Investigation of particle-functionalized tissue engineering scaffolds using X-ray tomographic microscopy.

A low-density, porous chitosan/poly-(dl-lactide-co-glycolide) (PLGA) microparticle composite scaffold was produced by thermally induced phase separation followed by lyophilization, to provide a bicontinuous microstructure potentially suitable for tissue engineering and locally controlled drug release. PLGA particles were mixed into the chitosan solution and subsequent phase separation during chitosan solidification forced PLGA particles into chitosan phase (Plateau borders). The distributions of volume, surface area, and elongation of 15,422 inclusions of agglomerated PLGA particles were calculated and approximated with log-normal distribution functions from nanotomography reconstructions. Cluster analysis revealed a homogenous inclusion distribution throughout the scaffold. The spatial location and orientation of individual inclusions within the Plateau borders of the scaffold were determined and from these the nearest-neighbor inter-inclusion distance distribution calculated, showing a mean of 2.5 microm. The depth of the inclusions in Plateau borders peaks at 700 or 125 nm, respectively, indicating a step-wise drug release from inclusions successively exposed during scaffold decomposition. Particle diameter ranged from 400 nm to 3 microm and inclusion Feret lengths ranged from 800 nm to 12 microm. These findings on composite morphology and distribution of inclusions are fundamental for predicting scaffold deterioration and particle-mediated drug release during ex vivo and in vivo cell cultivation.

Surgical repair of annulus defect with biomimetic multilamellar nano/microfibrous scaffold in a porcine model.

Annulus defect is associated with reherniation and disc degeneration after discectomy; currently there is no effective treatment that addresses this problem. The annulus is a hierarchical lamellar structure, where each lamella consists of aligned collagen fibres, which are parallel and tilted at 30° to the spinal axis. In this study, a biomimetic biodegradable scaffold consisting of multilamellar nano/microfibres, sharing nanotopography and microporosity similar to the native lamellar structure, was assessed in a porcine model, aided by sealing with fascia and medical glue and subsequent suture fixation. After 6- and 12-week observation, we found that this treatment restored nucleus volume and slowed down disc degeneration, as indicated by magnetic resonance imaging of T1/T2-weighted, T2-mapping, T1-ρ imaging. Histological analysis showed aligned collagen fibres organized in the scaffold and integrated with surrounding native annulus tissue. The autologous bone marrow concentrate-seeded scaffolds showed slightly earlier collagen fibre formation at 6 weeks. This novel treatment could efficiently close the annulus defect with newly formed, organized and integrated collagen fibres in a porcine model. Copyright © 2016 John Wiley & Sons, Ltd.

Deletion of the tissue response against alginate-pll capsules by temporary release of co-encapsulated steroids.

Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases. Large-scale application of the technique, however, is hampered by inflammatory responses against the capsules. In the present study, we investigate whether tissue responses against alginate-PLL-alginate capsules can be modulated by co-encapsulation and temporary release of immunomodulating factors such as dexamethasone. Such an approach may be mandatory in order to increase the function and survival of encapsulated tissue since it has been shown that the tissue response can be caused by many, insurmountable factors. In an in vitro assay, we demonstrated an antiproliferative effect of dexamethasone-containing capsules on L929-mouse-fibroblasts. Subsequently, capsules prepared of purified alginate with or without solved dexamethasone were implanted in the peritoneal cavity of rats and retrieved one month later for histological evaluation. Most of the capsules without dexamethasone proved to be overgrown and adherent to the abdominal organs whereas with co-encapsulated dexamethasone the majority of the capsules were found freely floating in the peritoneal cavity without overgrowth. We conclude that co-encapsulation of dexamethasone has a profound effect on fibroblasts and macrophages adherence to immunoisolating capsules.

Improved bone anchorage of hydroxypatite coated implants compared with tricalcium-phosphate coated implants in trabecular bone in dogs.

