Bone-biomaterial Interface Research Articles

Histologic and Ultrastructural Analysis of Regenerated Bone in Maxillary Sinus Augmentation Using a Porcine Bone–Derived Biomaterial

The purpose of the present study was the histologic and ultrastructural evaluation of a biomaterial composed of cortical pig bone in the form of granules. After maxillary sinus augmentation using this biomaterial, 10 specimens were retrieved after 5 months in 10 patients using this biomaterial. The specimens were processed to be observed under light microscopy (LM) and transmission electron microscopy (TEM). Histomorphometric measurements were presented by means +/- standard deviations. LM showed that most of the particles were surrounded by newly formed bone. In some areas, the osteoid matrix was present; however, mainly compact bone was present at the interface. There was no evidence of an acute inflammatory infiltrate. The newly formed bone was 36% +/- 2.8% and marrow spaces were 38% +/- 1.6%, whereas residual grafted material was 31% +/- 1.6%. Under TEM, all phases of bone formation (osteoid matrix, woven, and lamellar bone) were observed in proximity with the biomaterial particles. The bone-biomaterial interface showed a close contact between the porcine bone particles and the surrounding bone that had mainly features of mature bone with numerous osteocytes. A lamina limitans was sometimes present at this interface. According to our knowledge, this is the first study presenting data on TEM of a porcine bone-derived biomaterial used in sinus augmentation procedures in humans. Our findings show that this is a biocompatible biomaterial that can be used for maxillary sinus augmentation procedures without interfering with the normal reparative bone processes.

Maxillary Sinus Augmentation Using an Engineered Porous Hydroxyapatite: A Clinical, Histological, and Transmission Electron Microscopy Study in Man

Porous hydroxyapatite (HA) is a calcium-phosphate-based material that is biocompatible, nonimmunological, and osteoconductive, and has a macroporosity of about 200 to 800 microm. The pores seem to be able to induce migration, adhesion, and proliferation of osteoblasts inside the pore network and to promote angiogenesis inside the pore system. The aim of this study was to evaluate the clinical behavior and the histological and ultrastructural aspects of porous HA in maxillary sinus augmentation procedures. Twenty-four patients (19 men, 5 women; average age 53.4 years) in good general physical and mental health and with partially or completely edentulous maxillae were selected for this study. Six months after sinus floor elevation, at the time of dental implant placement, biopsies were carried out under local anesthesia. These bone cores were cut in half and were processed for light and transmission electron microscopy. After a mean 3 years after implantation, all implants are clinically in function and no surgical or prosthetic complications have occurred. Under light microscopy, newly formed bone was 38.5% +/- 4.5%, whereas the residual biomaterial represented 12% +/- 2.3% and the marrow spaces represented 44.6% +/- 4.2%. In addition, in the majority of cases, the biomaterial particles were in close contact with the bone, which appeared compact with the characteristic features of well-organized lamellar bone. A cement-like line was slightly visible at the bone-biomaterial interface, but there were no gaps or interposed connective tissue in between. A high quantity (about 40%) of newly formed bone was present. Bone was closely apposed to the biomaterials particles as shown in light microscopy and transmission electron microscopy. Moreover, no signs of inflammatory cell infiltrate or foreign body reaction were present. Also, most of the biomaterial was resorbed and only a small quantity (a little more than 10%) was still present. The results of our study show that porous HA can be a suitable synthetic material for bone regeneration in maxillary sinus augmentation procedures.

A method for immunohistochemical detection of osteogenic markers in undecalcified bone sections

To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials.

Maxillary sinus augmentation with Bio‐Oss® particles: A light, scanning, and transmission electron microscopy study in man

Biological interactions occurring at the bone-biomaterial interface are critical for long-term clinical success. Bio-Oss is a deproteinized, sterilized bovine bone that has been extensively used in bone regeneration procedures. The aim of the present study was a comparative light, scanning, and electron microscopy evaluation of the interface between Bio-Oss and bone in specimens retrieved after sinus augmentation procedures. Under light microscopy, most of the particles were surrounded by newly formed bone, while in a few cases, at the interface of some particles it was possible to observe marrow spaces and biological fluids. Under scanning electron microscopy, in most cases, the particle perimeter appeared lined by bone that was tightly adherent to the biomaterial surface. Transmission electron microscopy showed that the bone tissue around the biomaterial showed all the phases of the bone healing process. In some areas, randomly organized collagen fibers were present, while in other areas, newly formed compact bone was present. In the first bone lamella collagen fibers contacting the Bio-Oss surface were oriented at 243.73 +/- 7.12 degrees (mean +/- SD), while in the rest of the lamella they were oriented at 288.05 +/- 4.86 degrees (mean +/- SD) with a statistically significant difference of 44.32 degrees (p < 0.001). In the same areas the intensity of gray value was 172.56 +/- 18.15 (mean +/- SD) near the biomaterial surface and 158.71 +/- 21.95 (mean +/- SD) in the other part of the lamella with an unstatistically significant difference of 13.79 (p = 0.071). At the bone-biomaterial interface there was also an electron-dense layer similar to cement lines. This layer had a variable morphology being, in some areas, a thin line, and in other areas, a thick irregular band. The analyses showed that Bio-Oss particles do not interfere with the normal osseous healing process after sinus lift procedures and promote new bone formation. In conclusion, this study serves as a better understanding of the morphologic characteristics of Bio-Oss and its interaction with the surrounding tissues.

