The effect of pores and connections geometries on bone ingrowth into titanium scaffolds: an assessment based on 3D microCT images.

In order to support bone tissue regeneration, porous biomaterial implants (scaffolds) must offer chemical and mechanical properties, besides favorable fluid transport. Titanium implants provide these requirements, and depending on their microstructural parameters, the osteointegration process can be stimulated. The pore structure of scaffolds plays an essential role in this process, guiding fluid transport for neo-bone regeneration. The objective of this work was to analyze geometric and morphologic parameters of the porous microstructure of implants and analyze their influences in the bone regeneration process, and then discuss which parameters are the most fundamental. Bone ingrowths into two different sorts of porous titanium implants were analyzed after 7, 14, 21, 28, and 35 incubation days in experimental animal models. Measurements were accomplished with x-ray microtomography image analysis from rabbit tibiae, applying a pore-network technique. Taking into account the most favorable pore sizes for neo-bone regeneration, a novel approach was employed to assess the influence of the pore structure on this process: the analyses were carried out considering minimum pore and connection sizes. With this technique, pores and connections were analyzed separately and the influence of connectivity was deeply evaluated. This investigation showed a considerable influence of the size of connections on the permeability parameter and consequently on the neo-bone regeneration. The results indicate that the processing of porous scaffolds must be focused on deliver pore connections that stimulate the transport of fluids throughout the implant to be applied as a bone replacer.

Synthesis of silver @hydroxyapatite nanoparticles based biocomposite and their assessment for viability of Osseointegration for rabbit knee joint anterior cruciate ligament rehabilitation.

In this examination, chitosan-silk fibroin/polyethylene terephthalate (CTS-SF/PET), chitosan-silk fibroin/polyethylene terephthalate/hydroxyapatite (CTS-SF/PET/HAP) and chitosan-silk fibroin/polyethylene terephthalate/Silver @hydroxyapatite (CTS-SF/PET/Ag@HAP) scaffolds were prepared by utilizing the plasma splashing procedure. Field emission scanning electron microscopy (FESEM) results demonstrated that the outside of the PET covered with HAP nanoparticles. The cell viability results demonstrated that the number of Mesenchymal stem cells (MSCs) primarily spread out on CTS-SF/PET/Ag@HAP. RT-PCR results demonstrated that there was an upregulated mRNA articulation of osseous development-related properties in the CTS-SF/PET/Ag@HAP composite. The in vivo rabbit animal assessment scores of the CTS-SF/PET/Ag@HAP composite were significantly better than those of the CTS-SF/PET at 1 to 3 months. Both in-vivo and in-vitro results exhibited in this investigation recommend that the cytocompatibility and osseointegration of CTS-SF/PET/Ag@HAP tendon were fundamentally improved by expanding the multiplication of cells and up-regulating the outflow of tendon development-related properties. In conclusion, the CTS-SF/PET/Ag@HAP tendon is a promising candidate for Anterior Cruciate Ligament (ACL) replacement in the future.

Engineering a multifunctional 3D-printed PLA-collagen-minocycline-nanoHydroxyapatite scaffold with combined antimicrobial and osteogenic effects for bone regeneration.

