability. A good purity and quality of RNA was achieved according to A260/280 and A260/230 ratios, and RIN values. TBP, YWHAZ, and PGK1 were the most stable reference genes that should be used for gene expression analysis. In summary, the method proposed is suitable for gene expression evaluation in human bone and a set of reliable reference genes has been identified.

High Serum Fractalkine is Associated with Lower Trabecular Bone Score in Premenopausal Women with Graves' Disease.

Chemokine CX3CL1 (fractalkine) may be an important factor linking thyroid status and bone remodeling, through tetrac, a derivative of thyroxine. This study explores the relationship between serum fractalkine levels and parameters of thyroid status and bone in premenopausal women with Graves' disease (GD) in comparison to healthy controls. This cross-sectional study included three premenopausal female groups: active GD; cured GD, and healthy age-, gender-, and BMI-matched controls. Measurement of serum fractalkine levels (Quantikine® ELISA), total amino-terminal peptide of procollagen type 1 (P1NP), CTx, thyroid hormones, BMD and trabecular bone score (TBS) were performed in all study subjects. Sixty women (21, 16, and 23 in active GD, cured GD, and healthy control groups, respectively) were included. Serum fractalkine levels were higher (p<0.05) in active and cured GD subjects compared to healthy controls (mean 0.7±0.14; 0.93±0.15, and 0.48±0.13 ng/ml, respectively). Lumbar spine BMD was lowest in the cured GD group in comparison to active GD and control group subjects (0.926±0.03; 1.016±0.03; 1.051±0.03 g/cm2; p<0.05, respectively). TBS was lower (p<0.05) in both GD groups than controls being lowest in those with active GD (1.395±0.02; 1.402±0.02, 1.469±0.02, respectively). Serum fractalkine concentration was positively correlated with fT4, and negatively correlated with TBS values. GD in pre-menopausal females is associated with increased serum fractalkine concentration and decreased TBS. Fractalkine may be a currently unappreciated link between hyperthyroidism and bone; further research into this possibility is needed.

Laser-induced plasma spectroscopy (LIPS): use of a geological tool in assessing bone mineral content.

Bone may be similar to geological formulations in many ways. Therefore, it may be logical to apply laser-based geological techniques in bone research. The mineral and element oxide composition of bioapatite can be estimated by mathematical models. Laser-induced plasma spectrometry (LIPS) has long been used in geology. This method may provide a possibility to determine the composition and concentration of element oxides forming the inorganic part of bones. In this study, we wished to standardize the LIPS technique and use mathematical calculations and models in order to determine CaO distribution and bone homogeneity using bovine shin bone samples. We used polished slices of five bovine shin bones. A portable LIPS instrument using high-power Nd++YAG laser pulses has been developed (OpLab, Budapest). Analysis of CaO distribution was carried out in a 10 × 10 sampling matrix applying 300-µm sampling intervals. We assessed both cortical and trabecular bone areas. Regions of interest (ROI) were determined under microscope. CaO peaks were identified in the 200-500 nm wavelength range. A mathematical formula was used to calculate the element oxide composition (wt%) of inorganic bone. We also applied two accepted mathematical approaches, the Bartlett's test and frequency distribution curve-based analysis, to determine the homogeneity of CaO distribution in bones. We were able to standardize the LIPS technique for bone research. CaO concentrations in the cortical and trabecular regions of B1-5 bones were 33.11 ± 3.99% (range 24.02-40.43%) and 27.60 ± 7.44% (range 3.58-39.51%), respectively. CaO concentrations highly corresponded to those routinely determined by ICP-OES. We were able to graphically demonstrate CaO distribution in both 2D and 3D. We also determined possible interrelations between laser-induced craters and bone structure units, which may reflect the bone structure and may influence the heterogeneity of CaO distributions. By using two different statistical methods, we could confirm if bone samples were homogeneous or not with respect to CaO concentration distribution. LIPS, a technique previously used in geology, may be included in bone research. Assessment of element oxide concentrations in the inorganic part of bone, as well as mathematical calculations may be useful to determine the content of CaO and other element oxides in bone, further analyze bone structure and homogeneity and possibly apply this research to normal, as well as diseased bones.