Histomorphometric, ultrastructural and microhardness evaluation of the osseointegration of a nanostructured titanium oxide coating by metal-organic chemical vapour deposition: an in vivo study

Over the past decade the increase of elderly population has determined a rise in the incidence of bone fractures, and the improvement of the implant–bone interface remains an open problem. Metal-organic chemical vapour deposition (MOCVD) has recently been proposed as a technique to coat orthopaedic and dental prostheses with metal nanostructured oxide films either through the decomposition of oxygenated compounds (single-source precursors) or the reaction of oxygen-free metal compounds with oxygenating agents. The present study was performed to assess the in vivo biocompatibility of commercially pure Ti (control material: TI/MA) implants (∅ 2 mm×5 mm length) coated with nanostructured TiO2 films by MOCVD (Ti/MOCVD) and then inserted into rabbit femoral cortical (middhiaphysis) and cancellous (distal epiphysis) bone. Histomorphometric, ultrastructural and microhardness investigations were carried out. Four and 12 weeks after surgery, significant (p<0.0005) increases in AI of Ti/MOCVD implants were observed as compared to Ti/MA implants (distal femoral epiphysis: 4 weeks=8.2%, ns; 12 weeks=52.3%, p<0.005; femoral diaphysis: 4 weeks=20.2%, p<0.0005; 12 weeks=10.7%, p<0.005). Bone microhardness results showed significant increases for the Ti/MOCVD versus Ti/MA implants at 200 μm in the femoral diaphysis (4 weeks=14.2, p<0.005) and distal femoral epiphysis (12 weeks=14.5, p<0.01) at 4 and 12 weeks, respectively. In conclusion, the current findings demonstrate that the nanostructured TiO2 coating positively affects the osseointegration rate of commercially pure Ti implants and the bone mineralization at the bone–biomaterial interface in both cortical and cancellous bone.

Histomorphometric, ultrastructural and microhardness evaluation of the osseointegration of a nanostructured titanium oxide coating by metal-organic chemical vapour deposition: an in vivo study

Over the past decade the increase of elderly population has determined a rise in the incidence of bone fractures, and the improvement of the implant–bone interface remains an open problem. Metal-organic chemical vapour deposition (MOCVD) has recently been proposed as a technique to coat orthopaedic and dental prostheses with metal nanostructured oxide films either through the decomposition of oxygenated compounds (single-source precursors) or the reaction of oxygen-free metal compounds with oxygenating agents. The present study was performed to assess the in vivo biocompatibility of commercially pure Ti (control material: TI/MA) implants (∅ 2 mm×5 mm length) coated with nanostructured TiO 2 films by MOCVD (Ti/MOCVD) and then inserted into rabbit femoral cortical (middhiaphysis) and cancellous (distal epiphysis) bone. Histomorphometric, ultrastructural and microhardness investigations were carried out. Four and 12 weeks after surgery, significant ( p<0.0005) increases in AI of Ti/MOCVD implants were observed as compared to Ti/MA implants (distal femoral epiphysis: 4 weeks=8.2%, ns; 12 weeks=52.3%, p<0.005; femoral diaphysis: 4 weeks=20.2%, p<0.0005; 12 weeks=10.7%, p<0.005). Bone microhardness results showed significant increases for the Ti/MOCVD versus Ti/MA implants at 200 μm in the femoral diaphysis (4 weeks=14.2, p<0.005) and distal femoral epiphysis (12 weeks=14.5, p<0.01) at 4 and 12 weeks, respectively. In conclusion, the current findings demonstrate that the nanostructured TiO 2 coating positively affects the osseointegration rate of commercially pure Ti implants and the bone mineralization at the bone–biomaterial interface in both cortical and cancellous bone.

