Engineering human bone grafts with new macroporous calcium phosphate cement scaffolds.

Bone engineering opens the possibility to grow large amounts of tissue products by combining patient-specific cells with compliant biomaterials. Decellularized tissue matrices represent suitable biomaterials, but availability, long processing time, excessive cost, and concerns on pathogen transmission have led to the development of biomimetic synthetic alternatives. We recently fabricated calcium phosphate cement (CPC) scaffolds with variable macroporosity using a facile synthesis method with minimal manufacturing steps and demonstrated long-term biocompatibility in vitro. However, there is no knowledge on the potential use of these scaffolds for bone engineering and whether the porosity of the scaffolds affects osteogenic differentiation and tissue formation in vitro. In this study, we explored the bone engineering potential of CPC scaffolds with two different macroporosities using human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MP) or isolated from bone marrow (BMSC). Biomimetic decellularized bone scaffolds were used as reference material in all experiments. The results demonstrate that, irrespective of their macroporosity, the CPC scaffolds tested in this study support attachment, viability, and growth of iPSC-MP and BMSC cells similarly to decellularized bone. Importantly, the tested materials sustained differentiation of the cells as evidenced by increased expression of osteogenic markers and formation of a mineralized tissue. In conclusion, the results of this study suggest that the CPC scaffolds fabricated using our method are suitable to engineer bone grafts from different cell sources and could lead to the development of safe and more affordable tissue grafts for reconstructive dentistry and orthopaedics and in vitro models for basic and applied research.

Excessive dietary intake of vitamin A reduces skull bone thickness in mice.

Calvarial thinning and skull bone defects have been reported in infants with hypervitaminosis A. These findings have also been described in humans, mice and zebrafish with loss-of-function mutations in the enzyme CYP26B1 that degrades retinoic acid (RA), the active metabolite of vitamin A, indicating that these effects are indeed caused by too high levels of vitamin A and that evolutionary conserved mechanisms are involved. To explore these mechanisms, we have fed young mice excessive doses of vitamin A for one week and then analyzed the skull bones using micro computed tomography, histomorphometry, histology and immunohistochemistry. In addition, we have examined the effect of RA on gene expression in osteoblasts in vitro. Compared to a standard diet, a high dietary intake of vitamin A resulted in a rapid and significant reduction in calvarial bone density and suture diastasis. The bone formation rate was almost halved. There was also increased staining of tartrate resistant acid phosphatase in osteocytes and an increased perilacunar matrix area, indicating osteocytic osteolysis. Consistent with this, RA induced genes associated with bone degradation in osteoblasts in vitro. Moreover, and in contrast to other known bone resorption stimulators, vitamin A induced osteoclastic bone resorption on the endocranial surfaces.

Fabrication of macroporous cement scaffolds using PEG particles: In vitro evaluation with induced pluripotent stem cell-derived mesenchymal progenitors.

Calcium phosphate cements (CPCs) have been extensively used in reconstructive dentistry and orthopedics, but it is only recently that CPCs have been combined with stem cells to engineer biological substitutes with enhanced healing potential. In the present study, macroporous CPC scaffolds with defined composition were fabricated using an easily reproduced synthesis method, with minimal fabrication and processing steps. Scaffold pore size and porosity, essential for cell infiltration and tissue ingrowth, were tuned by varying the content and size of polyethylene glycol (PEG) particles, resulting in 9 groups with different architectural features. The scaffolds were characterized for chemical composition, porosity and mechanical properties, then tested in vitro with human mesenchymal progenitors derived from induced pluripotent stem cells (iPSC-MPs). Biomimetic decellularized bone scaffolds were used as reference material in this study. Our manufacturing process resulted in the formation of macroporous monetite scaffolds with no residual traces of PEG. The size and content of PEG particles was found to affect scaffold porosity, and thus mechanical properties. Irrespective of pore size and porosity, the CPC scaffolds fabricated in this study supported adhesion and viability of human iPSC-MPs similarly to decellularized bone scaffolds. However, the architectural features of the scaffolds were found to affect the expression of bone specific genes, suggesting that specific scaffold groups could be more suitable to direct human iPSC-MPs in vitro toward an osteoblastic phenotype. Our simplistic fabrication method allows rapid, inexpensive and reproducible construction of macroporous CPC scaffolds with tunable architecture for potential use in dental and orthopedic applications.

