Poly(dl-lactide) Polymer Blended with Mineral Phases for Extrusion 3D Printing-Studies on Degradation and Biocompatibility.

A promising therapeutic option for the treatment of critical-size mandibular defects is the implantation of biodegradable, porous structures that are produced patient-specifically by using additive manufacturing techniques. In this work, degradable poly(DL-lactide) polymer (PDLLA) was blended with different mineral phases with the aim of buffering its acidic degradation products, which can cause inflammation and stimulate bone regeneration. Microparticles of CaCO3, SrCO3, tricalcium phosphates (α-TCP, ß-TCP), or strontium-modified hydroxyapatite (SrHAp) were mixed with the polymer powder following processing the blends into scaffolds with the Arburg Plastic Freeforming 3D-printing method. An in vitro degradation study over 24 weeks revealed a buffer effect for all mineral phases, with the buffering capacity of CaCO3 and SrCO3 being the highest. Analysis of conductivity, swelling, microstructure, viscosity, and glass transition temperature evidenced that the mineral phases influence the degradation behavior of the scaffolds. Cytocompatibility of all polymer blends was proven in cell experiments with SaOS-2 cells. Patient-specific implants consisting of PDLLA + CaCO3, which were tested in a pilot in vivo study in a segmental mandibular defect in minipigs, exhibited strong swelling. Based on these results, an in vitro swelling prediction model was developed that simulates the conditions of anisotropic swelling after implantation.

Antioxidant flavonoid-loaded nano-bioactive glass bone paste: in vitro apatite formation and flow behavior.

Non-cement pastes in the form of injectable materials have gained considerable attention in non-invasive regenerative medicine. Different osteoconductive bioceramics have been used as the solid phase of these bone pastes. Mesoporous bioactive glass can be used as an alternative bioceramic for paste preparation because of its osteogenic qualities. Plant-derived osteogenic agents can also be used in paste formulation to improve osteogenesis; however, their side effects on physical and physicochemical properties should be investigated. In this study, nano-bioactive glass powder was synthesized by a sol-gel method, loaded with different amounts of quercetin (0, 100, 150, and 200 µM), an antioxidant flavonoid with osteogenesis capacity. The loaded powder was then homogenized with a mixture of hyaluronic acid and sodium alginate solution to form a paste. We subsequently evaluated the rheological behavior, injectability, washout resistance, and in vitro bioactivity of the quercetin-loaded pastes. The washout resistance was found to be more than 96% after 14 days of immersion in simulated body fluid (SBF) as well as tris-buffered and citric acid-buffered solutions at 25 °C and 37 °C. All pastes exhibited viscoelastic behavior, in which the elastic modulus exceeded the viscous modulus. The pastes displayed shear-thinning behavior, in which viscosity was more influenced by angular frequency when the quercetin content increased. Results indicated that injectability was much improved using quercetin and the injection force was in the range 20-150 N. Following 14 days of SBF soaking, the formation of a nano-structured apatite phase on the surfaces of quercetin-loaded pastes was confirmed through scanning electron microscopy, X-ray diffractometry, and Fourier-transform infrared spectroscopy. Overall, quercetin, an antioxidant flavonoid osteogenic agent, can be loaded onto the nano-bioactive glass/hyaluronic acid/sodium alginate paste system to enhance injectability, rheological properties, and bioactivity.

A comparative analysis of 3D printed scaffolds consisting of poly(lactic-co-glycolic) acid and different bioactive mineral fillers: aspects of degradation and cytocompatibility.

Their excellent mechanical properties, degradability and suitability for processing by 3D printing technologies make the thermoplastic polylactic acid and its derivatives favourable candidates for biomaterial-based bone regeneration therapies. In this study, we investigated whether bioactive mineral fillers, which are known to promote bone healing based on their dissolution products, can be integrated into a poly(L-lactic-co-glycolic) acid (PLLA-PGA) matrix and how key characteristics of degradation and cytocompatibility are influenced. The polymer powder was mixed with particles of CaCO3, SrCO3, strontium-modified hydroxyapatite (SrHAp) or tricalcium phosphates (α-TCP, ß-TCP) in a mass ratio of 90 : 10; the resulting composite materials have been successfully processed into scaffolds by the additive manufacturing method Arburg Plastic Freeforming (APF). Degradation of the composite scaffolds was investigated in terms of dimensional change, bioactivity, ion (calcium, phosphate, strontium) release/uptake and pH development during long-term (70 days) incubation. The mineral fillers influenced the degradation behavior of the scaffolds to varying degrees, with the calcium phosphate phases showing a clear buffer effect and an acceptable dimensional increase. The amount of 10 wt% SrCO3 or SrHAp particles did not appear to be appropriate to release a sufficient amount of strontium ions to exert a biological effect in vitro. Cell culture experiments with the human osteosarcoma cell line SAOS-2 and human dental pulp stem cells (hDPSC) indicated the high cytocompatibility of the composites: For all material groups cell spreading and complete colonization of the scaffolds over the culture period of 14 days as well as an increase of the specific alkaline phosphatase activity, typical for osteogenic differentiation, were observed.