3D-printing and additive manufacturing can be powerful techniques to design customized structures and produce synthetic bone grafts with multifunctional effects suitable for bone repair. In our work we aimed the development of novel multifunctionalized 3D printed poly(lactic acid) (PLA) scaffolds with bioinspired surface coatings able to reduce bacterial biofilm formation while favoring human bone marrow-derived mesenchymal stem cells (hMSCs) activity. For that purpose, 3D printing was used to prepare PLA scaffolds that were further multifunctionalized with collagen (Col), minocycline (MH) and bioinspired citrate- hydroxyapatite nanoparticles (cHA). PLA-Col-MH-cHA scaffolds provide a closer structural support approximation to native bone architecture with uniform macroporous, adequate wettability and an excellent compressive strength. The addition of MH resulted in an adequate antibiotic release profile that by being compatible with local drug delivery therapy was translated into antibacterial activities against Staphylococcus aureus, a main pathogen associated to bone-related infections. Subsequently, the hMSCs response to these scaffolds revealed that the incorporation of cHA significantly stimulated the adhesion, proliferation and osteogenesis-related gene expression (RUNX2, OCN and OPN) of hMSCs. Furthermore, the association of a bioinspired material (cHA) with the antibiotic MH resulted in a combined effect of an enhanced osteogenic activity. These findings, together with the antibiofilm activity depicted strengthen the appropriateness of this 3D-printed PLA-Col-MH-cHA scaffold for future use in bone repair. By targeting bone repair while mitigating the typical infections associated to bone implants, our 3D scaffolds deliver an integrated strategy with the combined effects further envisaging an increase in the success rate of bone-implanted devices.

Assessment of osteoinduction using a porous hydroxyapatite coating prepared by micro-arc oxidation on a new titanium alloy.

Surface modification and material improvement is now an important way to improve the osseointegration between bone and uncemented prothesis. The purpose of this study was to investigate the bone ingrowth potential of porous hydroxyapatite (HA) coatings prepared by micro-arc oxidation (MAO) on Ti-3Zr-2Sn-3Mo-25Nb, a new titanium alloy. HA-coated specimens were implanted in the left proximal femoral medullary canal of beagles for 4, 12, and 24 weeks, and uncoated specimens were implanted in the right as a control. The surface morphology and phase composition were investigated with environmental scanning electron microscopy and X-ray diffractometry. The bone ingrowth was assessed by histomorphometry. A pull-out test was performed to assess the mechanical performance of the bone-implant interface. A porous coating was well prepared on the new titanium alloy by using the MAO method. The bone-to-implant contact was significantly higher for the HA-coated group compared to that in the uncoated group. Mechanical tests showed that the HA-coated group had significantly higher maximum force at the bone-implant interface compared to the uncoated specimens. MAO is a suitable coating approach for this new titanium alloy. The HA coating prepared by this approach can significantly promote bone ingrowth and the mechanical performance of the bone-implant interface.

Mechanical assessment of grit blasting surface treatments of dental implants.

This paper investigates the influence of surface preparation treatments of dental implants on their potential (mechanical) fatigue failure, with emphasis on grit-blasting. The investigation includes limited fatigue testing of implants, showing the relationship between fatigue life and surface damage condition. Those observations are corroborated by a detailed failure analysis of retrieved fracture dental implants. In both cases, the negative effect of embedded alumina particles related to the grit-blasting process is identified. The study also comprises a numerical simulation part of the grit blasting process that reveals, for a given implant material and particle size, the existence of a velocity threshold, below which the rough surface is obtained without damage, and beyond which the creation of significant surface damage will severely reduce the fatigue life, thus increasing fracture probability. The main outcome of this work is that the overall performance of dental implants comprises, in addition to the biological considerations, mechanical reliability aspects. Fatigue fracture is a central issue, and this study shows that uncontrolled surface roughening grit-blasting treatments can induce significant surface damage which accelerate fatigue fracture under certain conditions, even if those treatments are beneficial to the osseointegration process.

Assessment of cellular reactions to magnesium as implant material in comparison to titanium and to glyconate using the mouse tail model.