Compressive fatigue limit of four types of dental restorative materials.

The purpose of this study was to evaluate the quasi-static compressive strength and the compressive fatigue limit of four different dental restorative materials, before and after aging in distilled water for 30 days. A conventional glass ionomer cement (Fuji IX GP; IG), a zinc-reinforced glass ionomer cement (Chemfil rock; CF), a light curable resin-reinforced glass ionomer cement (Fuji II LC; LC) and a resin-based composite (Quixfil; QF) were investigated. Cylindrical specimens (4mm in diameter and 6mm in height) were prepared according to the manufacturer׳s instructions. The compressive fatigue limit was obtained using the staircase method. Samples were tested in distilled water at 37°C, at a frequency of 10Hz with 10(5) cycles set as run-out. 17 fatigue samples were tested for each group. Two-way ANOVA and one-way ANOVA followed by Tukey׳s post-hoc test were used to analyze the results. Among the four types of materials, the resin-based composite exhibited the highest compressive strength (244±13.0MPa) and compressive fatigue limit (134±7.8MPa), followed by the light-cured resin reinforced glass ionomer cement (168±8.5MPa and 92±6.6MPa, respectively) after one day of storage in distilled water. After being stored for 30 days, all specimens showed an increase in compressive strength. Aging showed no effect on the compressive fatigue limit of the resin-based composite and the light-cured resin reinforced glass ionomer cement, however, the conventional glass ionomer cements showed a drastic decrease (37% for IG, 31% for CF) in compressive fatigue limit. In conclusion, in the present study, resin modified GIC and resin-based composite were found to have superior mechanical properties to conventional GIC.

Changes in the drug release pattern of fresh and set simvastatin-loaded brushite cement.

Calcium phosphate cements are synthetic bone graft substitutes able to set at physiological conditions. They can be applied by minimally invasive surgery and can also be used as drug delivery systems. Consequently, the drug release pattern from the cement paste (fresh cement) is of high clinical interest. However, previous studies have commonly evaluated the drug release using pre-set cements only. Therefore, the aim of this work was to determine if the time elapsed from cement preparation until immersion in the solution (3 min for fresh cements, and 1h and 15 h for pre-set cements) had an influence on its physical properties, and correlating these to the drug release profile. Simvastatin was selected as a model drug, while brushite cement was used as drug carrier. This study quantified how the setting of a material reduces the accessibility of the release media to the material, thus preventing drug release. A shift in the drug release pattern was observed, from a burst-release for fresh cements to a sustained release for pre-set cements.

Long-Term In Vitro Degradation of a High-Strength Brushite Cement in Water, PBS, and Serum Solution.

Bone loss and fractures may call for the use of bone substituting materials, such as calcium phosphate cements (CPCs). CPCs can be degradable, and, to determine their limitations in terms of applications, their mechanical as well as chemical properties need to be evaluated over longer periods of time, under physiological conditions. However, there is lack of data on how the in vitro degradation affects high-strength brushite CPCs over longer periods of time, that is, longer than it takes for a bone fracture to heal. This study aimed at evaluating the long-term in vitro degradation properties of a high-strength brushite CPC in three different solutions: water, phosphate buffered saline, and a serum solution. Microcomputed tomography was used to evaluate the degradation nondestructively, complemented with gravimetric analysis. The compressive strength, chemical composition, and microstructure were also evaluated. Major changes from 10 weeks onwards were seen, in terms of formation of a porous outer layer of octacalcium phosphate on the specimens with a concomitant change in phase composition, increased porosity, decrease in object volume, and mechanical properties. This study illustrates the importance of long-term evaluation of similar cement compositions to be able to predict the material's physical changes over a relevant time frame.

Evaluation of a porosity measurement method for wet calcium phosphate cements.

