A polymethyl methacrylate method for large specimens of mineralized bone with implants

A simple modified polymethyl methacrylate method is described for large mineralized bone specimens with implants and bioactive materials which produces consistently good histological preservation of the interface between bone and implant. Human femoral heads, whole rabbit condyles and canine tibias and femurs containing implants consisting of hydroxyapatite, smooth polyethylene, porous polyethylene and carbon were dehydrated in ascending grades of ethanol and cleared with xylene on an automated tissue processor which alternated vacuum and pressure for 22 hr. Infiltration was done with washed polymethyl methacrylate at 4 C under vacuum for 13 days. Polymerization was carried out in wide-mouth glass jars at 38 C for 36 hr so that the total processing time was less than 20 days. The only important modification was in the polymethyl methacrylate, which had less plasticizer than usual in order to give a harder block. This enabled production of 4 micron sections with good preservation of mineralized and cellular areas for the study of metabolic bone diseases, morphometry, fluorochrome labelling and interface analysis with the implant in situ.     

A bone preservation protocol that enables evaluation of osseointegration of implants with micro- and nanotextured surfaces

Development of surface treatments has enabled secure attachment of dental implants in less than 1 month. Consequently, it is necessary to characterize accurately the osseointegration of the implant surface in the region of the bone-implant contact (BIC). We developed a method for sample preparation that preserves both bone and BIC to permit analysis of the contact interface. We prepared eight nanotextured implants and implanted them in rabbit tibias. After healing for 30 days, outcomes were analyzed using both our bone preservation protocol and routine decalcification followed by preparation of histological sections stained by hematoxylin and eosin (H & E). Pull-out tests for implant osseointegration were performed after healing. Non-implanted samples of rabbit mandible were used as a control for assessing organic and mineralized bone characteristics and bone structure. Our bone preservation protocol enabled evaluation of many of the same bone characteristics as histological sections stained with H & E. Our protocol enables analysis of implant samples, implant surfaces and osseointegration without risk of BIC damage.     

Morphological and histochemical comparison of the cells elicited by ectopic bone implants and tibial osteoclasts.

Pellets of mineralized and demineralized bone and a composite mixture of mineralized and demineralized, devitalized bone particles were implanted subcutaneously on the dorsal body wall of young adult rats. Two weeks post-implantation, the pellets were removed and processed for histochemical and morphological analyses. Rat proximal tibia was also processed for evaluation. The levels of tartrate-resistant acid phosphatase (TRAP) activity in the multinucleated giant cells (MNGCs) from each of the three implants and from osteoclasts were assessed using an image analyzer. The osteoclasts from the proximal tibia and the majority of MNGCs from the demineralized implants demonstrated high levels of TRAP activity. MNGCs from the mineralized implants showed either a low level or absence of TRAP activity. Most MNGCs from the composite implants exhibited a low level of TRAP activity; however, there was a population of cells that demonstrated a high level of reaction product, similar to that seen in the tibia and demineralized implant. Morphologically, osteoclasts from the proximal tibia and from the osteogenic demineralized implant exhibited ruffled borders. A small population of MNGCs from the composite implant also revealed osteoclastic features. In summary, MNGCs from the mineralized implant did not exhibit a level of TRAP reaction product or morphology similar to osteoclasts, while the majority of cells from the demineralized implant and a subpopulation of the MNGCs elicited by the composite implant did demonstrate TRAP expression and morphology similar to osteoclasts. The expression of osteoclastic characteristics in cells at an ectopic site may be dependent on accessory signals from the skeletal microenvironment; such signals appear to be absent from or incomplete in the mineralized implants but appear to be present when demineralized bone particles are implanted.     