Effect of the trabecular bone microstructure on measuring its thermal conductivity: A computer modeling-based study.

The objective of this work is to quantify the relation between the value of the effective thermal conductivity of trabecular bone and its microstructure and marrow content. The thermal conductivity of twenty bovine trabecular bone samples was measured prior to and after defatting at 37, 47, and 57 °C. Computer models were built including the microstructure geometry and the gap between the tissue and measurement probe. The thermal conductivity (k) measured was 0.39 ±â€¯0.06 W m-1 K-1 at 37 °C, with a temperature dependence of + 0.2%°C-1. Replacing marrow by phosphate-buffered saline (defatting) increased both the computer simulations and measurement results by 0.04 W m-1 K-1. The computer simulations showed that k increases by 0.02-0.04 W m-1 K-1 when the model includes a gap filled by phosphate-buffered saline between the tissue and measurement probe. In the presence of microstructure and fatty red marrow, k varies by ±â€¯0.01 W m-1 K-1 compared with the case considering matrix only, which suggests that there are no significant differences between cortical and trabecular bone in terms of k. The computer results showed that the presence of a gap filled by phosphate-buffered saline around the energy applicator changes maximum temperature by < 0.7 °C, while including the bone microstructure involved a variation of < 0.2 mm in the isotherm location. Future experimental studies on measuring the value of k involving the insertion of a probe into the bone through a drill hole should consider the bias found in the simulations. Thermal models based on a homogeneous geometry (i.e. ignoring the microstructure) could provide sufficient accuracy.

Bone and subcutaneous adipose tissue pharmacokinetics of vancomycin in total knee replacement patients.

Background and purpose - The incidence of orthopedic methicillin-resistant Staphylococcus aureus (MRSA) infections is increasing. Vancomycin may therefore play an increasingly important role in orthopedic perioperative antimicrobial prophylaxis. Studies investigating perioperative bone and soft tissue concentrations of vancomycin are sparse and challenged by a lack of appropriate methods. We assessed single-dose plasma, subcutaneous adipose tissue (SCT) and bone concentrations of vancomycin using microdialysis in male patients undergoing total knee replacement. Methods - 1,000 mg of vancomycin was administered postoperatively intravenously over 100 minutes to 10 male patients undergoing primary total knee replacement. Vancomycin concentrations in plasma, SCT, cancellous, and cortical bone were measured over the following 8 hours. Microdialysis was applied for sampling in solid tissues. Results - For all solid tissues, tissue penetration of vancomycin was significantly impaired. The time to a mean clinically relevant minimal inhibitory concentration (MIC) of 2 mg/L was 3, 36, 27, and 110 min for plasma, SCT, cancellous, and cortical bone, respectively. As opposed to the other compartments, a mean MIC of 4 mg/L could not be reached in cortical bone. The area under the concentration-time curve from 0 to the last measured value and peak drug concentrations (Cmax) for SCT, cancellous, and cortical bone was lower than that of free plasma. The time to Cmax was higher for all tissues compared with free plasma. Interpretation - Postoperative penetration of vancomycin to bone and SCT was impaired and delayed in male patients undergoing total knee replacement surgery. Adequate perioperative vancomycin concentrations may not be reached using standard prophylactic dosage.

The effect of tannic acid on bone mechanical and geometric properties, bone density, and trabecular histomorphometry as well as the morphology of articular and growth cartilages in rats co-exposed to cadmium and lead is dose dependent.