Tricalcium phosphate (TCP) and hydroxyapatite (HA) ceramic coatings are bioactive coatings that have been shown to stimulate bone apposition onto ceramic-coated implants. TCP and HA ceramics have well-documented differences in physical properties, but both types of ceramics are used for stimulation of bone ongrowth to cementless endo-prosthetic components clinically. However, little is known about the difference in osteoconductive properties between these coatings when inserted into trabecular bone in a controlled experimental situation. Unloaded cylindrical gritblasted titanium (Ti-6A1-4V) implants (6 x 10 mm) coated with either hydroxyapatite (HA) or tricalcium phosphate (TCP) ceramic were inserted into the proximal humerus of 20 skeletally mature dogs. The implants were initially surrounded by a 2 mm gap. Each animal received one HA-coated implant and one TCP-coated implant. All dogs were sacrificed 6 weeks after surgery. Results were evaluated by histomorphometry and mechanical push-out test. Push-out tests demonstrated that HA-coated implants were 10-fold stronger fixated in comparison to TCP-coated implant. Bone ongrowth was significantly higher for HA-coated implants compared to TCP-coated implants. Bone volume in the gap showed a tendency to less bone volume around HA-coated implants compared to TCP-coated implants but this difference was insignificant. As expected almost all of the TCP coating were resorbed after 6 weeks and almost none of the HA coating. HA-coated implants with a grit-blasted surface provide a favorable early mechanical implant anchorage most likely due to superior ceramic stability compared to TCP-coated implants.

Biocompatibility and surface structure of chemically modified immunoisolating alginate-PLL capsules.

Grafting of encapsulated living cells has the potential to cure a wide variety of diseases. Large-scale application of the technique, however, is hampered by insufficient biocompatibility of the capsules. A major factor in the biocompatibility of capsules is inadequate covering of the inflammatory poly-L-lysine (PLL) on the capsules' surface. In the present study, we investigate whether tissue responses against alginate-PLL capsules can be reduced by crosslinking the surface of the capsules with heparin or polyacrylic acid. Our transplant study in rats shows a tissue response composed of fibroblasts and macrophages on alginate-PLL-alginate and alginate-PLL-heparin capsules that was completely absent on alginate-PLL-polyacrylic acid capsules. Atomic force microscopy analyses of the capsules demonstrates that the improved biocompatibility of alginate-PLL-capsules by polyacrylic acid coating should not only be explained by a more adequate binding of PLL but also by the induction of a smoother surface. This study shows for the first time that biologic responses against capsules can be successfully deleted by chemically crosslinking biocompatible molecules on the surface of alginate-PLL capsules.

Resorption of hydroxyapatite and fluorapatite coatings in man. An experimental study in trabecular bone.

The clinical use of hydroxyapatite (HA) coating is controversial especially in regard to the long-term performance of the coating and the effects of resorption. In each of 15 consenting patients we inserted two implants, coated with either HA or fluorapatite (FA) into the iliac crest. They were harvested at a mean of 13.6 +/- 0.6 months after surgery. Histological examination showed that bone ongrowth on the HA-coated implants was significantly greater (29%) than that on the FA-coated implants. When bone was present on the coating surface the HA coating was significantly thicker than the FA coating. When bone marrow was present, the HA coating was significantly thinner than the FA coating. The reduction in coating thickness when covered by bone or bone marrow was 23.1 +/- 9.7 microm for HA and 5.1 +/- 1.7 microm for FA (p < 0.01) suggesting that FA is more stable than HA against resorption by bone marrow. The findings suggest that in man the osteoconductive properties of HA coating are superior to those of FA. Resorption rates for both coatings were approximately 20% of the coating thickness per year. Bone ongrowth appears to protect against resorption whereas bone marrow seems to accelerate resorption. No adverse reaction was seen in the surrounding bone.

The influence of crystallinity of the hydroxyapatite coating on the fixation of implants. Mechanical and histomorphometric results.

We inserted two hydroxyapatite (HA)-coated implants with crystallinities of either 50% (HA-50%) or 75% (HA-75%) bilaterally into the medial femoral condyles of the knees of 16 dogs. The implants were allocated to two groups with implantation periods of 16 and 32 weeks. They were weight-bearing and subjected to controlled micromovement of 250 microm during each gait cycle. After 16 weeks, mechanical fixation of the HA-50% implants was increased threefold as compared with the HA-75% implants. After 32 weeks there was no difference between HA-50% and HA-75%. Fixation of HA-75% increased from 16 to 32 weeks whereas that of HA-50% was unchanged. HA-50% implants had 100% more bone ingrowth than HA-75% implants after 16 weeks. More HA coating was removed on HA-50% implants compared with HA-75% implants after both 16 and 32 weeks. No further loss of the HA coating was shown from 16 to 32 weeks. Our study suggests that the crystallinity of the HA coating is an important factor in its bioactivity and resorption during weight-bearing conditions. Our findings suggest two phases of coating resorption, an initial rapid loss, followed by a slow loss. Resorbed HA coating was partly replaced by bone ingrowth, suggesting that implant fixation will be durable.

Sealing effect of hydroxyapatite coating on peri-implant migration of particles. An experimental study in dogs.