Transformation of the viscoelastic functions of calcified tissues and interfacial biomaterials into a common representation

Since both connective and calcified tissues are markedly viscoelastic in nature, an understanding of the behavior of these tissues intrinsically as materials on their own, as well as in composite formation with synthetic implants, is of prime importance in order to predict and anticipate materials' design and function. Thus considerable interest has developed in recent years with respect to measurements of the viscoelastic properties of biological materials. However, attempts to characterize the viscoelasticity of calcified tissues have involved many different experimental procedures; hence results appear in terms of different functions, e.g. relaxation modulus, creep compliance. Since this diversity precludes a simple useful comparison of the results, the present study was initiated so that measured functions could be cast into a common representation, and thus compared. Linear viscoelasticity theory implies definite exact relationships between the functions. Using these relations, experimental results on bone, dentin and implant materials presently used to interface to the natural tissues, e.g. polymethyl methacrylate and high density linear polyethylene, were transformed into the complex dynamic modulus representation. Analysis shows that the results of experiments on bone are not in agreement as to dispersion (i.e. change of modulus with frequency) and its variation with strain. Further, analysis of the internal consistency of some experiments demonstrates a violation of the Boltzmann integral which indicates that linear viscoelasticity (almost invariably assumed by workers in the field) fails for bone in compression. It is concluded that the dynamic behavior of bone is not as well understood as has been thought heretofore; direction is given for future experiments. Contribution No. 59 from the Laboratory for Crystallographic Biophysics; supported by USPHS through NIDR Grant Number 5T1-DE-117-10.     

Influence of the stiffness of bone defect implants on the mechanical conditions at the interface--a finite element analysis with contact

The study focused on the influence of the implant material stiffness on stress distribution and micromotion at the interface of bone defect implants. We hypothesized that a low-stiffness implant with a modulus closer to that of the surrounding trabecular bone would yield a more homogeneous stress distribution and less micromotion at the interface with the bony bed. To prove this hypothesis we generated a three-dimensional, non-linear, anisotropic finite element (FE) model. The FE model corresponded to a previously developed animal model in sheep. A prismatic implant filled a standardized defect in the load-bearing area of the trabecular bone beneath the tibial plateau. The interface was described by face-to-face contact elements, which allow press fits, friction, sliding, and gapping. We assumed a physiological load condition and calculated contact pressures, shear stresses, and shear movements at the interface for two implants of different stiffness (titanium: E=110GPa; composite: E=2.2GPa). The FE model showed that the stress distribution was more homogeneous for the low-stiffness implant. The maximum pressure for the composite implant (2.1 MPa) was lower than for the titanium implant (5.6 MPa). Contrary to our hypothesis, we found more micromotion for the composite (up to 6 microm) than for the titanium implant (up to 4.5 microm). However, for both implants peak stresses and micromotion were in a range that predicts adequate conditions for the osseointegration. This was confirmed by the histological results from the animal studies.     

过盈植入对种植体-骨界面应力分布影响的三维有限元分析（英文）

目的：应用三维有限元分析法研究牙种植体过盈植入对种植体-骨界面接触压力的影响。方法：选择直径为3.3 mm的ITI种植体和成人离体下颌骨,模拟种植体植入下颌骨内,过盈量为0.5 mm,建立三维有限元模型,应用ANSYS软件分析种植体-骨界面的应力分布情况。结果：种植体周围骨最大应力为48.796 MPa,应力分布均匀。种植体所受应力主要集中于颈部,最大应力值为87.832 MPa。结论：过盈量为0.5 mm时,种植体-骨界面所产生的应力值在骨组织所能承受的最大应力值范围内,种植体所受到的应力值远远小于钛的屈服强度,从生物力学角度,周围骨所受应力在骨组织能够承受范围,种植体也不会断裂,过盈联结在临床种植时有其可行性。     

Prognosis of implant longevity in terms of annual bone loss: a methodological finite element study