The porosity of a calcium phosphate cement is a key parameter as it affects several important properties of the cement. However, a successful, non-destructive porosity measurement method that does not include drying has not yet been reported for calcium phosphate cements. The aim of this study was to evaluate isopropanol solvent exchange as such a method. Two different types of calcium phosphate cements were used, one basic (hydroxyapatite) and one acidic (brushite). The cements were allowed to set in an aqueous environment and then immersed in isopropanol and stored under three different conditions: at room temperature, at room temperature under vacuum (300 mbar) or at 37℃. The specimen mass was monitored regularly. Solvent exchange took much longer time to reach steady state in hydroxyapatite cements compared to brushite cements, 350 and 18 h, respectively. Furthermore, the immersion affected the quasi-static compressive strength of the hydroxyapatite cements. However, the strength and phase composition of the brushite cements were not affected by isopropanol immersion, suggesting that isopropanol solvent exchange can be used for brushite calcium phosphate cements. The main advantages with this method are that it is non-destructive, fast, easy and the porosity can be evaluated while the cements remain wet, allowing for further analysis on the same specimen.

Porosity prediction of calcium phosphate cements based on chemical composition.

The porosity of calcium phosphate cements has an impact on several important parameters, such as strength, resorbability and bioactivity. A model to predict the porosity for biomedical cements would hence be a useful tool. At the moment such a model only exists for Portland cements. The aim of this study was to develop and validate a first porosity prediction model for calcium phosphate cements. On the basis of chemical reaction, molar weight and density of components, a volume-based model was developed and validated using calcium phosphate cement as model material. 60 mol% ß-tricalcium phosphate and 40 mol% monocalcium phosphate monohydrate were mixed with deionized water, at different liquid-to-powder ratios. Samples were set for 24 h at 37°C and 100% relative humidity. Thereafter, samples were dried either under vacuum at room temperature for 24 h or in air at 37 °C for 7 days. Porosity and phase composition were determined. It was found that the two drying protocols led to the formation of brushite and monetite, respectively. The model was found to predict well the experimental values and also data reported in the literature for apatite cements, as deduced from the small absolute average residual errors (<2.0%). In conclusion, a theoretical model for porosity prediction was developed and validated for brushite, monetite and apatite cements. The model gives a good estimate of the final porosity and has the potential to be used as a porosity prediction tool in the biomedical cement field.

Enhanced drug delivery of antibiotic-loaded acrylic bone cements using calcium phosphate spheres.

BACKGROUND: Local infection near an implant may pose a serious problem for patients. Antibiotic delivery from acrylic (poly(methyl methacrylate)-based) cements is commonly used to prevent and treat infections in the proximity of, e.g., hip joint implants. However, at present, the drug release properties of PMMA cements are not optimal. An initial burst followed by very slow release means that an unnecessarily large amount of antibiotic needs to be added to the cement, increasing the risk of bacterial resistance. The main purpose of this study was to enhance drug delivery from PMMA cements without influencing the mechanical properties. METHODS: We incorporated strontium-doped calcium phosphate spheres (SCPS) into PMMA cement to enhance the antibiotic release and potentially improve the bone-cement integration. The release of strontium and vancomycin was investigated using inductively coupled plasma atomic emission spectroscopy and UV spectrophotometry, respectively. RESULTS: It was found that incorporating SCPS into PMMA could enhance the antibiotic release and deliver strontium ions to the surroundings. The incorporation of SCPS also increased the radiopacity as well as the working time of the cement. The compressive strength and Young's modulus were not affected. CONCLUSIONS: Our results showed that SCPS/PMMA antibiotic-loaded cement had enhanced antibiotic release, delivered strontium ions and maintained mechanical properties, indicating that the SCPS additive could be a good alternative for controlling the drug-delivery properties of PMMA cement.

Human bone hardness seems to depend on tissue type but not on anatomical site in the long bones of an old subject.

It has been hypothesised that among different human subjects, the bone tissue quality varies as a function of the bone segment morphology. The aim of this study was to assess and compare the quality, evaluated in terms of hardness of packages of lamellae, of cortical and trabecular bones, at different anatomical sites within the human skeleton. The contralateral six long bones of an old human subject were indented at different levels along the diaphysis and at both epiphyses of each bone. Hardness value, which is correlated to the degree of mineralisation, of both cortical and trabecular bone tissues was calculated for each indentation location. It was found that the cortical bone tissue was harder (+18%) than the trabecular one. In general, the bone hardness was found to be locally highly heterogeneous. In fact, considering one single slice obtained for a bone segment, the coefficient of variation of the hardness values was up to 12% for cortical bone and up to 17% for trabecular bone. However, the tissue hardness was on average quite homogeneous within and among the long bones of the studied donor, although differences up to 9% among levels and up to 7% among bone segments were found. These findings seem not to support the mentioned hypothesis, at least not for the long bones of an old subject.