We have studied the beneficial effects of a hydroxyapatite (HA) coating on the prevention of the migration of wear debris along the implant-bone interface. We implanted a loaded HA-coated implant and a non-coated grit-blasted titanium alloy (Ti) implant in each distal femoral condyle of eight Labrador dogs. The test implant was surrounded by a gap communicating with the joint space and allowing access of joint fluid to the implant-bone interface. We injected polyethylene (PE) particles into the right knee three weeks after surgery and repeated this weekly for the following five weeks. The left knee received sham injections. The animals were killed eight weeks after surgery. Specimens from the implant-bone interface were examined under plain and polarised light. Only a few particles were found around HA-coated implants, but around Ti implants there was a large amount of particles. HA-coated implants had approximately 35% bone ingrowth, whereas Ti implants had virtually no bone ingrowth and were surrounded by a fibrous membrane. Our findings suggest that HA coating of implants is able to inhibit peri-implant migration of PE particles by creating a seal of tightly-bonded bone on the surface of the implant.

Transforming growth factor-beta 1 stimulates bone ongrowth to weight-loaded tricalcium phosphate coated implants: an experimental study in dogs.

Bone growth into cementless prosthetic components is compromised by osteoporosis, by any gap between the implant and the bone, by micromotion, and after the revision of failed prostheses. Recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) has recently been shown to be a potent stimulator of bone healing and bone formation in various models in vivo. We have investigated the potential of rhTGF-beta 1, adsorbed on to weight-loaded tricalcium phosphate (TCP) coated implants, to enhance bone ongrowth and mechanical fixation. We inserted cylindrical grit-blasted titanium alloy implants bilaterally into the weight-bearing part of the medial femoral condyles of ten skeletally mature dogs. The implants were mounted on special devices which ensured stable weight-loading during each gait cycle. All implants were initially surrounded by a 0.75 mm gap and were coated with TCP ceramic. Each animal received two implants, one with 0.3 microgram rhTGF-beta 1 adsorbed on the ceramic surface and the other without growth factor. Histological analysis showed that bone ongrowth was significantly increased from 22 +/- 5.6% bone-implant contact in the control group to 36 +/- 2.9% in the rhTGF-beta 1 stimulated group, an increase of 59%. The volume of bone in the gap was increased by 16% in rhTGF-beta1-stimulated TCP-coated implants, but this difference was not significant. Mechanical push-out tests showed no difference in fixation of the implant between the two groups. Our study suggests that rhTGF-beta 1 adsorbed on TCP-ceramic-coated implants can enhance bone ongrowth.

Mechanical and histological analysis of bone-pedicle screw interface in vivo: titanium versus stainless steel.

OBJECTIVE: To investigate the differences in bone interface between titanium and stainless steel pedicle screws in the lumbar spine. METHODS: Eighteen adult mini-pigs that underwent total laminectomy, posterolateral spinal fusion (L4-L5) were randomly selected to receive stainless steel (9) or titanium pedicle screw devices (9). In both groups, the devices were CCD (Sofamore Danek) type with the same size and shape. The postoperative observation time was 3 months. Screws from L4 were harvested along their long axis of pedicle for histomorphometric study. Bone-screw interface and bone volume from thread were examined using linear intercept techniques. Mechanical testing (torsional test and pull-out test) was performed on the screws from L5. RESULTS: The titanium screw group had a significantly higher maximum torque (P < 0.05) and angle related stiffness (P < 0.05) measured by torsional test. In the pull-out tests, no differences were found between the two groups in relation to the maximum load, stiffness and energy to failure. Direct bone contact with the screw in percentage was 29.4% for stainless steel and 43.8% for titanium (P < 0.05). No differences in the bone purchase between the vertebral body part and pedicle part were found. CONCLUSION: Pedicle screws made of titanium have a better bone-screw interface binding than screws made of stainless steel. Torsional tests are more informative for bone-screw interface study. Pull-out tests seem less valuable when comparing bone purchase of screws made from different materials.

A review of ceramic coatings for implant fixation.

