In vitro and in vivo evaluation of akermanite bioceramics for bone regeneration

This study investigated the effects of a calcium magnesium silicate bioceramic (akermanite) for bone regeneration in vitro and in vivo, with β-tricalcium phosphate (β-TCP) as a control. In vitro, the human bone marrow-derived mesenchymal stromal cells (hBMSCs) were cultured in an osteogenic medium supplemented with a certain concentration of two bioceramics' extracts for 20 days. An MTT assay showed that akermanite extract promoted proliferation of hBMSC significantly more than did β-TCP extract. The results of alkaline phosphatase (ALP) activity test and the expression of osteogenic marker genes such as ALP, osteopontin (OPN), osteocalcin (OCN) and bone sialoprotein (BSP) demonstrated that the osteogenic differentiation of hBMSC was enhanced more by akermanite extract than by β-TCP extract. In vivo, a histomorphology analysis and histomorphometry of the two porous bioceramics implants in rabbit femur defect models indicated that both in early- and late-stage implantations, akermanite promoted more osteogenesis and biodegradation than did β-TCP; and in late-stage implantations, the rate of new bone formation was faster in akermanite than in β-TCP. These results suggest that akermanite might be a potential and attractive bioceramic for tissue engineering.     

Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC–chitosan and CPC–chitosan–fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC–chitosan–fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC–chitosan–fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.     

Understanding the influence of MgO and SrO binary doping on the mechanical and biological properties of β-TCP ceramics

The objective of this study was to evaluate the influence of MgO and SrO doping on the mechanical and biological properties of β-tricalcium phosphate (β-TCP). β-TCP was doped with two different binary compositions, 0.25 and 1.0 wt.% SrO along with 1.0 wt.% MgO. MgO and SrO doping increased the β phase stability at a sintering temperature of 1250 °C and marginally decreased the compressive strength of β-TCP. An in vitro cell–material interaction study, using human fetal osteoblast cells (hFOB), indicated that doped β-TCP was non-toxic, and MgO/SrO dopants improved cell attachment and growth. β-TCP implants doped with 1.0 wt.% MgO and 1.0 wt.% SrO showed good in vivo biocompatibility when tested in male Sprague–Dawley rats for 16 weeks. Histomorphology analysis indicated that MgO/SrO-doped β-TCP promoted more osteogenesis than pure β-TCP. In vivo osteocalcin and type I collagen assay also revealed faster bone formation in rats with doped β-TCP implant compared to rats with pure β-TCP implant. Low Ca²⁺ concentration in the urine of rats with doped β-TCP implant confirmed slower degradation of MgO/SrO-doped β-TCP than pure β-TCP.     

beta-Sitosterol modulation of monocyte-endothelial cell interaction: a comparison to female hormones

ObjectiveTo investigate the effect of β-sitosterol, 17β-estradiol and progesterone on oxidized LDL (oxLDL)-stimulated human umbilical venous endothelial cell (HUVEC) expression of intercellular adhesion molecule-1 (ICAM-1), THP-1 monocyte chemotactic activity, migration and adhesion of THP-1 cells co-cultured with HUVECs.MethodsICAM-1 expression was determined by immunofluorescence in HUVEC monolayers treated with LDL or oxLDL and 17β-estradiol, progesterone or β-sitosterol. Monocyte chemotactic activity was performed in Transwell chambers by culturing HUVECs with different stimuli and steroids, THP-1 cells labeled with [³H] thymidine were added to the upper chamber and the radioactivity was measured. Migration assays were performed using Transwell chambers but monocytes were labeled with BCECF-AM and THP-1 cells adhered to HUVECs were visualized by fluorescence microscopy. MCP-1 was quantified by ELISA.ResultsICAM-1 expression was inhibited by β-sitosterol alone, when combined with 17β-estradiol or progesterone, or with both hormones. It was shown that 7.5 μM β-sitosterol decreased migration and adhesion of THP-1 cells to HUVECs cultured in the presence of oxLDL. This effect was also observed in HUVEC cultures in the presence of β-sitosterol, the 17β-estradiol and progesterone mixture, and in the presence of the two hormones. It was shown that 7.5 μM β-sitosterol significantly inhibited chemotaxis of [³H] thymidine labeled THP-1 cells in oxLDL-stimulated HUVEC cultures. MCP-1 concentrations in the supernatants of oxLDL-stimulated HUVEC cultures were inhibited by 7.5 μM β-sitosterol as well as by progesterone and the mixture of the two female hormones.     