Compressive behaviour of child and adult cortical bone.

In this study, cortical bone tissue from children was investigated. It is extremely difficult to obtain human child tissue. Therefore, the only possibility was to use bone tissue, free from any lesion, collected from young bone cancer patients. The compressive mechanical behaviour of child bone tissue was compared to the behaviour of adult tissue. Moreover, two hypotheses were tested: 1) that the mechanical behaviour of both groups is correlated to ash density; 2) that yield strain is an invariant. Small parts of the diaphysis of femora or tibiae from 12 children (4-15 years) and 12 adults (22-61 years) were collected. Cylindrical specimens were extracted from the cortical wall along the longitudinal axis of the diaphysis. A total of 107 specimens underwent compressive testing (strain rate: 0.1 s(-1)). Only the specimens showing a regular load-displacement curve (94) were considered valid and thereafter reduced to ash. It was found that the child bone tissue had significant lower compressive Young's modulus (-34%), yield stress (-38%), ultimate stress (-33%) and ash density (-17%) than the adult tissue. Conversely, higher compressive ultimate strain was found in the child group (+24%). Despite specimens extracted from both children and adults, ash density largely described the variation in tissue strength and stiffness (R(2)=in the range of 0.86-0.91). Furthermore, yield strain seemed to be roughly an invariant to subject age and tissue density. These results confirm that the mechanical properties of child cortical bone tissue are different from that of adult tissue. However, such differences are correlated to differences in tissue ash density. In fact, ash density was found to be a good predictor of strength and stiffness, also for cortical bone collected from children. Finally, the present findings support the hypothesis that compressive yield strain is an invariant.

Reduced tissue hardness of trabecular bone is associated with severe osteoarthritis.

This study investigated whether changes in hardness of human trabecular bone are associated with osteoarthritis. Twenty femoral heads extracted from subjects without musculoskeletal diseases (subject age: 49-83 years) and twenty femoral heads extracted from osteoarthritic subjects (subject age: 42-85 years) were tested. Sixty indentations were performed along the main trabecular direction of each sample at a fixed relative distance. Two microstructures were found on the indenting locations: packs of parallel-lamellae (PL) and secondary osteons (SO). A 25gf load was applied for 15s and the Vickers Hardness (HV) was assessed. Trabecular tissue extracted from osteoarthritic subjects was found to be about 13% less hard compared to tissue extracted from non-pathologic subjects. However, tissue hardness was not significantly affected by gender or age. The SO was 10% less hard than the PL for both pathologic and non-pathologic tissues. A hardness of 34.1HV for PL and 30.8HV for SO was found for the non-pathologic tissue. For osteoarthritic tissue, the hardness was 30.2HV for PL and 27.1HV for SO. In the bone tissue extracted from osteoarthritic subjects the occurrence of indenting a SO (28%) was higher than that observed in the non-pathological tissue (15%). Osteoarthritis is associated with reduced tissue hardness and alterations in microstructure of the trabecular bone tissue. Gender does not significantly affect trabecular bone hardness either in non-pathological or osteoarthritic subjects. A similar conclusion can be drawn for age, although a larger donor sample size would be necessary to definitively exclude the existence of a slight effect.

Volume to density relation in adult human bone tissue.