A new austenitic stainless steel with negligible nickel content: an in vitro and in vivo comparative investigation

New nickel (Ni)-reduced stainless-steel metals have recently been developed to avoid sensitivity to Ni. In the present study, an austenitic Ni-reduced SSt named P558 (P558, Böhler, Milan, Italy) was studied in vitro on primary osteoblasts and in vivo after bone implantation in the sheep tibia, and was compared to ISO 5832-9 SSt (SSt) and Ti6Al4V. Cells were cultured directly on P558 and Ti6Al4V. Cells cultured on polystyrene were used as controls. Osteoblast proliferation, viability and synthetic activity were evaluated at 72 h by assaying WST1, alkaline phosphatase activity (ALP), nitric oxide, pro-collagen I (PICP), osteocalcin (OC), transforming growth factor- β1 (TGF β-1) and interleukin-6 (IL-6) after 1.25(OH) 2D 3 stimulation. Under general anaesthesia, four sheep were submitted for bilateral tibial implantation of P558, SSt and Ti6Al4V rods. In vitro results demonstrated that the effect of P558 on osteoblast viability, PICP, TGF β-1, tumor necrosis factor- α production did not significantly differ from that exerted by Ti6Al4V and controls. Furthermore, P558 enhanced osteoblast differentiation, as confirmed by ALP and OC levels, and reduced IL-6 production. At 26 weeks, the bone-to-implant contact was higher in P558 than in SSt (28%, p<0.005) and Ti6Al4V (4%, p<0.05), and was higher in Ti6Al4V than in SSt (22%, p<0.005). The tested materials did not affect bone microhardness in pre-existing host bone as evidenced by the measurements taken at 1000 μm from the bone–biomaterial interface ( F=1.89, ns). At the bone–biomaterial interface the lowest HV value was found for SSt, whereas no differences in HV were observed between materials ( F=1.55, ns). The current findings demonstrate P558 biocompatibility both in vitro and in vivo, and osteointegration processes are shown to be significantly improved by P558 as compared to the other materials tested.

Synchrotron X-ray microtomography (on a micron scale) provides three-dimensional imaging representation of bone ingrowth in calcium phosphate biomaterials

This study used synchrotron X-ray microtomography on a micron scale to compare three-dimensional (3D) bone ingrowth after implantation of various calcium phosphate bone substitutes in a rabbit model. The advantage of using this new method for the study of biomaterials was then compared with histomorphometry for analysis of interconnection and bone ingrowth. The study focused on the newly formed bone-biomaterial interface. Macroporous Biphasic Calcium Phosphate (MBCP™) ceramic blocks and two different injectable calcium phosphate biomaterials [an injectable bone substitute (IBS) consisting of a biphasic calcium phosphate granule suspension in hydrosoluble polymer and a calcium phosphate cement material (CPC)] were studied after in vivo implantation. Absorption or phase-contrast microtomography was performed with the dedicated set-up at beamline ID22. Experimental spatial resolution was between 1 and 1.4 μm, depending on experimental radiation. All calcium phosphates tested showed osteoconduction. IBS observations after 3D reconstruction showed interconnected bioactive biomaterial with total open macroporosity and complete bone ingrowth as early as 3 weeks after implantation. This experimentation was consistent with two-dimensional histomorphometric analysis, which confirmed its suitability for biomaterials. This 3D study relates the different types of bone substitution to biomaterial architecture. As porosity and interconnection increase, bone ingrowth becomes greater at the expense of the bone substitute: IBS>MBCP>CPC.

FINITE ELEMENT SIMULATION OF ELASTIC COMPLIANCE TECHNIQUE FOR FORMULATING A TEST METHOD TO DETERMINE THE FRACTURE TOUGHNESS OF BONE

Compact Sandwich Specimen (CSS) is the best specimen to test the fracture toughness of bone from small laboratory animals. There exists a direct method to calculate the stress intensity factor (K) of bone using CSS. However, energy release rate (G) is the fundamental measure of fracture toughness of a material. The direct relation between K, G, Young's Modulus (E), and Poisson's ratio (ν), defined for homogeneous materials may not apply to bone, which has a non-homogeneous microstucture. Therefore, a direct test method is required to calculate G of bone using CSS. Elastic compliance technique is used to measure G of a material. This paper presents an experimentally tested and reliable procedure to simulate the elastic compliance technique by finite element analysis. The procedure may eventually be used in formulating the direct test method to calculate G of bone and bone-biomaterial interface, using CSS.