PURPOSE: Nowadays, research in magnesium alloys as a biodegradable implant material has increased. The aim of this study was to examine osteoinductive properties and tissue responses to pure magnesium in comparison to conventional permanent (titanium) and to degradable (glyconate) implant materials. METHODS: Magnesium wires (0.4 mm in diameter, 10 mm length) were implanted into tail veins of mice and examined after 2, 4, 8, 16 and 32 weeks. Titanium and glyconate as controls were assessed after 2, 4, 8 and 24 weeks. µ-computed tompgraphy, histology and SEM examinations were performed. RESULTS: Magnesium implants showed increasing structural losses over time with fragmentation after an observation period of 32 weeks. Glyconate was fully degraded and titanium remained almost unaffected after 24 weeks. In contrast to some titanium and glyconate implants, first calcium and phosphate precipitations could be observed around magnesium implants after two weeks. However, ossification could not be observed even after 32 weeks, whereas enchondral ossification was found partially in the sourrounding of glyconate and titanium implants after eight weeks. Nevertheless, magnesium implants showed less inflammatory responses and fibrosing properties than the conventional implant materials. CONCLUSIONS: Although the assumed osteoinductive properties could not be detected, magnesium appears to be a promising degradable implant material because of the low sensitizing and inflammatory potential.

Osseointegration assessment of chairside argon-based nonthermal plasma-treated Ca-P coated dental implants.

This study investigated the effect of an Argon-based nonthermal plasma (NTP) surface treatment-operated chairside at atmospheric pressure conditions applied immediately prior to dental implant placement in a canine model. Surfaces investigated comprised: Calcium-Phosphate (CaP) and CaP + NTP (CaP-Plasma). Surface energy was characterized by the Owens-Wendt-Rabel-Kaelble method and chemistry by X-ray photoelectron spectroscopy (XPS). Six adult beagles dogs received 2 plateau-root form implants (n = 1 each surface) in each radii, providing implants that remained 1 and 3 weeks in vivo. Histometric parameters assessed were bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO). Statistical analysis was performed by Kruskall-Wallis (95% level of significance) and Dunn's post-hoc test. The XPS analysis showed peaks of Ca, C, O, and P for the CaP and CaP-Plasma surfaces. Both surfaces presented carbon primarily as hydrocarbon (C-C, C-H) with lower levels of oxidized carbon forms. The CaP surface presented atomic percent values of 38, 42, 11, and 7 for C, O, Ca, and P, respectively, and the CaP-Plasma presented increases in O, Ca, and P atomic percent levels at 53, 12, and 13, respectively, in addition to a decrease in C content at 18 atomic percent. At 1 week no difference was found in histometric parameters between groups. At 3 weeks significantly higher BIC and BAFO were observed for CaP-Plasma treated surfaces. Surface elemental chemistry was modified by the Ar-based NTP. Ar-based NTP improved bone formation around plateau-root form implants at 3 weeks compared with CaP treatment alone.

Assessment of combination techniques in enhancing the regenerative potential of tricalcium phosphate graft in treatment of infrabony periodontal defects.

AIMS: The purpose of the present study was to evaluate and compare the clinical outcome of infrabony defects following reconstructive surgery with the use of tricalcium phosphate (TCP) alone; TCP and citric acid (CA) root conditioning; and TCP, CA, and oxidized regenerated cellulose (ORC) membrane. MATERIALS AND METHODS: Thirty-nine systemically healthy subjects with vertical infrabony defect were initially selected based on intraoral periapical radiographs and clinical examination to record probing pocket depth (PPD) and clinical attachment level (CAL). Only 21 defects revealed two-walled configuration on surgical debridement. These defects were selected and randomly allotted to the study groups. Group 1 defects were treated with TCP, group 2 with TCP+CA, and group 3 with TCP+CA+ORC. PPD, CAL, defect depth (DD), and level of alveolar crest (AC) were evaluated at the time of initial surgery and after 6 months at surgical re-entry. These measurements were utilized to calculate PPD reduction, CAL gain, defect fill (DF), %defect fill (%DF), and crestal resorption (CR). STATISTICAL ANALYSIS: A paired t-test was used for assessing changes in each group. Unpaired t-test was used for intergroup comparisons. RESULTS: All three groups showed statistically significant PPD reduction, CAL gain, DF, and %DF, but insignificant CR at the end of 6 months. On intergroup comparison, no statistically significant differences were noted between the groups for all the parameters. CONCLUSION: Efficacy of combination techniques using TCP+CA; TCP+CA+ORC in treatment of periodontal infrabony defects is at least equal to that of TCP alone.