Three-Dimensional Plotted Calcium Phosphate Scaffolds for Bone Defect Augmentation-A New Method for Regeneration.

For sinus grafting, different methods and materials are available. One possible shortcoming of particulate bone grafts is either overfilling or augmenting the planned implant area insufficiently. To overcome this risk and to determine the implant position prior augmentation, we present an approach using three-dimensional printed scaffolds. A patient with a remaining anterior dentition and bilateral severely atrophied posterior maxilla was seeking oral rehabilitation. The cone beam computed tomography (CBCT) showed residual bone heights between one and two millimeters. Following the three-dimensional reconstruction of the CBCT data, the positions of the implants were determined in areas 16 and 26. Three-dimensional scaffolds adapted to the topography of the sinus were virtually designed and printed using a calcium phosphate cement paste. Bilateral sinus floor augmentation applying the printed scaffolds with an interconnecting porosity followed. After nine months, a satisfying integration of the scaffolds was obvious. At the re-entry, vital bone with sufficient blood supply was found. One implant could be placed in positions 16 and 26, respectively. After five months, the implants could be uncovered and were provided with a temporary denture. The application of three-dimensionally printed scaffolds from calcium phosphate cement paste seems to be a promising technique to graft the severely atrophied posterior maxilla for the placement of dental implants.

Confirmation of Calcium Phosphate Cement Biodegradation after Jawbone Augmentation around Dental Implants Using Three-Dimensional Visualization and Segmentation Software.

The use of autologous bone graft for oral rehabilitation of bone atrophy is considered the gold standard. However, the available grafts do not allow a fast loading of dental implants, as they require a long healing time before full functionality. Innovative bioactive materials provide an easy-to-use solution to this problem. The current study shows the feasibility of calcium phosphate cement paste (Paste-CPC) in the sinus. Long implants were placed simultaneously with the cement paste, and provisional prosthetics were also mounted in the same sessions. Final prosthetics and the full loading took place within the same week. Furthermore, the study shows for the first time the possibility to monitor not only healing progression using Cone Beam Computer tomography (CBCT) but also material retention, over two years, on a case study example. The segmented images showed a 30% reduction of the cement size and an increased mineralized tissue in the sinus. Mechanical testing was performed qualitatively using reverse torque after insertion and cement solidification to indicate clinical feasibility. Both functional and esthetic satisfaction remain unchanged after one year. This flowable paste encourages the augmentation procedure with less invasive measure through socket of removed implants. However, this limitation can be addressed in future studies.

Performance of Calcium Phosphate Cements in the Augmentation of Sheep Vertebrae-An Ex Vivo Study.

Oil-based calcium phosphate cement (Paste-CPC) shows not only prolonged shelf life and injection times, but also improved cohesion and reproducibility during application, while retaining the advantages of fast setting, mechanical strength, and biocompatibility. In addition, poly(L-lactide-co-glycolide) (PLGA) fiber reinforcement may decrease the risk for local extrusion. Bone defects (diameter 5 mm; depth 15 mm) generated ex vivo in lumbar (L) spines of female Merino sheep (2-4 years) were augmented using: (i) water-based CPC with 10% PLGA fiber reinforcement (L3); (ii) Paste-CPC (L4); or (iii) clinically established polymethylmethacrylate (PMMA) bone cement (L5). Untouched (L1) and empty vertebrae (L2) served as controls. Cement performance was analyzed using micro-computed tomography, histology, and biomechanical testing. Extrusion was comparable for Paste-CPC(-PLGA) and PMMA, but significantly lower for CPC + PLGA. Compressive strength and Young's modulus were similar for Paste-CPC and PMMA, but significantly higher compared to those for empty defects and/or CPC + PLGA. Expectedly, all experimental groups showed significantly or numerically lower compressive strength and Young's modulus than those of untouched controls. Ready-to-use Paste-CPC demonstrates a performance similar to that of PMMA, but improved biomechanics compared to those of water-based CPC + PLGA, expanding the therapeutic arsenal for bone defects. O, significantly lower extrusion of CPC + PLGA fibers into adjacent lumbar spongiosa may help to reduce the risk of local extrusion in spinal surgery.