Laser Technology in Orthopedics: Preliminary Study on Low Power Laser Therapy to Improve the Bone-Biomaterial Interface

Low Power Laser (LPL) seems to enhance the healing of bone defects and fractures. The effect of LPL in other orthopedic areas such as osteointegration of implanted prosthetic bone devices is still unclear. In the present study, 12 rabbits were used to evaluate whether Ga-Al-As (780 nm) LPL stimulation has positive effects on osteointegration. Hydroxyapatite (HA) cylindrical nails were drilled into both distal femurs of rabbits. From postoperative day 1 and for 5 consecutive days, the left femura of all rabbits were given LPL treatment (Laser Group-LG) with the following parameters: 300 Joule/cm2, 1 Watt, 300 Hertz, pulsating emission, 10 minutes. The right femura were sham-treated (Control Group-CG). At 4 and 8 weeks after implantation, histologic and histomorphometric investigations evaluated bone-biomaterial-contact. Histomorphometry showed a higher degree of osteointegration at the HA-bone interface in the LG Group at 4 (p < 0.0005) and 8 weeks (p < 0.001). These preliminary positive results seem to support the hypothesis that LPL treatment can be considered a good tool to enhance the bone-implant interface in orthopedic surgery.

Understanding and controlling the bone–implant interface

A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, endosseous implants should result in formation of a characteristic interfacial layer and bone matrix with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the interface and of the effects biomaterials have on bone and bone cells is needed. Such knowledge is essential for developing strategies to optimally control osseointegration. This paper reviews current knowledge of the bone–biomaterial interface and methods being investigated for controlling it. Morphological studies have revealed the heterogeneity of the bone–implant interface. One feature often reported, regardless of implant material, is an afibrillar interfacial zone, comparable to cement lines and laminae limitantes at natural bone interfaces. These electron-dense interfacial layers are rich in noncollagenous proteins, such as osteopontin and bone sialoprotein. Several approaches, involving alteration of surface physicochemical, morphological, and/or biochemical properties, are being investigated in an effort to obtain a desirable bone–implant interface. Of particular interest are biochemical methods of surface modification, which immobilize molecules on biomaterials for the purpose of inducing specific cell and tissue responses or, in other words, to control the tissue–implant interface with biomolecules delivered directly to the interface. Although still in its infancy, early studies indicate the value of this methodology for controlling cell and matrix events at the bone–implant interface.

In Vivo Bone Response to Biomechanical Loading at the Bone/Dental-Implant Interface

Since dental implants must withstand relatively large forces and moments in function, a better understanding of in vivo bone response to loading would aid implant design. The following topics are essential in this problem. (1) Theoretical models and experimental data are available for understanding implant loading as an aid to case planning. (2) At least for several months after surgery, bone healing in gaps between implant and bone as well as in pre-existing damaged bone will determine interface structure and properties. The ongoing healing creates a complicated environment. (3) Recent studies reveal that an interfacial cement line exists between the implant surface and bone for titanium and hydroxyapatite (HA). Since cement lines in normal bone have been identified as weak interfaces, a cement line at a bone-biomaterial interface may also be a weak point. Indeed, data on interfacial shear and tensile "bond" strengths are consistent with this idea. (4) Excessive interfacial micromotion early after implantation interferes with local bone healing and predisposes to a fibrous tissue interface instead of osseointegration. (5) Large strains can damage bone. For implants that have healed in situ for several months before being loaded, data support the hypothesis that interfacial overload occurs if the strains are excessive in interfacial bone. While bone "adaptation" to loading is a long-standing concept in bone physiology, researchers may sometimes be too willing to accept this paradigm as an exclusive explanation of in vivo tissue responses during experiments, while overlooking confounding variables, alternative (non-mechanical) explanations, and the possibility that different types of bone (e.g., woven bone, Haversian bone, plexiform bone) may have different sensitivities to loading under healing vs. quiescent conditions.

Analysis of push-out test data based on interfacial fracture energy.

Push-out testing is frequently used to assess the interfacial shear strength developed at a bone-biomaterial interface during in vivo experiments. The aim of the present research was to assess the in vivo performance of a novel substrate/coating combination and to introduce a more rigorous fracture mechanics analysis of the push-out test data. An adhesively bonded hydroxyapatite (HA), and a Ti-6Al-4V alloy plasma sprayed with HA, were implanted in female New Zealand white rabbits for up to 6 months in duration. After death, push-out tests were carried out and the shear strength was calculated in the conventional way, together with microscopical examination of crack paths. A finite element model was drawn up representing four potential failure mechanisms. The measured "failure shear strengths" in conventional analysis were approximately equal for the two coatings. However, JC at failure calculated from the model was 210 J m(-2) at the novel adhesively bonded HA/bone interface and 5 J m(-2) at a conventional titanium/plasma-sprayed HA interface. The conventional shear strength approach is strongly test dependent, and we believe that the fracture energy approach represents a more rigorous analysis of the real failure criterion in the implant/host tissue structure.