The present series of eight studies was performed in order to investigate the effect of various clinically relevant factors on bone ingrowth in relation to hydroxyapatite (HA) and titanium-alloy (Ti) coating when subjected to pathological and mechanical conditions mimicking the clinical situation. HA- and Ti-coated implants were inserted into the femoral condyles of mature dogs and one study was performed on humans. The observation period ranged from 4 to 52 weeks, and the results were evaluated by mechanical push-out testing, histomorphometric analysis, polarized light microscopy, UV fluorescence microscopy and collagen analysis. There were no complications related to the operative procedures, and all dogs were killed according to the original time schedule. Two studies focused on in vivo mechanisms and factors influencing resorption of HA coating. The overall conclusions from these studies are that HA coatings do resorb in vivo, that micromotion accelerates resorption, and that resorbed HA is partly replaced by newly formed bone, suggesting that implants fixation is durable. The other studies focused on the significance of mechanical stabilization and loading conditions of the implant immediately after surgery. From these studies, it can be concluded that HA-coating has a positive effect on bone-implant fixation in various situations, i.e., under stable loaded conditions and under unstable mechanical conditions. The most striking effect of HA coating was that it enhanced bone growth across a gap around the implant both during stable and unstable mechanical conditions; it even converted a motion-induced fibrous membrane to bony anchorage.

Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 µm for surfaces with small pillar sizes of 1 and 2 µm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 µm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine.

Osteopontin functionalization of hydroxyapatite nanoparticles in a PDLLA matrix promotes bone formation.

We studied the osteoconductive tissue response of hydroxyapatite (HA) nanoparticles functionalized with osteopontin (OPN) in a matrix of poly-D,L-lactic-acid (PDLLA). In a canine endosseus 0.75-mm gap implant model, we tested the osteointegrative impact of the OPN functionalized composite as an implant coating, and a non-functionalized composite was used as reference control. During the four weeks of observation, the OPN functionalized composite coating significantly increased the formation of new bone in the porosities of the implant, but no differences were observed in the gap. The study provides evidence of its potential use either alone or in combination with other osteoconductive compounds.

Osteogenic protein-1 increases the fixation of implants grafted with morcellised bone allograft and ProOsteon bone substitute: an experimental study in dogs.

Impacted bone allograft is often used in revision joint replacement. Hydroxyapatite granules have been suggested as a substitute or to enhance morcellised bone allograft. We hypothesised that adding osteogenic protein-1 to a composite of bone allograft and non-resorbable hydroxyapatite granules (ProOsteon) would improve the incorporation of bone and implant fixation. We also compared the response to using ProOsteon alone against bone allograft used in isolation. We implanted two non-weight-bearing hydroxyapatite-coated implants into each proximal humerus of six dogs, with each implant surrounded by a concentric 3 mm gap. These gaps were randomly allocated to four different procedures in each dog: 1) bone allograft used on its own; 2) ProOsteon used on its own; 3) allograft and ProOsteon used together; or 4) allograft and ProOsteon with the addition of osteogenic protein-1. After three weeks osteogenic protein-1 increased bone formation and the energy absorption of implants grafted with allograft and ProOsteon. A composite of allograft, ProOsteon and osteogenic protein-1 was comparable, but not superior to, allograft used on its own. ProOsteon alone cannot be recommended as a substitute for allograft around non-cemented implants, but should be used to extend the volume of the graft, preferably with the addition of a growth factor.

A method for bone-cement interface thermometry. An in vitro comparison between low temperature curing cement Palavit, and Surgical Simplex P.

Previous temperature measurements at the bone-cement intreface have all shown large variations. To evaluate more precisely the temperature profiles during cement curing, a new experimental model was developed. Eight thermocouple electrodes at the bone-cement interface for each test specimen were used for continuous temperature recordings. Temperature profiles of Palavit was compared with those of Surgical Simplex P in an in vitro model using isolated pig femurs. Defects of 12 x 17 mm in the femoral metaphysis were filled with cement. In six tests with each type of cement, Palavit peaked at a temperature of 50 +/- 0.5 degrees C, whereas Surgical Simplex P peaked at 60 +/- 0.7 degrees C. Core temperatures reached peak values of 70 +/- 0.8 degrees C and 95 +/- 2.2 degrees C for Palavit and Surgical Simplex P, respectively.

Polyethylene particles stimulate monocyte chemotactic and activating factor production in synovial mononuclear cells in vivo. An immunohistochemical study in rabbits.

We report that polyethylene particles can activate mononuclear cells within the joint to produce the monocyte chemotactic and activating factor (MCAF) and to a lesser degree interleukin 8 (IL-8) as judged by immunohistological staining. Polyethylene particles suspended in hyaluronic acid were injected weekly for 12 weeks into the right knee joint of New Zealand white rabbits. The average size of the particles was 7 (3-12) microns in diameter. The left knee joint was injected with hyaluronic acid as the control. The animals were killed after 13 weeks. On the control side, the synovial membrane was histologically normal, without signs of inflammation. In the knees that were injected with polyethylene particles, histological analysis showed a weak inflammatory response, consisting of mononuclear cells, multi-nucleated giant cells and polyethylene particles. In the vicinity of the particles, the presence of mononuclear cells that were highly positive for MCAF was noted, whereas IL-8 was present in endothelial cells and in the lining layer, but not in cells in the vicinity of polyethylene particles, suggesting that polyethylene particles are able to activate cytokine metabolism in a differentiated way in the synovial monocytes.