Calcium Phosphate Bone Graft Substitutes with High Mechanical Load Capacity and High Degree of Interconnecting Porosity.

Bone graft substitutes in orthopedic applications have to fulfill various demanding requirements. Most calcium phosphate (CaP) bone graft substitutes are highly porous to achieve bone regeneration, but typically lack mechanical stability. This study presents a novel approach, in which a scaffold structure with appropriate properties for bone regeneration emerges from the space between specifically shaped granules. The granule types were tetrapods (TEPO) and pyramids (PYRA), which were compared to porous CaP granules (CALC) and morselized bone chips (BC). Bulk materials of the granules were mechanically loaded with a peak pressure of 4 MP; i.e., comparable to the load occurring behind an acetabular cup. Mechanical loading reduced the volume of CALC and BC considerably (89% and 85%, respectively), indicating a collapse of the macroporous structure. Volumes of TEPO and PYRA remained almost constant (94% and 98%, respectively). After loading, the porosity was highest for BC (46%), lowest for CALC (25%) and comparable for TEPO and PYRA (37%). The pore spaces of TEPO and PYRA were highly interconnected in a way that a virtual object with a diameter of 150 µm could access 34% of the TEPO volume and 36% of the PYRA volume. This study shows that a bulk of dense CaP granules in form of tetrapods and pyramids can create a scaffold structure with load capacities suitable for the regeneration of an acetabular bone defect.

Organically modified hydroxyapatite (ormoHAP) nanospheres stimulate the differentiation of osteoblast and osteoclast precursors: a co-culture study.

Isolated nanospheres consisting of organically modified hydroxyapatite (ormoHAP), prepared by an electric field-assisted ion double migration process, were embedded in foamed gelatin to form a composite scaffold. Degradation rates have been demonstrated to correlate with the crosslinking degree (40%, 80%) as well as with the mineral content of the scaffolds (0%, 20%, 40%). A human co-culture model of osteoblasts and osteoclasts, derived from bone marrow stromal cells and monocytes, respectively, without external addition of the factors RANKL and M-CSF, was run for up to 42 d in order to characterize the action of the ormoHAP-gelatin scaffolds on the co-culture. Examination was performed by quantitative biochemical methods (DNA, LDH, ALP, TRAP5b), gene expression analysis (ALP, BSP II, RANKL, IL-6, VTNR, CTSK, TRAP, OSCAR, CALCR) and confocal laser scanning microscopy (cell nuclei, actin, CD68, TRAP). Results confirm that ormoHAP embedded in the gelatin matrix enhanced TRAP 5b activity. As a feedback, ALP activity and gene expression of BSP II of osteoblasts increased. Finally, a sequence of cell cross-talk actions is suggested, which can explain the behavior of the formed vital co-culture and moreover the influence of the presence and concentration of ormoHAP.

Effect of bone sialoprotein coated three-dimensional printed calcium phosphate scaffolds on primary human osteoblasts.

The combination of the two techniques of rapid prototyping 3D-plotting and bioactive surface functionalization is presented, with emphasis on the in vitro effect of Bone Sialoprotein (BSP) on primary human osteoblasts (hOBs). Our primary objective was to demonstrate the BSP influence on the expression of distinctive osteoblast markers in hOBs. Secondary objectives included examinations of the scaffolds' surface and the stability of BSP-coating as well as investigations of cell viability and proliferation. 3D-plotted calcium phosphate cement (CPC) scaffolds were coated with BSP via physisorption. hOBs were seeded on the coated scaffolds, followed by cell viability measurements, gene expression analysis and visualization. Physisorption is an effective method for BSP-coating. Coating with higher BSP concentrations leads to enhanced BSP release. Two BSP concentrations (50 and 200 µg/mL) were examined in this study. The lower BSP concentration (50 µg/mL) decreased ALP and SPARC expression, whereas the higher BSP concentration (200 µg/mL) did not change gene marker expression. Enhanced cell viability was observed on BSP-coated scaffolds on day 3. hOBs developed a polygonal shape and connected in an intercellular network under BSP influence. Quantitative cell morphology analyses demonstrated for BSP-coated CPCs an enhanced cell area and reduced circularity. The strength of the above-mentioned effects of BSP-coated scaffolds in vivo is unknown, and future work is focusing on bone ingrowth and vascularization in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2565-2575, 2018.