Cadmium (Cd) and lead (Pb) are toxic elements that accumulate to the largest extent in bones. Rats were used to investigate whether tannic acid (TA; 0.5%, 1.0%, 1.5%. 2.0%, or 2.5%) would have a protective effect on the structure and properties of bones in the case of exposure to Cd and Pb (diet: 7 mg Cd/kg and 50 mg Pb/kg) for 6 weeks. The effects of administration of TA in Cd- and Pb-poisoned rats on bone characteristics and the morphology of articular and growth cartilages were determined. All the rats administered Cd and Pb had an enhanced Cd and Pb concentration in blood plasma and bone and reduced bone Ca content irrespective of the TA administration. Cd and Pb alone reduced the mechanical endurance and histomorphometric parameters of trabecular bone and the thickness of the growth plate and articular cartilage. Tannic acid improved cancellous bone parameters in the rat exposed to Cd and Pb. A diet rich in TA improved articular cartilage constituents in heavy metal-poisoned rats. These results suggest that alimentary TA supplementation can counteract in a dose-dependent manner some of the destructive changes evoked by Cd and Pb possibly by reducing the exposure.

Fabrication and development of artificial osteochondral constructs based on cancellous bone/hydrogel hybrid scaffold.

Using tissue engineering techniques, an artificial osteochondral construct was successfully fabricated to treat large osteochondral defects. In this study, porcine cancellous bones and chitosan/gelatin hydrogel scaffolds were used as substitutes to mimic bone and cartilage, respectively. The porosity and distribution of pore size in porcine bone was measured and the degradation ratio and swelling ratio for chitosan/gelatin hydrogel scaffolds was also determined in vitro. Surface morphology was analyzed with the scanning electron microscope (SEM). The physicochemical properties and the composition were tested by using an infrared instrument. A double layer composite scaffold was constructed via seeding adipose-derived stem cells (ADSCs) induced to chondrocytes and osteoblasts, followed by inoculation in cancellous bones and hydrogel scaffolds. Cell proliferation was assessed through Dead/Live staining and cellular activity was analyzed with IpWin5 software. Cell growth, adhesion and formation of extracellular matrix in composite scaffolds blank cancellous bones or hydrogel scaffolds were also analyzed. SEM analysis revealed a super porous internal structure of cancellous bone scaffolds and pore size was measured at an average of 410 ± 59 µm while porosity was recorded at 70.6 ± 1.7 %. In the hydrogel scaffold, the average pore size was measured at 117 ± 21 µm and the porosity and swelling rate were recorded at 83.4 ± 0.8 % and 362.0 ± 2.4 %, respectively. Furthermore, the remaining hydrogel weighed 80.76 ± 1.6 % of the original dry weight after hydration in PBS for 6 weeks. In summary, the cancellous bone and hydrogel composite scaffold is a promising biomaterial which shows an essential physical performance and strength with excellent osteochondral tissue interaction in situ. ADSCs are a suitable cell source for osteochondral composite reconstruction. Moreover, the bi-layered scaffold significantly enhanced cell proliferation compared to the cells seeded on either single scaffold. Therefore, a bi-layered composite scaffold is an appropriate candidate for fabrication of osteochondral tissue.

A novel methodological approach to simultaneously extract high-quality total RNA and proteins from cortical and trabecular bone.

Molecular differences between cortical and trabecular bone, of relevance to understanding the pathophysiological basis of bone diseases, can be determined only through effective isolation methods for RNA and proteins. Here we present a TRIzol-based method, which combines bone pulverization and homogenization to extract simultaneously total RNA and proteins from human cortical and trabecular bone from the same carrot. RNA integrity and purity were determined as the 260/280 nm and 260/230 nm absorbance ratios and the 28S/18S rRNA ratio. Protein integrity and quality were evaluated by Coomassie blue staining. Reverse transcription quantitative polymerase chain reaction and immunoblotting for bone-specific genes and proteins were performed to verify the suitability of the isolated material in downstream applications. The 260/280 nm and 260/230 nm absorbance ratios were, on average, less than or equal to 1.8. Bands on agarose gel were consistent with intact RNA, with mean 28S/18S ratios of 1.68 ± 0.35 and 1.88 ± 0.10 for cortical and trabecular bone, respectively. Band patterns after Coomassie blue staining confirmed protein integrity. Successful gene and protein expression analysis, with relevant differences between the two compartments, highlighted the suitability of the material in downstream applications. The method presented here is appropriate and effective for the study of human bone.