Chondrogenic pre-induction of human mesenchymal stem cells on β-TCP: Enhanced bone quality by endochondral heterotopic bone formation

New techniques to heal bone defects include the combination of bone substitute materials with mesenchymal stem cells (MSC). To find solutions not hampered by low material resorbability or high donor variability of human MSC, the potency of such composites is usually evaluated by heterotopic bone formation assays in immunocompromised animals. The aim of this study was to investigate whether resorbable phase-pure β-tricalcium-phosphate (β-TCP) could support heterotopic bone formation by MSC comparable to partially resorbable hydroxyapatite/tricalcium-phosphate (HA/TCP). Furthermore, in light of disappointing results with osteogenic in vitro priming of MSC, we tested whether chondrogenic pre-induction of constructs may allow for enhanced bone formation by triggering the endochondral pathway. β-TCP granules of three different sizes and HA/TCP were seeded with MSC and transplanted subcutaneously into immunocompromised mice either immediately or after a chondrogenic pre-induction for 6 weeks. After 8 weeks, explants were analysed by histology. β-TCP seeded with unprimed MSC revealed intramembranous bone formation without haematopoietic marrow with 3.8-fold more bone formed with granules smaller than 0.7 mm than with 0.7–1.4 mm particles (p ? 0.018). Chondrogenic pre-induction of β-TCP/MSC composites resulted in collagen type II and proteoglycan-rich cartilage-like tissue which, after transplantation, underwent endochondral ossification, yielding ectopic bone produced by human cells while haematopoietic marrow was derived from the mouse. Transdifferentiation of MSC-derived chondrocytes to osteoblasts or direct osteogenesis of cartilage-resident MSC is postulated to explain the human origin of new bone. In conclusion, β-TCP was significantly more osteo-permissive (p = 0.004) than HA/TCP for human MSC, and chondrogenic priming of β-TCP/MSC represented a superior approach capable of supporting full bone formation, including marrow organization.     

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone–tricalcium phosphate (PCL–TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2×, p < 0.01), and maintained high cellular viability in the core, which was 2000 μm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5× p < 0.01), calcium deposition (5.5×, p < 0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2×, p < 0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.     

Creation of bony microenvironment with CaP and cell-derived ECM to enhance human bone-marrow MSC behavior and delivery of BMP-2

Extracellular matrix (ECM) comprises a rich meshwork of proteins and proteoglycans, which not only contains biological cues for cell behavior, but is also a reservoir for binding growth factors and controlling their release. Here we aimed to create a suitable bony microenvironment with cell-derived ECM and biodegradable β-tricalcium phosphate (β-TCP). More specifically, we investigated whether the ECM produced by bone marrow-derived mesenchymal stem cells (hBMSC) on a β-TCP scaffold can bind bone morphogenetic protein-2 (BMP-2) and control its release in a sustained manner, and further examined the effect of ECM and the BMP-2 released from ECM on cell behaviors. The ECM was obtained through culturing the hBMSC on a β-TCP porous scaffold and performing decellularization and sterilization. SEM, XPS, FTIR, and immunofluorescent staining results indicated the presence of ECM on the β-TCP and the amount of ECM increased with the incubation time. BMP-2 was loaded onto the β-TCP with and without ECM by immersing the scaffolds in the BMP-2 solution. The loading and release kinetics of the BMP-2 on the β-TCP/ECM were significantly slower than those on the β-TCP. The β-TCP/ECM exhibited a sustained release profile of the BMP-2, which was also affected by the amount of ECM. This is probably because the β-TCP/ECM has different binding mechanisms with BMP-2. The β-TCP/ECM promoted cell proliferation. Furthermore, the BMP-2-loaded β-TCP/ECM stimulated reorganization of the actin cytoskeleton, increased expression of alkaline phosphatase and calcium deposition by the cells compared to those without BMP-2 loading and the β-TCP with BMP-2 loading.     