Assessment of angiogenesis in osseointegration of a silica-collagen biomaterial using 3D-nano-CT.

Bony integration of biomaterials is a complex process in which angiogenesis plays a crucial role. We evaluated micro- and nano-CT imaging to demonstrate and quantify neovascularization in bony integration of a biomaterial and to give an image based estimation for the needed resolution for imaging angiogenesis in an animal model of femora defect healing. In 8 rats 5mm full-size defects were created at the left femur that was filled with silica-collagen bone substitute material and internally fixed with plate osteosynthesis. After 6 weeks the femora were infused in situ with Microfil, harvested and scanned for micro-CT (9 µm)(3) and nano-CT (3 µm)(3) imaging. Using those 3D images, the newly formed blood vessels in the area of the biomaterial were assessed and the total vascular volume fraction, the volume of the bone substitute material and the volume of the bone defect were quantitatively characterized. Results were complemented by histology. Differences were statistically assessed using (ANOVA). High-resolution nano-CT demonstrated new blood vessel formation surrounding the biomaterial in all animals at capillary level. Immunohistochemistry confirmed the newly formed blood vessels surrounding the bone substitute material. The mean vascular volume fraction (VVF) around the implant was calculated to be 3.01 ± 0.4%. The VVF was inversely correlated with the volume of the bone substitute material (r=0.8) but not with the dimension of the fracture zone (r=0.3). Nano-CT imaging is feasible for quantitative analysis of angiogenesis during bony integration of biomaterials and a promising tool in this context for the future.

Establishment of an in vivo model for molecular assessment of titanium implant osseointegration in compromised bone.

Shortening of the healing time before loading risks impeding successful titanium implant anchorage into compromised bone. A thorough understanding at the genetic scale of the early phases of bone regeneration at the implant interface is required before the development of strategies to enhance implant osseointegration. In this study a new in vivo implant model to explore the mechanism by which titanium implant osseointegration is affected by the host bone properties is presented. An implant was conceptualized enabling standardized harvesting of peri-implant tissue for quantitative molecular analysis while preserving the mimicking of the clinical setting. The implant is partly indented to provide a well-defined healing compartment from where tissue differentiation and de novo bone formation can be investigated and partly screw-threaded to provide a good implant anchorage into the bone. The feasibility of the implant design was assessed in osteopenic bone conditions, evoked by simulated weightlessness. Wistar rats were either hindlimb unloaded by tail suspension (HU) for 9 days or acted as controls (CTL). The status of compromised bone tissue through 9-days HU was confirmed by micro-X-ray computed tomography. The implant was installed in the proximal tibial bone 7 days after the onset of HU or CTL. Two days postimplantation, the peri-implant regenerating tissue responses were recorded by measuring expression of inflammatory, angiogenic, and bone resorption parameters (hypoxia-inducible factor 1, alpha subunit; vascular endothelial growth factor A; angiopoietin 1; endothelial PAS domain protein 1; fibroblast growth factor 2; tumor necrosis factor; interleukin 11; acid phosphatase 5, tartrate resistant; tumor necrosis factor (ligand) superfamily, member 11/RANKL). We successfully demonstrated that HU-associated bone conditions evoked a significant alteration of expression of the angiogenic markers in the peri-implant regenerative tissue during initial implant osseointegration, whereas the expression levels of the inflammatory and bone resorption parameters remained unchanged. We concluded that this in vivo implant model provides a well-designed and controlled method to examine molecular responses in implant osseointegration to impaired bone conditions. This model may serve to explore the application of anabolic strategies in peri-implant osteogenesis.

An in vitro assessment of titanium functionalized with polysaccharides conjugated with vascular endothelial growth factor for enhanced osseointegration and inhibition of bacterial adhesion.