Dental implant failure is mainly the consequence of bone loss at peri-implant area. It usually begins in crestal bone. Due to this gradual loss, implants cannot withstand functional force without bone overload, which promotes complementary loss. As a result, implant lifetime is significantly decreased. To estimate implant success prognosis, taking into account 0.2 mm annual bone loss for successful implantation, ultimate occlusal forces for the range of commercial cylindrical implants were determined and changes of the force value for each implant due to gradual bone loss were studied. For this purpose, finite element method was applied and von Mises stresses in implant–bone interface under 118.2 N functional occlusal load were calculated. Geometrical models of mandible segment, which corresponded to Type II bone (Lekholm & Zarb classification), were generated from computed tomography images. The models were analyzed both for completely and partially osseointegrated implants (bone loss simulation). The ultimate value of occlusal load, which generated 100 MPa von Mises stresses in the critical point of adjacent bone, was calculated for each implant. To estimate longevity of implants, ultimate occlusal loads were correlated with an experimentally measured 275 N occlusal load (Mericske-Stern & Zarb). These findings generally provide prediction of dental implants success.     

Coating with a Modular Bone Morphogenetic Peptide Promotes Healing of a Bone-Implant Gap in an Ovine Model

Despite the potential for growth factor delivery strategies to promote orthopedic implant healing, there is a need for growth factor delivery methods that are controllable and amenable to clinical translation. We have developed a modular bone growth factor, herein termed “modular bone morphogenetic peptide (mBMP)”, which was designed to efficiently bind to the surface of orthopedic implants and also stimulate new bone formation. The purpose of this study was to coat a hydroxyapatite-titanium implant with mBMP and evaluate bone healing across a bone-implant gap in the sheep femoral condyle. The mBMP molecules efficiently bound to a hydroxyapatite-titanium implant and 64% of the initially bound mBMP molecules were released in a sustained manner over 28 days. The results demonstrated that the mBMP-coated implant group had significantly more mineralized bone filling in the implant-bone gap than the control group in C-arm computed tomography (DynaCT) scanning (25% more), histological (35% more) and microradiographic images (50% more). Push-out stiffness of the mBMP group was nearly 40% greater than that of control group whereas peak force did not show a significant difference. The results of this study demonstrated that mBMP coated on a hydroxyapatite-titanium implant stimulates new bone formation and may be useful to improve implant fixation in total joint arthroplasty applications.     

Stress transfer properties of different commercial dental implants: a finite element study

Dental implantology has high success rates, and a suitable estimation of how stresses are transferred to the surrounding bone sheds insight into the correct design of implant features. In this study, we estimate stress transfer properties of four commercial implants (GMI, Lifecore, Intri and Avinent) that differ significantly in macroscopic geometry. Detailed three-dimensional finite element models were adopted to analyse the behaviour of the bone-implant system depending on the geometry of the implant (two different diameters) and the bone-implant interface condition. Occlusal static forces were applied and their effects on the bone, implant and bone-implant interface were evaluated. Large diameters avoided overload-induced bone resorption. Higher stresses were obtained with a debonded bone-implant interface. Relative micromotions at the bone-implant interface were within the limits required to achieve a good osseointegration. We anticipate that the methodology proposed may be a useful tool for a quantitative and qualitative comparison between different commercial dental implants.     

运用偏振光观测聚乙烯磨屑颗粒诱导人工关节假体无菌性松动

目的:探讨运用偏振光显微镜来观测无菌性松动人工关节假体周围的聚乙烯颗粒分布,评估其在研究磨屑颗粒诱导假体无菌性松动机制及防治措施等实验研究中的可行性。方法:我们用雌性新西兰大白兔建立动物模型,在左侧胫骨髓腔内植入羟基磷灰石(hydroxyapatite,HA)涂层假体。并分别于假体表面和膝关节腔内植入0.5×107超高分子量聚乙烯(Ultra-high molecular weight polyethylene,UHMWPE)颗粒。术后行四环素荧光双标记。膝关节滑膜组织苏木精-伊红(hematoxylin-eosin,HE)染色、骨组织改良丽春红染色后分别用普通光镜和偏振光镜观察,未染色的骨组织行荧光显微镜和偏振光镜观察。结果:在聚乙烯颗粒刺激下,膝关节滑膜组织增生明显,骨-假体结合差,假体周围骨小梁稀疏,偏振光显微镜可清晰显示双折光性的聚乙烯颗粒在膝关节分布于滑膜及其深层结缔组织中,在骨-假体间隙间大量充填,阻碍骨-假体整合。结论:运用偏振光显微镜可以清晰而简便地观察滑膜和假体周围的聚乙烯颗粒分布,与传统实验方法相比,更加直观、简便和经济。     