Superior mineralization and neovascularization capacity of adult human metaphyseal periosteum-derived cells for skeletal tissue engineering applications

Bone tissue engineering is a promising cell-based strategy to treat bone defects. Mesenchymal stem cells from adult human bone marrow (hBMSCs) are a frequently used cellular source for bone tissue generation. However, the low frequency of these stem cells in adult bone marrow and their limited proliferation restrict their clinical utility. An alternative source of MSCs is the periosteum-derived cells, and these cells appear to be easy to harvest and expand ex vivo. We isolated human metaphyseal periosteum-derived cells (hMPCs) and hBMSCs from the same donors and compared their osteogenic capacity both in vitro and in vivo. After osteogenic induction in monolayer cultures, hMPCs resulted in more robust mineralization and expressed higher mRNA levels of BMP-2, osteopontin and osteocalcin than hBMSCs. Eight weeks after implantation of cellular-β-TCP scaffolds in immunodeficient mice, hMPC implantation showed higher neovascularization and higher percentage of mature bone formation than hBMSC implantation. In conclusion, hMPCs represent a promising cellular candidate for bone tissue engineering.     

In vitro assessment of three-dimensionally plotted nagelschmidtite bioceramic scaffolds with varied macropore morphologies

It is known that porous scaffolds play an important role in bone/periodontal tissue engineering. A new nagelschmidtite (NAGEL, Ca₇Si₂P₂O₁₆) ceramic has recently been prepared which shows excellent apatite mineralization ability and osteo-/cementostimulation properties in vitro. However, up to now porous NAGEL scaffolds have not been developed yet. There has been no systematic study of the effect of macropore morphology of bioceramic scaffolds on their physico-chemical and biological properties. The aim of this study was to prepare NAGEL scaffolds for bone tissue engineering applications. We applied a modified three-dimensional (3-D) plotting method to prepare highly controllable NAGEL scaffolds and investigated the effect of macropore morphology on the physico-chemical and biological properties. The results showed that the macropore size and morphology of 3-D plotted NAGEL scaffolds could be effectively controlled. Compared with β-tricalcium phosphate (β-TCP) scaffolds NAGEL scaffolds possess a significantly enhanced compressive strength, a higher modulus and better degradability. Nagel scaffolds with a square pore morphology presented a higher compressive strength, a higher modulus and greater weight loss rate than those with triangular and parallelogram pore morphologies. In addition, all of the NAGEL scaffolds with the three macropore morphologies supported the attachment and proliferation of MC3T3 cells. The proliferation of MC3T3 cells on NAGEL scaffolds with triangular and parallelogram structures was higher than that on β-TCP scaffolds with the same pore structure. Cells on all three groups of NAGEL scaffolds revealed higher alkaline phosphatase (ALP) activity compared with cells on β-TCP scaffolds, and among the three NAGEL scaffolds groups those with a parallelogram pore structure showed the highest ALP activity. Furthermore, the angiogenic cell experiments showed that the ionic products from NAGEL scaffolds promoted tube formation, expression of pro-angiogenic factors and their receptors on human umbilical vein endothelial (HUVECs) compared with β-TCP scaffolds, indicating that NAGEL scaffolds possessed improved angiogenesis capacity. Our results suggest that 3-D plotted NAGEL scaffolds are a promising bioactive material for bone tissue engineering by virtue of their highly controllable macropore structure, excellent mechanical strength, degradability and in vitro biological response to osteogenic/angiogenic cells.     

Mechanical properties,electronic structure and bonding of α- and β-tricalcium phosphates with surface characterization