Uniformity of tissue mineralisation is a strongly debated issue, due to its relation with bone mechanical behaviour. Bone mineral density (BMD) is measured in the clinical practice and is applied in computational application to derive material properties of bone tissue. However, BMD cannot identify if the variation in bone density is related to a modification of tissue mineral density (TMD), a change in bone volume or a combination of the two. This study was aimed to investigate whether TMD can be assumed as a constant in adult human bone (trabecular and cortical). A total number of 115 cylindrical bone specimens were collected. An inter-site analysis (96 specimens, 2 donors) was performed on cortical and trabecular specimens extracted from different anatomical sites. An intra-site study (19 specimens, 19 donors) was performed on specimens extracted from femoral heads. Bone volume fraction (BV/TV) was computed by means of a micro-computed tomography. Furthermore, ash density (ρ(ash)) was measured. TMD was computed as the ratio between ρ(ash) and BV/TV. It was found that the TMD of trabecular (1.24 ± 0.16 g/cm(3)) and cortical (1.19 ± 0.06 g/cm(3)) bone were not statistically different (p=0.31). Furthermore, the linear regression between ρ(ash) and BV/TV was statistically significant (r(2)=0.99, p<0.001). Intra- and inter-site analyses demonstrated that the mineral distribution was independent of the extraction site. The present study suggests that TMD can be assumed reasonably constant in non-pathological adult bone tissue. Consequently, it is suggested that TMD can be managed as a constant in computational models, varying only BV in relation to clinical densitometric analysis.

Anisotropy and inhomogeneity of the trabecular structure can describe the mechanical strength of osteoarthritic cancellous bone.

Osteoarthritic cancellous bone was studied to investigate the development of this pathology, and the functional changes it induces in the bone. In order to predict how the morphological alterations of the tissue induced by the pathology can change the mechanical properties of the structure, two different strategies have been used in the literature: (1) emphasising the influence of structural anisotropy; (2) stressing the highly inhomogeneous characteristics of cancellous bone. The aim of the present study was to verify the theory that mechanical strength of osteoarthritic cancellous bone depends both on tissue anisotropy and inhomogeneity. Twenty-five specimens were extracted from osteoarthritic femoral heads, along selected directions, and analysed by means of a microtomograph. The same specimens were mechanically tested in compression to determine the mechanical strength. The most representative structural parameters, confirmed by a stepwise analysis, were used to define four models to describe the measured mechanical strength. The models were applied neglecting (global analysis) or considering (local analysis) tissue inhomogeneities to verify whether the correlation with ultimate stress could be improved. The coefficient of determination increased from 0.53, considering only bone volume fraction, up to 0.88, combining it with off-axis angle and normalised eigenvalue. A further improvement was found performing a local analysis (R(2)=0.90), which corresponded to a decrease of 17% in the residual error. The proposed approach of considering both tissue anisotropy and inhomogeneity improved the accuracy in predicting the mechanical behaviour of cancellous bone tissue and should be suitable for more general loading conditions.

Repeatability of experimental procedures to determine mechanical behaviour of ligaments.

The aim of this study was to investigate intra- and interspecimen repeatability of an experimental procedure, which determines elastic and viscoelastic properties of knee ligaments. The collateral ligaments from sheep were used and the repeatability was evaluated in terms of the coefficient of variation. The results indicated a good intraspecimen repeatability (the coefficient of variation generally less than 5%), whereas the interspecimen repeatability was lower (coefficient of variation of about 50%). In conclusion, since the intraspecimen coefficient of variation was low the test procedure was assumed to be repeatable.

The effects of embalming using a 4% formalin solution on the compressive mechanical properties of human cortical bone.

BACKGROUND: The use of formalin fixed bone tissue is often avoided because of its assumed influence on the mechanical properties of bone. Fixed bone tissue would minimise biological risks and eliminate preservation issues for long duration experimental tests. This study aimed to determine the short- and long-term effects of embalming, using a solution with 4% formalin concentration, on the mechanical properties of human cortical bone. METHODS: Three-millimetre cylindrical specimens of human cortical bone were extracted from two femoral diaphyses and divided in four groups. The first group was used as control, the remaining three groups were left in the embalming solution for 48 h, 4 week, and 8 week, respectively. Compressive mechanical properties, hardness and ash density were assessed. The last was used to check the homogeneity among the four groups. FINDINGS: No significant differences were found among the four groups in yield stress, ultimate stress and hardness. The specimens stored for 8 week in the embalming solution had significant lower Young's modulus (-24%), higher yield strain (+20%) and ultimate strain (+53%) compared to the other groups. INTERPRETATION: On a short-term perspective, embalming did not affect the compressive mechanical properties, nor hardness of human cortical bone, whereas a long-term preservation (8 week) did significantly affect Young's modulus, yield strain and ultimate strain in compression. Preserving bone segments for up to 4 week in an embalming solution with low formalin concentration seems to be an interesting alternative when collecting and/or managing fresh or fresh-frozen bone segments for biomechanical experiments is not possible.