Strontium(II) and mechanical loading additively augment bone formation in calcium phosphate scaffolds.

Calcium phosphate cements (CPCs) are widely used for bone-defect treatment. Current developments comprise the fabrication of porous scaffolds by three-dimensional plotting and doting using biologically active substances, such as strontium. Strontium is known to increase osteoblast activity and simultaneously to decrease osteoclast resorption. This study investigated the short- and long-term in vivo performances of strontium(II)-doted CPC (SrCPC) scaffolds compared to non-doted CPC scaffolds after implantation in unloaded or load-bearing trabecular bone defects in sheep. After 6 weeks, both CPC and SrCPC scaffolds exhibited good biocompatibility and osseointegration. Fluorochrome labeling revealed that both scaffolds were penetrated by newly formed bone already after 4 weeks. Neither strontium doting nor mechanical loading significantly influenced early bone formation. In contrast, after 6 months, bone formation was significantly enhanced in SrCPC compared to CPC scaffolds. Energy dispersive X-ray analysis demonstrated the release of strontium from the SrCPC into the bone. Strontium addition did not significantly influence material resorption or osteoclast formation. Mechanical loading significantly stimulated bone formation in both CPC and SrCPC scaffolds after 6 months without impairing scaffold integrity. The most bone was found in SrCPC scaffolds under load-bearing conditions. Concluding, these results demonstrate that strontium doting and mechanical loading additively stimulated bone formation in CPC scaffolds and that the scaffolds exhibited mechanical stability under moderate load, implying good clinical suitability. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:106-117, 2018.

Drug Release as a function of bioactivity, incubation regime, liquid, and initial load: Release of bortezomib from calcium phosphate-containing silica/collagen xerogels.

The ability of silica-/collagen-based composite xerogels to act as drug delivery systems was evaluated by taking into account the initial drug concentration, bioactivity of the xerogels, liquid, and incubation regime. The proteasome inhibitor bortezomib was chosen as a model drug, used for the systemic treatment of multiple myeloma. Incubation during 14 days in phosphate-buffered saline (PBS) or simulated body fluid (SBF) showed a weak initial burst and was identified to be of first order with subsequent release being independent from the initial load of 0.1 or 0.2 mg bortezomib per 60 mg monolithic sample. Faster drug release occurred during incubation in SBF compared to PBS, and during static incubation without changing the liquid, compared to dynamic incubation with daily liquid changes. Drug-loaded xerogels with hydroxyapatite as a third component exhibited enhanced bioactivity retarding drug release, explained by formation of a surface calcium phosphate layer. The fastest release of 50% of the total drug load was observed for biphasic xerogels after 7 days during dynamic incubation in SBF. As a result, the presented concept is suitable for the intended combination of the advantageous bone substitution properties of xerogels and local application of drugs exemplified by bortezomib. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1165-1173, 2018.

Performance of bioactive PMMA-based bone cement under load-bearing conditions: an in vivo evaluation and FE simulation.

In the past, bioactive bone cement was investigated in order to improve the durability of cemented arthroplasties by strengthening the bone-cement interface. As direct bone-cement bonding may theoretically lead to higher stresses within the cement, the question arises, whether polymethylmethacrylate features suitable mechanical properties to withstand altered stress conditions? To answer this question, in vivo experiments and finite element simulations were conducted. Twelve rabbits were divided into two groups examining either bioactive polymethylmethacrylate-based cement with unchanged mechanical properties or commercially available polymethylmethacrylate cement. The cements were tested under load-bearing conditions over a period of 7 months, using a spacer prosthesis cemented into the femur. For the finite element analyses, boundary conditions of the rabbit femur were simulated and analyses were performed with respect to different loading scenarios. Calculations of equivalent stress distributions within the cements were applied, with a completely bonded cement surface for the bioactive cement and with a continuously interfering fibrous tissue layer for the reference cement. The bioactive cement revealed good in vivo bioactivity. In the bioactive cement group two failures (33 %), with complete break-out of the prosthesis occurred, while none in the reference group. Finite element analyses of simulated bioactive cement fixation showed an increase in maximal equivalent stress by 49.2 to 109.4 % compared to the simulation of reference cement. The two failures as well as an increase in calculated equivalent stress highlight the importance of fatigue properties of polymethylmethacrylate in general and especially when developing bioactive cements designated for load-bearing conditions.