The mechanical properties and electronic structure of α- and β-tricalcium phosphate (TCP) crystals are studied by using two ab initio density functional methods, the Vienna Ab initio Simulation Package (VASP) and the orthogonalized linear combination of atomic orbitals method. Based on the VASP optimized crystal structures, the elastic constants of α- and β-TCP are obtained using an effective stress–strain computational scheme. From the calculated elastic constants, the bulk modulus, shear modulus, Young’s modulus and Poisson’s ratios are obtained. The results show that the mechanical properties of the two crystals are comparable and that α-TCP is somewhat softer than β-TCP. Comparison with experimental extrapolations of the elastic constants shows significant differences, which attest to the difficulty of obtaining single crystal samples. The calculated electronic structure results show that both crystals are large gap insulators with a direct band gap of 4.89 eV for α-TCP and 5.25 eV for β-TCP. Effective charge calculations show that, on average, β-TCP has slightly less charge transfer per Ca than α-TCP. The (0 1 0) ((0 0 1)) surface model for α-TCP (β-TCP) is studied using a supercell slab geometry and fully relaxed to obtain the optimized structures. The estimated surface formation energies are 0.777 and 0.842 J m^?2 for α-TCP and β-TCP, respectively. The electronic structures of the two surface models are compared with the bulk models. Charge density analysis shows that the surfaces of both TCP crystals are positively charged overall owing to the presence of Ca ions near the surfaces.     

In vitro osteogenic potential of human bone marrow stromal cells cultivated in porous scaffolds from mineralized collagen

Porous 3D structures from mineralized collagen were fabricated applying a procedure in which collagen fibril reassembly and precipitation of nanocrystalline hydroxyapatite (HA) occur simultaneously. The resulting matrices were evaluated in vitro with respect to their suitability as scaffolds for bone tissue engineering. We found a high capacity of the material to bind serum proteins as well as to absorb Ca2+ ions, which could be advantageous to promote cell attachment, growth, and differentiation. Human bone marrow stromal cells (hBMSCs) were seeded onto the 3D scaffolds and cultivated for 4 weeks in the presence and absence of osteogenic supplements. We studied viability, proliferation, and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content, and alkaline phosphatase (ALP) activity. Furthermore, the expression for bone-related genes (ALP, bone sialo protein II (BSP II), and osteocalcin) was analyzed. In our investigation we found a 2.5-fold to 5-fold raise in DNA content and an increase of ALP activity for osteogenic induced hBMSC on collagen HA scaffolds. The expression of ALP and BSP II in these cells was also stimulated in the course of cultivation; however, we did not detect an upregulation of osteocalcin gene expression. These data suggest, that porous collagen HA scaffolds are suitable for the expansion and osteogenic differentiation of hBMSC and are therefore promising candidates for application as bone grafts.     

Flexible and elastic porous poly(trimethylene carbonate) structures for use in vascular tissue engineering

Biocompatible and elastic porous tubular structures based on poly(1,3-trimethylene carbonate), PTMC, were developed as scaffolds for tissue engineering of small-diameter blood vessels. High-molecular-weight PTMC (M_n = 4.37 × 10⁵) was cross-linked by gamma-irradiation in an inert nitrogen atmosphere. The resulting networks (50–70% gel content) were elastic and creep resistant. The PTMC materials were highly biocompatible as determined by cell adhesion and proliferation studies using various relevant cell types (human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs)). Dimensionally stable tubular scaffolds with an interconnected pore network were prepared by particulate leaching. Different cross-linked porous PTMC specimens with average pore sizes ranging between 55 and 116 μm, and porosities ranging from 59% to 83% were prepared. These scaffolds were highly compliant and flexible, with high elongations at break. Furthermore, their resistance to creep was excellent and under cyclic loading conditions (20 deformation cycles to 30% elongation) no permanent deformation occurred. Seeding of SMCs into the wall of the tubular structures was done by carefully perfusing cell suspensions with syringes from the lumen through the wall. The cells were then cultured for 7 days. Upon proliferation of the SMCs, the formed blood vessel constructs had excellent mechanical properties. Their radial tensile strengths had increased from 0.23 to 0.78 MPa, which is close to those of natural blood vessels.     

Mesenchymal stem cell proliferation and differentiation on load-bearing trabecular Nitinol scaffolds