A new cancellous bone material of silk fibroin/cellulose dual network composite aerogel reinforced by nano-hydroxyapatite filler.

Composites materials comprised of biopolymeric aerogel matrices and inorganic nano-hydroxyapatite (n-HA) fillers have received considerable attention in bone engineering. Although with significant progress in aerogel-based biomaterials, the brittleness and low strengths limit the application. The improvements in toughness and mechanical strength of aerogel-based biomaterials are in great need. In this work, an alkali urea system was used to dissolve, regenerate and gelate cellulose and silk fibroin (SF) to prepare composite aerosol. A dual network structure was shaped in the composite aerosol materials interlaced by sheet-like SF and reticular cellulose wrapping n-HA on the surface. Through uniaxial compression, the density of the composite aerogel material was close to the one of natural bone, and mechanical strength and toughness were high. Our work indicates that the composite aerogel has the same mechanical strength range as cancellous bone when the ratio of cellulose, n-HA and SF being 8:1:1. In vitro cell culture showed HEK-293T cells cultured on composite aerogels had high ability of adhesion, proliferation and differentiation. Totally, the presented biodegradable composite aerogel has application potential in bone tissue engineering.

Study on mechanical properties and permeability of elliptical porous scaffold based on the SLM manufactured medical Ti6Al4V.

In this paper, we take the elliptical pore structure which is similar to the microstructure of cancellous bone as the research object, four groups of bone scaffolds were designed from the perspective of pore size, porosity and pore distribution. The size of the all scaffolds were uniformly designed as 10 × 10 × 12 mm. Four groups of model samples were prepared by selective laser melting (SLM) and Ti6Al4V materials. The statics performance of the scaffolds was comprehensively evaluated by mechanical compression simulation and mechanical compression test, the manufacturing error of the scaffold samples were evaluated by scanning electron microscope (SEM), and the permeability of the scaffolds were predicted and evaluated by simulation analysis of computational fluid dynamics (CFD). The results show that the different distribution of porosity, pore size and pores of the elliptical scaffold have a certain influence on the mechanical properties and permeability of the scaffold, and the reasonable size and angle distribution of the elliptical pore can match the mechanical properties and permeability of the elliptical pore scaffold with human cancellous bone, which has great potential for research and application in the field of artificial bone scaffold.

Preparation of a novel bovine cancellous bone/poly-amino acid composite with low immunogenicity, proper strength, and cytocompatibility in vitro.

In this work, the delipidized and deproteinized bovine cancellous bone powder/poly-amino acid (DDBP/PAA) composite was fabricated by extrusion-injection molding method for the first time. After about 70% clearance rate by the delipidization and deproteinization procedures, the residual antigens of galactosyl α-(1, 3)-galactosyl ß-1,4-N-aeetylglueosaminyl (α-Gal) and major histocompatibility complex (MHC) II were basically eliminated by the extrusion-injection molding process, which may cause high titer of antibody and lead to hyperacute rejection or chronic immune toxicity. Meanwhile, the natural BMP II and apatite in bovine bone were kept in DDBP/PAA composite. After 26 weeks of immersion in simulated body fluid, the DDBP/PAA composite remained the intact appearance, 96.4% of weight, and 69.2% of compressive strength, and these showed sufficient degradation stability. The composite also exhibited excellent attachment and proliferation abilities of mouse bone marrow mesenchymal stem cells (mMSCs). The results herein suggested that the DDBP/PAA composite was expected to be a load-bearing transplant with some natural ingredients for hard tissue repair.

Effects of bovine cancellous bone powder/poly amino acid composites on cellular behaviors and osteogenic performances.