Effects of macroporous, strontium loaded xerogel-scaffolds on new bone formation in critical-size metaphyseal fracture defects in ovariectomized rats.

New bone formation was studied in a metaphyseal fracture-defect in ovariectomized rats stimulated by a plain and a strontium-enriched macroporous silica/collagen scaffold (ScB30 and ScB30Sr20) and a compact silica/collagen xerogel (B30). 45 female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) ScB30 (n=15), (2) ScB30Sr20 (n=15), and (3) B30 (n=15). 12 weeks after bilateral ovariectomy and multi-deficient diet, a 4 mm wedge-shaped fracture-defect was created at the metaphyseal area of the left femur. A 7-hole T-shaped plate at the lateral aspect of the femur stabilized the bone and the defect was filled with ScB30, ScB30Sr20 or B30 subsequently. After six weeks, histomorphometrical analysis revealed a statistically significant higher bone volume/tissue volume ratio in the ScB30Sr20 group compared to ScB30 (p=0.043) and B30 (p=0.0001) indicating an improved formation of new bone by the strontium-enriched macroporous silica/collagen scaffold. Furthermore, immunohistochemical results showed increased expression of BMP2 and OPG and a decreased RANKL expression in the ScB30Sr20 group. This was further confirmed with the gene expression analysis where an increase in prominent bone formation markers (ALP, OCN, Runx2, Col1a1 and Col10a1) was seen. No material remnants were found in the scaffold group indicating an almost complete degradation process of the biomaterials. This is confirmed by ToF-SIMS analysis that did not detect any strontium in the ScB30Sr20 group neither in the defect nor in the surrounding tissue. Taken together, this study shows the stimulating effects of strontium through increased bone formation by up regulation of osteoanabolic markers. This work also indicates the importance of material porosity, geometry and biodegradability in bone healing.

Collagen-lactoferrin fibrillar coatings enhance osteoblast proliferation and differentiation.

Lactoferrin is a milk-derived glycoprotein with anabolic effects on the bone tissue. In this study, artificial extracellular matrices (aECM) consisting of collagen type I fibrils formed in the presence of lactoferrin at two different concentrations (0.5 and 1 mg mL(-1) ) were prepared on the surface of poly(lactic-co-glycolic acid) (PLGA) foils. The aim of the study was to investigate the effects of aECM on the adhesion, growth and osteogenic differentiation of human osteoblast-like Saos-2 cells. On days 1 and 3 after seeding, higher numbers of cells were found on samples with collagen and collagen-lactoferrin coatings (particularly on those formed at the higher concentration of lacroferrin) than on control microscopic glass coverslips. Cells on coatings formed in the presence of lactoferrin had more numerous and better developed vinculin-containing focal adhesion plaques. On day 7, cells on coatings with and without lactoferrin produced significantly higher levels of osteocalcin than cells on control polystyrene cell culture dishes, the highest average values being found on samples with the lower concentration of lactoferrin. Expression of collagen I and alkaline phosphatase was on a similar level in cells on all tested samples and control polystyrene. Thus, lactoferrin promotes adhesion, growth and osteogenic differentiation of Saos-2 cells and is promising as a bone implant coating component.

Application of F-18-sodium fluoride (NaF) dynamic PET-CT (dPET-CT) for defect healing: a comparison of biomaterials in an experimental osteoporotic rat model.