Bone tissue regeneration in load-bearing regions of the body requires high-strength porous scaffolds capable of supporting angiogenesis and osteogenesis. 70% porous Nitinol (NiTi) scaffolds with a regular 3-D architecture resembling trabecular bone were produced from Ni foams using an original reactive vapor infiltration technique. The “trabecular Nitinol” scaffolds possessed a high compressive strength of 79 MPa and high permeability of 6.9 × 10^?6 cm². The scaffolds were further modified to produce a near Ni-free surface layer and evaluated in terms of Ni ion release and human mesenchymal stem cell (hMSC) proliferation (AlamarBlue), differentiation (alkaline phosphatase activity, ALP) and mineralization (Alizarin Red S staining). Scanning electron microscopy was employed to qualitatively corroborate the results. hMSCs were able to adhere and proliferate on both as-produced and surface-modified trabecular NiTi scaffolds, to acquire an osteoblastic phenotype and produce a mineralized extracellular matrix. Both ALP activity and mineralization were increased on porous scaffolds compared to control polystyrene plates. Experiments in a model coculture system of microvascular endothelial cells and hMSCs demonstrated the formation of prevascular structures in trabecular NiTi scaffolds. These data suggest that load-bearing trabecular Nitinol scaffolds could be effective in regenerating damaged or lost bone tissue.     

Freeform fabricated scaffolds with roughened struts that enhance both stem cell proliferation and differentiation by controlling cell shape

We demonstrate that freeform fabricated (FFF) scaffolds with a roughened surface topography can support hBMSC proliferation, while also inducing osteogenic differentiation, for maximized generation of calcified, bone-like tissue. Previously, hBMSCs rapidly proliferated, without osteogenic differentiation, during culture in FFF scaffolds. In contrast, hBMSCs underwent osteogenic differentiation, with slow proliferation, during culture in nanofiber scaffolds. Analysis of cell morphology showed that the topography presented by the nanofiber scaffolds drove hBMSC differentiation by guiding them into a morphology that induced osteogenic differentiation. Herein, we hypothesized that using the high-surface area architecture of FFF scaffolds to present a surface roughness that drives hBMSCs into a morphology that induces osteogenic differentiation would yield a maximum amount differentiated hBMSCs and bone-like tissue. Thus, a solvent etching method was developed that imparted a 5-fold increase in roughness to the surface of the struts of poly(ε-caprolactone) (PCL) FFF scaffolds. The etched scaffolds induced osteogenic differentiation of the hBMSCs while un-etched scaffolds did not. The etched scaffolds also supported the same high levels of hBMSC proliferation that un-etched scaffolds supported. Finally, hBMSCs on un-etched scaffolds had a large spread area, while hBMSCs on etched scaffolds has a smaller area and were more rounded, indicating that the surface roughness from the etched scaffolds dictated the morphology of the hBMSCs. The results demonstrate that FFF scaffolds with surface roughness can support hBMSC proliferation, while also inducing osteogenic differentiation, to maximize generation of calcified tissue. This work validates a rational approach to scaffold fabrication where the structure of the scaffold was designed to optimize stem cell function by controlling cell morphology.     

Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts

Nanotechnology has enabled the engineering of nanostructured materials to meet current challenges in bone replacement therapies. Biocomposite nanofibrous scaffolds of poly(l-lactic acid)-co-poly(?-caprolactone), gelatin and hydroxyapatite (HA) were fabricated by combining the electrospinning and electrospraying techniques in order to create a better osteophilic environment for the growth and mineralization of osteoblasts. Electrospraying of HA nanoparticles on electrospun nanofibers helped to attain rough surface morphology ideal for cell attachment and proliferation and also achieve improved mechanical properties than HA blended nanofibers. Nanofibrous scaffolds showed high pore size and porosity up to 90% with fiber diameter in the range of 200–700 nm. Nanofibrous scaffolds were characterized for their functional groups and chemical structure by FTIR and XRD analysis. Studies on cell–scaffold interaction were carried out by culturing human fetal osteoblast cells (hFOB) on both HA blended and sprayed PLACL/Gel scaffolds and assessing their growth, proliferation, mineralization and enzyme activity. The results of MTS, ALP, SEM and ARS studies confirmed, not only did HA sprayed biocomposite scaffolds showed better cell proliferation but also enhanced mineralization and alkaline phosphatase activity (ALP) proving that electrospraying in combination with electrospinning produced superior and more suitable biocomposite nanofibrous scaffolds for bone tissue regeneration.     