The long-term success of orthopedic implants may be compromised by defective osseointegration and bacterial infection. An effective approach to minimize implant failure would be to modify the surface of the implant to make it habitable for bone-forming cells and anti-infective at the same time. In this in vitro study, the surfaces of titanium (Ti) substrates were functionalized by first covalently grafting either dopamine followed by carboxymethyl chitosan (CMCS) or hyaluronic acid-catechol (HAC). Vascular endothelial growth factor (VEGF) was then conjugated to the polysaccharide-grafted surface. Antibacterial assay with Staphylococcus aureus (S. aureus) showed that the polysaccharide-modified substrates significantly decrease bacterial adhesion. The CMCS-functionalized Ti demonstrated better antibacterial property than the HAC-functionalized Ti since CMCS is bactericidal while HA only inhibits the adhesion of bacteria without killing them. Osteoblast attachment, as well as alkaline phosphatase (ALP) activity and calcium deposition were enhanced by the immobilized VEGF on the polysaccharide-grafted Ti. Thus, Ti substrates modified with polysaccharides conjugated with VEGF can promote osteoblast functions and concurrently reduce bacterial adhesion. Since VEGF is also known to enhance angiogenesis, the VEGF-polysaccharide functionalized substrates will have promising applications in the orthopedic field.

Correlation of computed tomography with histology in the assessment of periprosthetic defect healing.

Computed tomography (CT) may more accurately assess the healing of grafted osteolytic lesions around acetabular components compared with plain radiographs, although clinical validation is lacking. To determine whether clinical or micro-CT imaging could assess accurately the grafted lesion compared with histology, we therefore quantified bone healing and ingrowth to determine an effective rhBMP-2 dose and ratio to allograft bone when grafted adjacent to a cementless porous-coated component. We grafted surgically created acetabular defects in canines (n = 20) before uncemented total hip arthroplasty. At 6 weeks, embedded acetabula were imaged and the CT slice images matched to histology section images. The percentage of bone in the defect and growth into the porous surface was assessed quantitatively. Low-dose rhBMP-2 with allograft (1:5 ratio) resulted in a higher percentage of defect healing (43.8%) than rhBMP-2 alone (29.2%) and a higher percentage of bone ingrowth (15.7%) than allograft bone alone (1.1%) as measured by histology. Micro-CT measurements were similar to histologic measurements of defect healing, whereas clinical CT overestimated periprosthetic bone by 38%. Neither clinical CT nor micro-CT techniques are adequate for assessing ingrowth or the bone-implant interface with metal artifacts.

Biological assessment of porous-implant hydroxyapatite combined with periosteal grafting in maxillary defects.

PURPOSE: To investigate the use of porous hydroxyapatite (HA) combined with periosteal graft to repair an induced maxillary bone defect. MATERIALS AND METHODS: A defect was produced in the premaxillary bone of rats. Four groups were used: 1) those treated with the mucoperiosteal graft from the premaxilla; 2) those treated with HA combined with mucoperiosteal graft from the premaxilla; 3) those treated with HA combined with periosteal graft from the femur; and 4) those treated with periosteal graft from the femur. RESULTS: The radiographic aspects from all groups showed no signs of bone formation after 2 weeks. After 16 weeks, there was evidence of points of radiolucency inside the HA implants. Cell proliferation occurred from the periosteum covering the defect. Bone tissue grew from the defect margin to inside the defect in all cases. Mature bone was seen around the HA implants after 8 and 16 weeks. CONCLUSION: The periosteal graft provides satisfactory support to the HA implant, allowing the growth of new bone.

A prospective randomized study of posterolateral lumbar fusion using osteogenic protein-1 (OP-1) versus local autograft with ceramic bone substitute: emphasis of surgical exploration and histologic assessment.