Modulation of bone ingrowth of rabbit femur titanium implants by in vivo axial micromechanical loading.

Titanium implants commonly used in orthopedics and dentistry integrate into host bone by a complex and coordinated process. Despite increasingly well illustrated molecular healing processes, mechanical modulation of implant bone ingrowth is poorly understood. The objective of the present study was to determine whether micromechanical forces applied axially to titanium implants modulate bone ingrowth surrounding intraosseous titanium implants. We hypothesized that small doses of micromechanical forces delivered daily to the bone-implant interface enhance implant bone ingrowth. Small titanium implants were placed transcortically in the lateral aspect of the proximal femur in 15 New Zealand White rabbits under general anesthesia and allowed to integrate with the surrounding bone for 6 wk. Micromechanical forces at 200 mN and 1 Hz were delivered axially to the right femur implants for 10 min/day over 12 consecutive days, whereas the left femur implants served as controls. The average bone volume 1 mm from mechanically loaded implants (n = 15) was 73 +/- 12%, which was significantly greater than the average bone volume (52 +/- 21%) of the contralateral controls (n = 15) (P < 0.01). The average number of osteoblast-like cells per endocortical bone surface was 55 +/- 8 cells/mm(2) for mechanically loaded implants, which was significantly greater than the contralateral controls (35 +/- 6 cells/mm(2)) (P < 0.01). Dynamic histomorphometry showed a significant increase in mineral apposition rate and bone-formation rate of mechanically stressed implants (3.8 +/- 1.2 microm/day and 2.4 +/- 1.0 microm(3).microm(-2).day(-1), respectively) than contralateral controls (2.2 +/- 0.92 microm/day and 1.2 +/- 0.60 microm(3).microm(-2).day(-1), respectively; P < 0.01). Collectively, these data suggest that micromechanical forces delivered axially on intraosseous titanium implants may have anabolic effects on implant bone ingrowth.     

Multiscale design and multiobjective optimization of orthopedic hip implants with functionally graded cellular material

Revision surgeries of total hip arthroplasty are often caused by a deficient structural compatibility of the implant. Two main culprits, among others, are bone-implant interface instability and bone resorption. To address these issues, in this paper we propose a novel type of implant, which, in contrast to current hip replacement implants made of either a fully solid or a foam material, consists of a lattice microstructure with nonhomogeneous distribution of material properties. A methodology based on multiscale mechanics and design optimization is introduced to synthesize a graded cellular implant that can minimize concurrently bone resorption and implant interface failure. The procedure is applied to the design of a 2D left implanted femur with optimized gradients of relative density. To assess the manufacturability of the graded cellular microstructure, a proof-of-concept is fabricated by using rapid prototyping. The results from the analysis are used to compare the optimized cellular implant with a fully dense titanium implant and a homogeneous foam implant with a relative density of 50%. The bone resorption and the maximum value of interface stress of the cellular implant are found to be over 70% and 50% less than the titanium implant while being 53% and 65% less than the foam implant.     