Xenogeneic bone has good biological activity, but eliminating immunogenicity, while retaining osteogenic abilities, is a challenge. By combining xenogeneic bone with poly amino acid (PAA) that has an amide bond structure, a new type of composite conforming to bionics and low immunogenicity may be obtained. In this study, according to the principles of component bionics, three composites of delipidized cancellous bone powder (DCBP) and PAA were designed and obtained by anin situpolycondensation method, an extrusion molding (EM) method, and a solution-blend method. The three composites were all macroscopically uniform, non-cytotoxic, and demonstrated low immunogenicity by effective removal of residual antigens during preparation. Compared with PAA, mouse bone marrow mesenchymal stem cells (BMSCs) on the surfaces of three composites showed different cellular morphologies. The effects of different preparation methods and cellular morphology on cellular differentiation were confirmed by alkaline phosphatase activity, calcium nodule formation and the expression levels of osteogenic differentiation-related genes (bone morphogenetic protein 2, runt-related transcription factor 2, osteopontin and osteocalcin). Among these composites, DCBP/PAA EM showed best cell proliferation and osteogenic differentiationin vitro, and possessed greater bone formation than PAA in a rabbit femoral condyle study. This study may provide a new method for preparing bioactive bone repair materials with low immunogenicity and superior ability to stimulate differentiation of BMSCsin vitroand osteogenesisin vivo. DCBP/PAA EM might be a promising bone repair material for bone defect treatment.

Influence of processing parameters on mechanical properties of a 3D-printed trabecular bone microstructure.

Natural bone microstructure has shown to be the most efficient choice for the bone scaffold design. However, there are several process parameters involved in the generation of a microCT-based 3D-printed (3DP) bone. In this study, the effect of selected parameters on the reproducibility of mechanical properties of a 3DP trabecular bone structure is investigated. MicroCT images of a distal radial sample were used to reconstruct a 3D ROI of trabecular bone. Nine tensile tests on bulk material and 54 compression tests on 8.2 mm cubic samples were performed (9 cases × 6 specimens/case). The effect of input-image resolution, STL mesh decimation, boundary condition, support material, and repetition parameters on the weight, elastic modulus, and strength were studied. The elastic modulus and the strength of bulk material showed consistent results (CV% = 9 and 6%, respectively). The weight, elastic modulus, and strength of the cubic samples showed small intragroup variation (average CV% = 1.2, 9, and 5.5%, respectively). All studied parameters had a significant effect on the outcome variables with less effect on the weight. Utmost care to every step of the 3DP process and involved parameters is required to be able to reach the desired mechanical properties in the final printed specimen. © 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:38-47, 2020.

Inter and intra-individual variation in skeletal DNA preservation in buried remains.

Our ability to identify skeletal remains often relies on the quality and quantity of DNA extracted from bone and teeth. Current research on buried remains has been retrospective, and no study to our knowledge has comprehensively assessed both intra-individual and inter-individual variation in human skeletal DNA from all representative skeletal element types recovered from a burial. Three individuals were interred together in a single grave for four years. Following disinterment, skeletal DNA was extracted, quantified, and GlobalFiler™ results were produced from 49 bones per skeleton, representing all bone types. Multiple sites per bone were also tested to determine intra-bone variability. Co-extracted bacterial and fungal DNA were quantified to determine microbial loads in the bones. Results show that the small, cancellous bones of the feet outperformed other bones in terms of DNA yield, measured as nanograms per gram of bone powder, and short tandem repeat (STR) profile completeness. The cuneiforms, in particular, had consistently high human DNA yields for all three individuals. DNA yield varied by individual and depth within the grave, with the shallowest individual demonstrating the highest DNA yields While the feet exhibited the greatest variation in DNA yield across bone type and sampling site, they also demonstrated some of the highest DNA yields and the most complete STR profiles, evoking a re-evaluation of their use for skeletal DNA sampling and analysis.

Timing of Antimicrobial Prophylaxis and Tourniquet Inflation: A Randomized Controlled Microdialysis Study.