STUDY DESIGN: A prospective, randomized and controlled study. OBJECTIVES: To evaluate the osteoinductive property of Osteogenic Protein-1 (OP-1 or BMP-7) and fusion rate in human instrumented posterolateral lumbar fusion through radiographic examination, surgical exploration, and histologic assessment. SUMMARY OF BACKGROUND DATA: The use of osteoinductive agents is a current topic in spinal fusion. Numerous preclinical investigations have demonstrated efficacy of osteoinductive proteins in spinal fusion, but few human clinical studies have been reported. METHODS: Nineteen patients with L3-L4 or L4-L5 degenerative spondylolisthesis underwent posterolateral lumbar fusion using pedicle screw instrumentation. The patients were randomized to receive either OP-1 Putty (3.5 mg OP-1/g of collagen matrix per side) alone (n = 9), or local autograft with HA-TCP granules (n = 10). Fusion status was evaluated using plain radiography and CT scan. Radiographic fusion criteria included less than 5 degrees of angular motion, less than 2 mm of translation, and evidence of bridging bone in the posterolateral lumbar area in which the graft materials were placed following decortication. After a minimum 1-year follow-up, the patients who showed radiographic evidence of fusion underwent instrumentation removal and surgical exploration of the fusion site. Biopsy specimens were taken from the fusion mass and evaluated histologically. RESULTS: Radiographic fusion rate was 7 of 9 OP-1 patients and 9 of 10 control patients. Based on surgical exploration of these 16 patients, new bone formation was macroscopically observed in the posterolateral lumbar region in all cases; however, solid fusion was observed in 4 of 7OP-1 and 7 of 9 HA-TCP/autograft patients. Histologic assessment demonstrated viable bone in 6 of 7 OP-1 patients. All the control (HA-TCP/autograft) specimens contained viable bone and fibrous tissue surrounding ceramic granules, suggesting slow incorporation of the graft material. CONCLUSIONS: In a human posterolateral lumbar spine trial, OP-1 reliably induced viable amounts of new bone formation, but the fusion success rate evaluated by surgical exploration was only 4 of 7.

Biological assessment of the bone-screw interface after insertion of uncoated and hydroxyapatite-coated pedicular screws in the osteopenic sheep.

The sheep seems to be a promising model of osteoporosis and biomaterial osteointegration in osteopenic bone. The long-term ovariectomized sheep model was used for the biological investigation of bone healing around uncoated and hydroxyapatite (HA)-coated pedicle screws in osteopenic bone. Four sheep were ovariectomized and four sheep were sham-operated. Twenty-four months after surgery, the animals were implanted with uncoated and HA-coated stainless steel screws in the lumbar vertebral pedicles. Four months later, bone-to-implant contact, bone ingrowth, and bone hardness were measured around screws. Uncoated stainless steel presented significantly (p < 0.0005) lower bone-to-implant contact in healthy and osteopenic bone compared with HA-coated stainless steel. HA significantly improved bone ingrowth in healthy bone (p < 0.05) compared with uncoated stainless steel. Osteopenia significantly (p < 0.05) reduced the area of bone ingrowth around the screw threads for both types of implants. In the inner thread area, bone microhardness significantly increased (p < 0.05) in HA-coated surface versus uncoated for healthy and osteopenic bone. HA coating significantly enhances bone-to-implant contact also in osteopenic bone in comparison with uncoated stainless steel surfaces. Bone ingrowth and mineralization are ameliorated by the osteoconductive HA coating. However, osteopenia seems to greatly influence bone ingrowth processes around the implanted screws regardless of the characteristics of the material surface.

[The clinical assessment of the efficacy of using a biogenic composite material in dental implantology]. / Klinicheskaia otsenka éffektivnosti primeneniia biogennogo kompozitsionnogo materiala v dental'noi implantologii.

A new biogenic composite material based on hydroxyapatite and collagen with silamine was effectively used in dental implantology. Use of this material was conductive to a better course of the implant integration in the adjacent tissue structures and prevented the development of resorptive and atrophic changes in the alveolar process. Use of composite material in patients with relative indications for implantation precluded the development of complications in the early postoperative period and in remote periods of the follow-up.