Simultaneous Histological Preparation of Bone, Soft Tissue and Implanted Biomaterials for Light Microscopic Observations

Block specimens of formalin fixed bone, soft tissue and endosseous implanted biomaterials can be successfully embedded in polymethyl methacrylate by employing vacuum desiccation during the dehydration steps and refrigeration during the infiltration step. One-hundred-micrometer histological sections can be obtained from the cured polymethyl methacrylate blocks by cutting with a low concentration diamond wafering blade on a Buehler Isomet Circular Low Speed Saw using Buehler Isocut fluid. The sections can be readily stained and details of individual cells studied by light microscopy, thus allowing interpretation of the relationship between biomaterial and surrounding tissues. The advantage of this method is that it allows observation of the entire specimen in situ. The details of the procedure are presented.     

Silver Methenamine Staining for Scanning Electron Microscopy of Bone Sections Containing Biomaterials

Sections of tissue containing orthopedic materials are currently used to study the compatibility of those materials and to perform electron probe microanalysis at the material-tissue interface. Identification of the cells in contact with the material by Scanning electron microscopy (SEM) is of interest. We have developed a method for staining cells and tissue structures embedded in polymethyl methacrylate with silver methenamine once the sections have been obtained. Sections were prepared by grinding, and the silver methenamine was applied after oxidation with periodic acid. The procedure was carried out in a microwave oven. Backscatter SEM showed staining of the cell nucleus membrane, chromatin, the nuclear organizers, and the chromosomes of dividing cells. The cytoplasm and the cytoplasmic membrane were also stained. Collagen fibers of the extracellular matrix and the mineralized matrix of bone were labeled. Material particles in the macrophages were easily recognizable and Energy-Dispersive Spectrometer were not impaired by the presence of silver in the preparation.     

Implementation of contact definitions calculated by FEA to describe the healing process of basal implants

Aims: Bone structure around basal implants shows a dual healing mode: direct contact areas manifest primary osteonal remodeling, in the void osteotomy-induced spaces, the repair begins with woven bone formation. This woven bone is later converted into osteonal bone. The purpose of this study was to develop a model to accurately represent the interface between bone and basal implant throughout the healing process. The model was applied to the biological scenario of changing load distribution in a basal implant system over time. Methods: Computations were made through finite element analysis using multiple models with changing boneimplant contact definitions which reflected the dynamic nature of the interface throughout the bony healing process. Five stages of bony healing were calculated taking into account the changes in mineral content of bone in the vicinity of the load transmitting implant surfaces. Results: As the bony integration of basal implants proceeds during healing, peak stresses within the metal structure shift geographically. While bony repair may still weaken osteonal bone, woven bone has already matured. This leads to changes in the load distribution between and within the direct contact areas, and bone areas which make later contact with implant. Conclusions: This study shows that basal implants undergo an intrinsic shift of maximum stress regions during osseointegration. Fatigue testing methods in the case of basal implants must therefore take into account this gradual shift from early healing phase until full osseointegration is achieved.     

A paradigm for the development and evaluation of novel implant topologies for bone fixation: Implant design and fabrication

The future development of bio-integrated devices will improve the functionality of robotic prosthetic limbs. A critical step in the advancement of bio-integrated prostheses will be establishing long-term, secure fixation to the remnant bone. To overcome limitations associated with contemporary bone-anchored prosthetic limbs, we established a paradigm for developing and fabricating novel orthopedic implants undergoing specified loading. A topology optimization scheme was utilized to generate optimal implant macrostructures that minimize deformations near the bone-implant interface. Variations in implant characteristics and interfacial connectivity were investigated to examine how these variables influence the layout of the optimized implant. For enhanced tissue integration, the optimally designed macroscopic geometry of a titanium (Ti)-alloy implant was further modified by introducing optimized microstructures. The complex geometries of selected implants were successfully fabricated using selective laser sintering (SLS) technology. Fabrication accuracy was assessed by comparing volumes and cross-sectional areas of fabricated implants to CAD data. The error of fabricated volume to CAD design volume was less than 8% and differences in cross sectional areas between SEM images of fabricated implants and corresponding cross sections from CAD design were on average less than 9%. We have demonstrated that this computational design method, combined with solid freeform fabrication techniques, provides a versatile way to develop novel orthopedic implants.     