BACKGROUND: Tourniquets are widely used during extremity surgery. In order to prevent surgical site infection, correct timing of antimicrobial prophylaxis and tourniquet inflation is important. We aimed to evaluate the time for which the free drug concentration of cefuroxime is maintained above the minimum inhibitory concentration (t > MIC) in porcine subcutaneous adipose tissue and calcaneal cancellous bone during 3 clinically relevant tourniquet application scenarios. METHODS: Twenty-four female Danish Landrace pigs were included. Microdialysis catheters were placed bilaterally for sampling of cefuroxime concentrations in calcaneal cancellous bone and subcutaneous adipose tissue, and a tourniquet was applied to a randomly picked leg of each pig. Subsequently, the pigs were randomized into 3 groups to receive 1.5 g of cefuroxime by intravenous injection 15 minutes prior to tourniquet inflation (Group A), 45 minutes prior to tourniquet inflation (Group B), and at the time of tourniquet release (Group C). The tourniquet duration was 90 minutes in all groups. Dialysates and venous blood samples were collected for 8 hours after cefuroxime administration. Cefuroxime and various ischemic marker concentrations were quantified. RESULTS: Cefuroxime concentrations were maintained above the clinical breakpoint MIC for Staphylococcus aureus (4 µg/mL) in calcaneal cancellous bone and subcutaneous adipose tissue throughout the 90-minute tourniquet duration in Groups A and B. Cefuroxime administration at the time of tourniquet release (Group C) resulted in concentrations of >4 µg/mL for approximately of 3.5 hours in the tissues on the tourniquet side. Furthermore, tourniquet application induced ischemia (increased lactate:pyruvate ratio) and cell damage (increased glycerol) in subcutaneous adipose tissue and calcaneal cancellous bone. Tissue ischemia was sustained for 2.5 hours after tourniquet release in calcaneal cancellous bone. CONCLUSIONS: Administration of cefuroxime (1.5 g) in the 15 to 45-minute window prior to tourniquet inflation resulted in sufficient concentrations in calcaneal cancellous bone and subcutaneous adipose tissue throughout the 90-minute tourniquet application. Furthermore, tourniquet-induced tissue ischemia fully resolved 2.5 hours after tourniquet release. CLINICAL RELEVANCE: Cefuroxime administration 15 to 45 minutes prior to tourniquet inflation seems to be a safe window. If the goal is to maintain postoperative cefuroxime concentrations above relevant MIC values, our results suggest that a second dose of cefuroxime should be administered at the time of tourniquet release.

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.

Effect of chitosan infiltration on hydroxyapatite scaffolds derived from New Zealand bovine cancellous bones for bone regeneration.

Hydroxyapatite (HA) derived from bovine bones garnered wider interest as a bone substitute due to their abundant availability as meat wastes and similarities in morphology and mineral composition to human bone. In our previous work, we developed an easy and reproducible method to prepare xenograft HA scaffolds from NZ bovine cancellous bones (BHA). However, the processing methodology rendered the material mechanically weak. The present study investigated the infiltration of chitosan (CS) into the bovine HA scaffolds (CSHA) to improve the mechanical properties of BHA. The presence of characteristic functional groups of HA and CS as detected by infrared spectroscopy confirmed the infiltration of CS into the BHA scaffolds. X-ray Diffraction study confirmed the presence of the hydroxyapatite phase in both BHA and CSHA scaffolds. SEM and µCT analyses showed the CSHA scaffolds presented adequate porosity and an interconnected porous architecture required for cell migration and attachment. CSHA scaffolds presented good thermal, chemical and structural stability while demonstrating sustained biodegradability in simulated body fluid. CSHA scaffolds presented mechanical properties significantly higher than the BHA scaffolds. CSHA scaffolds were biocompatible with Saos-2 osteoblast cells and supported cell proliferation significantly better than the BHA scaffolds indicating their potential in bone tissue engineering.