The collagen component of biological bone graft substitutes promotes ectopic bone formation by human mesenchymal stem cells

Synthetic bone substitutes are attractive materials for repairing a variety of bone defects. They are readily available in unlimited quantities, have a defined composition without batch variability and bear no risk of disease transmission. When combined with mesenchymal stem cells (MSCs), bone healing can be further enhanced due to the osteogenic potential of these cells. However, human MSCs showed considerable donor variability in ectopic bone formation assays on synthetic bone substitutes, which may limit clinical success. This study addresses whether bone formation variability of MSCs is cell-intrinsic or biomaterial-dependent and may be improved using biological bone substitutes with and without collagen. Ectopic bone formation of MSCs from nine donors was tested in immune-deficient mice on biological bone substitutes of bovine and equine origin, containing collagen (bHA-C; eHA-C) or not (bHA; eHA). Synthetic β-TCP was used for comparison. Histology of 8-week explants demonstrated a significant influence of the bone graft substitute (BGS) on donor variability of ectopic bone formation with best results seen for eHA-C (15/17) and β-TCP (16/18). Bone was of human origin in all groups according to species-specific in situ hybridization, but MSCs from one donor formed no bone with any bone substitute. According to histomorphometry, most neo-bone was formed on eHA-C with significant differences to bHA, eHA and β-TCP (p < 0.001). Collagen-free biological BGSs were inferior to biological BGSs with collagen (p < 0.001), while species-origin was of little influence. In conclusion, BGS composition had a strong influence on ectopic bone formation ability of MSCs, and biological BGSs with a collagen component seem most promising to display the strong osteogenic potential of MSCs.     

Biofilm formation on bone grafts and bone graft substitutes: Comparison of different materials by a standard in vitro test and microcalorimetry

We analyzed the initial adhesion and biofilm formation of Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) on various bone grafts and bone graft substitutes under standardized in vitro conditions. In parallel, microcalorimetry was evaluated as a real-time microbiological assay in the investigation of biofilm formation and material science research. The materials β-tricalcium phosphate (β-TCP), processed human spongiosa (Tutoplast?) and poly(methyl methacrylate) (PMMA) were investigated and compared with polyethylene (PE). Bacterial counts (log₁₀ cfu per sample) were highest on β-TCP (S. aureus 7.67 ± 0.17; S. epidermidis 8.14 ± 0.05) while bacterial density (log₁₀ cfu per surface) was highest on PMMA (S. aureus 6.12 ± 0.2, S. epidermidis 7.65 ± 0.13). Detection time for S. aureus biofilms was shorter for the porous materials (β-TCP and processed human spongiosa, p < 0.001) compared to the smooth materials (PMMA and PE), with no differences between β-TCP and processed human spongiosa (p > 0.05) or PMMA and PE (p > 0.05). In contrast, for S. epidermidis biofilms the detection time was different (p < 0.001) between all materials except between processed human spongiosa and PE (p > 0.05). The quantitative analysis by quantitative culture after washing and sonication of the material demonstrated the importance of monitoring factors like specific surface or porosity of the test materials. Isothermal microcalorimetry proved to be a suitable tool for an accurate, non-invasive and real-time microbiological assay, allowing the detection of bacterial biomass without removing the biofilm from the surface.     

Growth kinetics of hexagonal sub-micrometric β-tricalcium phosphate particles in ethylene glycol

Recently, uniform, non-agglomerated, hexagonal β-tricalcium phosphate (β-TCP) platelets (diameter ≈ 400–1700 nm, h ≈ 100–200 nm) were obtained at fairly moderate temperatures (90–170 °C) by precipitation in ethylene glycol. Unfortunately, the platelet aspect ratios (diameter/thickness) obtained in the latter study were too small to optimize the strength of polymer–β-TCP composites. Therefore, the aim of the present study was to investigate β-TCP platelet crystallization kinetics, and based on this, to find ways to better control the β-TCP aspect ratio. For that purpose, precipitations were performed at different temperatures (90–170 °C) and precursor concentrations (4, 16 and 32 mM). Solution aliquots were retrieved at regular intervals (10 s–24 h), and the size of the particles was measured on scanning electron microscopy images, hence allowing the determination of the particle growth rates. The β-TCP platelets were observed to nucleate and grow very rapidly. For example, the first crystals were observed after 30 s at 150 °C, and crystallization was complete within 2 min. The crystal growth curves could be well-fitted with both diffusion- and reaction-controlled equations, but the high activation energies (∼100 kJ mol⁻¹) pointed towards a reaction-controlled mechanism. The results revealed that the best way to increase the diameter and aspect ratio of the platelets was to increase the precursor concentration. Aspect ratios as high as 14 were obtained, but the synthesis of such particles was always associated with the presence of large fractions of monetite impurities.     