An experimental study in X-ray spectroscopy of the zirconium (Ca-PSZ) - bone interface. Microanalytic evaluation of the osteogenetic response.

Ultrastructural difractometric and chemical evaluations of calcium partially stabilized zirconium (Ca-PSZ) implants were performed in an in vivo study on animals in order to evaluate its biological behaviour. The chemical-morphological investigations demonstrated the presence of an osteogenetic activity at the bone-biomaterial interface. The new-osteogenesis was preceded by the formation of a loose connective tissue around the implants. This mesenchymal-type tissue without a capsular organization, allowing modulation of the mechanical forces to which the implant is subject, could be considered a positive event in the osteogenetic process and not a sign of future failure of the implant. Finally, microanalytical investigations carried out on non-implanted and implanted Ca-PSZ tools suggested that the surface of this ceramic material does not undergo modification once it has been inserted in the biological environment (12 months).

Underlying mechanisms at the bone-biomaterial interface.

In order to understand how biomaterials influence bone formation in vivo, it is necessary to examine cellular response to materials in the context of wound healing. Four interrelated properties of biomaterials (chemical composition, surface energy, surface roughness, and surface topography) affect mesenchymal cells in vitro. Attachment, proliferation, metabolism, matrix synthesis, and differentiation of osteoblast-like cell lines and primary chondrocytes are sensitive to one or more of these properties. The nature of the response depends on cell maturation state. Rarely do differentiated osteoblasts or chondrocytes see a material prior to its modification by biological fluids, immune cells and less differentiated mesenchymal cells in vivo. Studies using the rat marrow ablation model of endosteal wound healing indicate that ability of osteoblasts to synthesize and calcify their extracellular matrix is affected by the local presence of the material. Changes in the morphology and biochemistry of matrix vesicles, extracellular organelles associated with matrix maturation and calcification, seen in normal endosteal healing, are altered by implants. Moreover, the material exerts a systemic effect on endosteal healing as well. This may be due to local effects on growth factor production and secretion into the circulation, as well as to the fact that the implant may serve as a bioreactor.

The Bone-Biomaterial Interface.

The Bone-Biomaterial Interface.

Ultrastructure of the Mineralized Tissue/Calcium Phosphate Interface in Vitro

ABSTRACTAn in vitro rat bone marrow cell (RBMC) system was used to examine the structure of the interface established between calcium phosphates (Ca-P) and mineralized tissue. The Ca-P used, varied either in chemical structure or crystallinity. Therefore, not only the influence of chemical composition, but also the effect of degradation of Ca-P ceramics could be studied. The interfaces were examined with scanning and transmission electron microscopy (SEM and TEM).SEM showed that deposition of mineralized extracellular matrix on the different materials examined varied both in time and morphology. Mineralization started with the formation of afibrillar globules with which collagen fibres became integrated. With TEM, three distinctly different interfacial structural arrangements were observed which were dependent on the presence or absence of an electron dense layer and/or an amorphous zone. The former was considered to be at least partially caused by protein adsorption, which would precede biological mineralization events, whereas the latter was considered to represent partial degradation of the ceramic surfaces.The results of this study showed that interfacial reactions were not only influenced by the chemical structure, but also by the crystallinity of Ca-P ceramics. Thus, characterisation of Ca-P implant materials is of critical importance in achieving a better understanding of the phenomena that occur at the bone-biomaterial interface.

The effects of pylon shape on bone-pylon interface performance in direct skeletal attachment.

The attachment of a prosthesis directly to the part of the skeleton remaining after an amputation offers many improvements over coupling schemes used in conventional prostheses. However, the stresses induced in the bone by the macrostructure of the attached prosthetic device must first be understood because they are a significant consideration in the design of a successful direct skeletal attachment (DSA) system. This investigation utilizes the finite element method of analysis to structurally model some possible DSA systems. Static stress-response models for an above-the-knee human femoral amputation are formulated to investigate the effect of variations in pylon macrostructure on system performance. The models approximate the initial stages of support following the insertion of a pylon into the medullary cavity of the bone and are also used to assess the bond strengths that must be developed between the bone and pylon for a "no-slip" condition to hold at their interface. The analyses indicate that either a shaped, marrow cavity-fit pylon or four 135 degree wedges with a complementary pylon are favorable geometries for a DSA system. The stress levels caused by these two geometries are on the order of 20% of the axial compressive strength of cortical bone. For each, local stress levels at the bone-biomaterial interface remain the critical parameters for investigation.