Hydroxyapatite coating modifies implant membrane formation. Controlled micromotion studied in dogs.

We studied the influence of controlled micromovements between bone and porous titanium alloy implants with and without hydroxyapatite coating. A dynamically loaded unstable device producing approximately 150-microns axial translation of knee implants during each gait cycle was developed. Stable implants served as controls. Matched stable and unstable implants with either porous titanium (Ti) or hydroxyapatite (HA) coating surrounded by a gap of 0.75 mm were inserted into the weight-bearing regions of the medial femoral condyles in 14 mature dogs. Histologic analysis after 4 weeks showed a fibrous membrane surrounding both types of implants subjected to micromovements, whereas various amounts of bone ingrowth was obtained in the stable implants. The membrane around unstable HA implants was thinner than that around unstable Ti implants. Islands of fibrocartilaginous tissue characterized the membrane around unstable HA implants, whereas fibrous connective tissue surrounded unstable Ti implants. The collagen concentration of the fibrous membranes was higher around unstable HA implants compared with Ti implants. Instability reduced the shear strength of the implants. However, the shear strength of unstable HA implants exceeded that of the Ti implants, both unstable and stable. The greatest shear strength was obtained by stable HA implants, i.e., tenfold greater than that of stable Ti implants. The gap-healing capacity around stable HA implants increased toward the HA surface, and was greater than that around Ti implants. Our study demonstrates that micromovements between bone and implant inhibit bone ingrowth and lead to the development of a fibrous membrane. The superior fixation of unstable HA implants compared with unstable Ti implants may be ascribed to the presence of fibrocartilage, a higher collagen concentration, and radiating orientation of collagen fibers in the membrane. The strongest mechanical anchorage and the greatest amount of bone ingrowth was obtained by stable implants coated with hydroxyapatite.

Transforming growth factor-beta stimulates bone ongrowth. Hydroxyapatite-coated implants studied in dogs.

Unloaded cylindrical grit-blasted titanium (Ti-6A-4V) implants (6 x 10 mm) coated with hydroxyapatite ceramic were inserted into the proximal part of the humerus of 20 skeletally mature Labrador dogs. The implants were initially surrounded by a 2 mm gap. In 10 dogs, HA-coated implants without growth factor were inserted in one humerus and implants with 0.3 microgram rhTGF-beta 1 adsorbed onto the HA coating were inserted in the contralateral humerus. In another group of 10 dogs, a dose of 3.0 micrograms rhTGF-beta 1 was tested in a similar design. All dogs were killed at 6 weeks after treatment. Results were evaluated by histomorphometry and mechanical push-out testing. Bone ongrowth was increased by one third, using the 0.3 mg rhTGF-beta 1 stimulation. Bone volume in the gap and mechanical testing showed no statistically significant differences between control and rhTGF-beta 1 stimulated implants. RhTGF-beta 1 only moderately enhanced bone ongrowth to hydroxyapatite-coated implants.

Hydroxyapatite and fluorapatite coatings for fixation of weight loaded implants.

Survivor analysis of total hip replacement recently has shown disappointing results in younger patients. To improve this, ceramic coatings have been applied to prostheses for cementless use. A new fluorine containing coating, fluorapatite, has been shown to increase bone ingrowth compared with hydroxyapatite in unloaded models. In a weight loaded model, the effects of hydroxyapatite and fluorapatite coated implants on implant fixation and bone ingrowth were evaluated. Eight hydroxyapatite and fluorapatite coated implants with porous surface were inserted into the medial femoral condyles of 8 mature dogs in a paired design. The implants initially were surrounded by a gap communicating with the joint space and were loaded during each gait cycle. After 25 weeks, no differences in pushout data or bone ingrowth between hydroxyapatite and fluorapatite coated implants were found. An important finding was the absence of foreign body reaction in the bone. Neither hydroxyapatite nor fluorapatite coatings delaminated during implantation or as a result of the pushout test. Bone repair activity remained in the initial gap zone, but most of the bone was of the lamellar type. No difference in bone remodeling between the hydroxyapatite and fluorapatite coated implants was found in the initial gap zone. Microprobe analysis showed no increase in fluorine content around the fluorapatite coated implants. The hydroxyapatite and fluorapatite coatings seem efficacious after a 25-week implantation period under weight loaded conditions.