Microporous calcium phosphate ceramics as tissue engineering scaffolds for the repair of osteochondral defects: Histological results

Treatment of defects in joint cartilage aims to re-establish normal joint function. In vitro experiments have shown that the application of synthetic scaffolds is a promising alternative to existing therapeutic options. A sheep study was conducted to test the suitability of microporous pure β-tricalcium phosphate (TCP) ceramics as tissue engineering scaffolds for the repair of osteochondral defects. Cylindrical plugs of microporous β-TCP (diameter: 7 mm; length: 25 mm; porosity: 43.5 ± 2.4%; pore diameter: ～5 μm) with interconnecting pores were used. Scaffolds were seeded with autologous chondrocytes in vitro and cultured for 4 weeks. A drill hole (diameter 7 mm) was placed in both medial femoral condyles of sheep. For the left knee the defect was filled with a TCP plug and for the right knee the defect was left empty. After 6, 12, 26 and 52 weeks, seven animals from each group were killed and studied. The samples were examined employing histological, histomorphometric and immunohistological methods as well as various imaging techniques (X-ray, microcomputer tomography and scanning electron microscopy). After explantation the cartilage defects were first assessed macroscopically. There were no signs of infection or inflammation. Histological grading scales were used for assessment of bony integration and cartilage repair. An increasing degradation (81% after 52 weeks) of the ceramic with concomitant bone formation was observed. The original structure of cancellous bone was almost completely restored. After 26 and 52 weeks, collagen II-positive hyaline cartilage was detected in several samples. New subchondral bone had formed. The formation of cartilage began at the outer edge and proceeded to the middle. According to the O’Driscoll score, values corresponding to healthy cartilage were not reached after 1 year. Integration of the newly formed cartilage tissue into the surrounding native cartilage was found. The formation of biomechanical stable cartilage began at the edge and progressed towards the centre of the defect. After 1 year this process was still not completed. Microporous β-TCP scaffolds seeded with chondrocytes are suitable for the treatment of osteochondral defects.     

Monocyte/macrophage cytokine activity regulates vascular smooth muscle cell function within a degradable polyurethane scaffold

Tissue engineering strategies rely on the ability to promote cell proliferation and migration into porous biomaterial constructs, as well as to support specific phenotypic states of the cells in vitro. The present study investigated the use of released factors from monocytes and their derived macrophages (MDM) and the mechanism by which they regulate vascular smooth muscle cell (VSMC) response in a VSMC–monocyte co-culture system within a porous degradable polyurethane (D-PHI) scaffold. VSMCs cultured in monocyte/MDM-conditioned medium (MCM), generated from the culture of monocytes/MDM on D-PHI scaffolds for up to 28 days, similarly affected VSMC contractile marker expression, growth and three-dimensional migration when compared to direct VSMC–monocyte co-culture. Monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) were identified as two cytokines present in MCM, at concentrations that have previously been shown to influence VSMC phenotype. VSMCs cultured alone on D-PHI scaffolds and exposed to MCP-1 (5 ng ml⁻¹) or IL-6 (1 ng ml⁻¹) for 7 days experienced a suppression in contractile marker expression (with MCP-1 or IL-6) and increased growth (with MCP-1) compared to no cytokine medium supplementation. These effects were also observed in VSMC–monocyte co-culture on D-PHI. Neutralization of IL-6, but not MCP-1, was subsequently shown to decrease VSMC growth and enhance calponin expression for VSMC–monocyte co-cultures on D-PHI scaffolds for 7 days, implying that IL-6 mediates VSMC response in monocyte–VSMC co-cultures. This study highlights the use of monocytes and their derived macrophages in conjunction with immunomodulatory biomaterials, such as D-PHI, as agents for regulating VSMC response, and demonstrates the importance of monocyte/MDM-released factors, such as IL-6 in particular, in this process.