The effect of tissue condition and applied load on Vickers hardness of human trabecular bone.

Hardness of trabecular human bone, evaluated by microindentation testing, has generally been measured on embedded tissues. It was known that this was not ideal but it had been preferred to other conditions (e.g. wet or dehydrated) as the trabeculae could withstand the applied load and the measurements were reliable. The aim of this study was to investigate if the tissue condition of the specimen and the applied load would alter the hardness values measured by Vickers microindentation. Vickers hardness values of human trabecular bone from the femoral head, prepared in three different ways (wet, dry and embedded) and tested with two different loads (50 and 25 gf), were measured. No significant difference was found between the two different loads. However, in several cases the 50 gf indentations had to be redone because they were too large or the trabecula broke locally. Even if the outlines of the indentations on wet bone were slightly less marked than the ones done on dehydrated or embedded bone, it was possible to measure the hardness. Significant differences of Vickers hardness values were found between the three preparations: the hardness increased passing from wet to dried (10%) and from wet to embedded (35%). Whereas the variation coefficient of the three tissue conditions were comparable. In conclusion, it is recommended to test human trabecular bone in a wet condition as it better represents the in vivo condition. Furthermore the use of a 25 gf load is suggested, allowing hardness measurements on almost all trabeculae without breaking them.

Mechanical testing of cancellous bone from the femoral head: experimental errors due to off-axis measurements.

The aim of this study was to verify whether a misalignment between the testing direction and the trabecular main direction has a significant effect on the compressive behaviour of cancellous bone. Ten cylindrical specimens were extracted from femoral heads with a misalignment to the trabecular main direction of approximately 20 degrees. Each specimen was paired with a specimen extracted aligned with the main direction of the trabeculae on the basis of the closest bone volume fraction, obtaining two groups, one 'aligned' and one 'misaligned'. The average off-axis angle was 6.1 degrees and 21.6 degrees for the 'aligned' and 'misaligned' group, respectively. The specimens underwent micro-tomographic analysis, compressive testing, micro-indentation testing and ashing. No significant differences were found in histomorphometric parameters, hardness and ash density between the two groups, whereas significant differences were found in Young's modulus and ultimate stress: both parameters, measured for the 'misaligned' group, were about 40% lower than those measured for the 'aligned' group. These results demonstrate a great effect of the angle between the testing direction and the main direction of the trabecular structure on the compressive behaviour of cancellous bone. This angle should be reduced as much as possible (in the present work the average value was 6.6+/-3.3 degrees), in any case measured, and always reported together with the mechanical parameters of cancellous bone.

Comparative study of different tendon grasping techniques for arthroscopic repair of the rotator cuff.

BACKGROUND: One cause of early failure of rotator cuff repairs is the pull out of the suture through the tendon. The aims of this study were to investigate the initial strength and failure mode of different tendon grasping techniques and to evaluate an alternative stitch proposed for arthroscopic repair of rotator cuff tendons. METHODS: Four different stitches were investigated: simple stitch, Mattress, modified Mason-Allen and simple stitch closed over a horizontal loop. The last stitch was proposed as an alternative to the modified Mason-Allen stitch since the former is simpler to sew arthroscopically than the latter. The experimental procedure was designed to assess the mechanical behaviour of the stitches. Tests were performed using sheep infraspinatus tendons. Two different non-absorbable sutures were used. Each specimen was preloaded with about 30 N and then loaded to failure. RESULTS: No significant difference was found in compliance among the four investigated stitches. Conversely, the tensile strength of the simple stitch and Mattress was lower than the tensile strength of the other two stitches, while no significant difference was observed between the modified Mason-Allen and the simple stitch closed over a horizontal loop. The maximum grasping power of these two 'reinforced' stitches was achieved only with the high-strength suture. INTERPRETATION: The simple stitch closed over a horizontal loop seems to be an attractive alternative to the modified Mason-Allen for arthroscopic repair of the rotator cuff and it seems recommendable instead of simple or Mattress stitches. The use of a high-strength suture would increase the tensile strength of the grasping in the case of good quality tendon tissue.