BACKGROUND: The aim of the current study was to measure and compare the effect of various biomaterials for the healing of osteoporotic bone defects in the rat femur using 18F-sodium fluoride dPET-CT. MATERIAL AND METHODS: Osteoporosis was induced by ovariectomy and a calcium-restricted diet. After 3 months, rats were operated on to create a 4-mm wedge-shaped defect in the distal metaphyseal femur. Bone substitution materials of calcium phosphate cement (CPC), composites of collagen and silica, and iron foams with interconnecting pores were inserted. Strontium or bisphosphonate, which are well known for having positive effects in osteoporosis treatment, were added into the materials. Eighteen weeks after osteoporosis induction and 6 weeks following femoral surgery, dPET-CT studies scan were performed with 18F-Sodium Fluoride. Standardized uptake values (SUVs) and a 2-tissue compartmental learning-machine model (K1-k4, vessel density [VB], influx [ki]) were used for quantitative analysis. RESULTS: k3, reflecting the formation of fluoroapatite, revealed a statistically significant increase at the biomaterial-bone interface due to the Sr release from strontium-modified calcium phosphate cement (SrCPC) compared to CPC, which demonstrated enhanced new bone formation. In addition, k3 as measured in the porous scaffold silica/collagen xerogel (Sc-B30), showed a significant increase based on Wilcoxon rank-sum test (p<0.05) as compared with monolithic silica/collagen xerogel (B30) in the defect region. Furthermore, ki, reflecting the net plasma clearance of tracer to bone mineral measured in the iron foam with coating of the bisphosphonate zoledronic acid (Fe-BP), was enhanced as compared with plain iron foam (Fe) in the defect region. CONCLUSIONS: k3 was the most significant parameter for the characterization of healing processes and revealed the best differentiation between the 2 different biomaterials. PET scanning using 18F-sodium fluoride seems to be a sensitive and useful method for evaluation of bone healing after replacement with these biomaterials.

Differences of bone healing in metaphyseal defect fractures between osteoporotic and physiological bone in rats.

Discrepancies in bone healing between osteoporotic and non-osteoporotic bone remain uncertain. The focus of the current work is to evaluate potential healing discrepancies in a metaphyseal defect model in rat femora. Female Sprague-Dawley rats were either ovariectomized (OVX, n=14) and combined with a calcium-, phosphorus- and vitamin D3-, soy- and phytoestrogen-free diet or received SHAM operation with standard diet rat (SHAM, n=14). Three months post-ovariectomy, DEXA measurement showed a reduction of bone mineral density reflecting an osteoporotic bone status in OVX rats. Rats then underwent a 3 mm wedge-shaped osteotomy at the distal metaphyseal area of the left femur stabilized with a T-shaped mini-plate and allowed to heal for 6 weeks. Biomechanical competence by means of a non-destructive three-point bending test showed significant lower flexural rigidity in the OVX rats at 3 mm lever span compared to SHAM animals (p=0.048) but no differences at 10 mm lever span. Microcomputer tomography (µCT) showed bridging cortices and consolidation of the defect in both groups, however, no measurable differences were found in either total ossified tissue or vascular volume fraction. Furthermore, histology showed healing discrepancies that were characterized by cartilaginous remnant and more unmineralized tissue presence in the OVX rats compared to more mature consolidation appearance in the SHAM group. In summary, bone defect healing in metaphyseal bone slightly differs between osteoporotic and non-osteoporotic bone in the current 3 mm defect model in both 3mm lever span biomechanical testing and histology.

Jellyfish collagen scaffolds for cartilage tissue engineering.

Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for potential application in cartilage regeneration. The influence of collagen concentration, salinity and temperature on fibril formation was evaluated by turbidity measurements and quantification of fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated glycosaminoglycans.

Bioactivity of xerogels as modulators of osteoclastogenesis mediated by connexin 43.

In order to investigate the effects of different degrees of bioactivity of xerogels on connexin 43 (cx43) signaling of osteoclasts a cell culture approach was developed. Cells isolated from peripheral blood mononuclear cells were cultured in combination with the xerogels and were harvested for further investigations on day 1, day 5, and day 10. By means of quantitative PCR increased cx43 mRNA levels and coincident decreasing mRNA levels of the calcium sensing receptor, TRAP, and Cathepsin K were detected with increasing bioactivity of the xerogel samples. Additionally, osteoclasts cultured on tissue culture plates were used to perform principle investigations on cell differentiation by means of transmission electron microscopy, life cell imaging, and immunofluorescence, and the results demonstrated that cx43-signaling could be attributed to migration and fusion of osteoclast precursors. Therefore, the positive correlation of cx43 expression with high xerogel bioactivity was caused by proceeding differentiation of the osteoclasts. Finally, the presently observed pattern of cx43 signaling refers to strong effects regarding bioactivity on cx43-associated cell differentiation of osteoclasts influenced by extracellular calcium ions.

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures.

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures.

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.