Importance of diameter-to-length ratio in selecting dental implants: a methodological finite element study

Implant dimensions greatly influence load transfer characteristics and the lifetime of a dental system. Excessive stresses at peri-implant area may result in bone failure. Finding the critical point at the implant–bone interface and evaluating the influence of implant diameter-to-length ratio on adjacent bone stresses makes it possible to select implant dimensions. For this, different cylindrical implants were numerically analysed using geometrical models generated from computed tomography images of mandible with osseointegrated implants. All materials were assumed to be linearly elastic and isotropic. Masticatory load was applied in its natural direction, oblique to occlusal plane. Maximum von Mises stresses were located around the implant neck at the critical point of its intersection with the plane of loading and were functions of implant diameter-to-length ratio. It was demonstrated that there exists a certain spectrum of diameter-to-length ratios, which will keep maximum bone stresses at a preset level chosen in accordance with patient's bone strength.     

Three-dimensional numerical simulation of stress induced by different lengths of osseointegrated implants in the anterior maxilla

Lower survival rates were observed for the implant placed in the anterior maxilla. The purpose of this study was to investigate the influence of different implant lengths on the stress distribution around osseointegrated implants under a static loading condition in the anterior maxilla using a three-dimensional finite element analysis. The diameter of 4.0 mm external type implants of different lengths (8.5 mm, 10.0 mm, 11.5 mm, 13.0 mm, 15.0 mm) was used in this study. The anterior maxilla was assumed to be D3 bone quality. All the material was assumed to be homogenous, isotropic and linearly elastic. The implant–bone interface was constructed using a rigid element for simulating the osseointegrated condition. Then, 176 N of static force was applied on the middle of the palatoincisal line angle of the abutment at a 120°angle to the long axis of abutment. The von Mises stress value was measured with an interval of 0.25 mm along the bone–implant interface. Incremental increase in implant length causes a gradual reduction of maximum and average von Mises stress at the labial portion within the implant. In the bone, higher stress was concentrated within cortical bone area and more distributed at the labial cortex, while cancellous bone showed relatively low stress concentration and even distribution. An increase in implant length reduced stress gradients at the cortical peri-implant region. Implant length affects the mechanisms of load transmission to the osseointegrated implant. On the basis of this study the biomechanical stress-based performance of implants placed in the anterior maxilla improves when using longer implants.     

Influence of interface condition and implant design on bone remodelling and failure risk for the resurfaced femoral head

Resurfacing of the femur has experienced a revival, particularly in younger and more active patients. The implant is generally cemented onto the reamed trabecular bone and theoretical remodelling for this configuration, as well as uncemented variations, has been studied with relation to component positioning for the most common designs. The purpose of this study was to investigate the influence of different interface conditions, for alternative interior implant geometries, on bone strains in comparison to the native femur, and its consequent remodelling. A cylindrical interior geometry, two conical geometries and a spherical cortex-preserving design were compared with a standard implant (ASR, DePuy International, Ltd., UK), which has a 3° cone. Cemented as well as uncemented line to line and press-fit conditions were modelled for each geometry. A patient-specific finite element model of the proximal femur was used with simulated walking loads. Strain energy density was compared between the reference and resurfaced femur, and input into a remodelling algorithm to predict density changes post-operatively. The common cemented designs (cylindrical, slightly conical) had strain shielding in the superior femoral head (>35% reduction) as well as strain concentrations (strain>5%) in the neck regions near the implant rim. The cortex-preserving (spherical) and strongly conical designs showed less strain shielding. In contrast to the cemented implants, line to line implants showed a density decrease at the centre of the femoral head, while all press-fit versions showed a density increase (>100%) relative to the native femur, which suggests that uncemented press-fit implants could limit bone resorption.     

Micromechanical evaluation of mineralized multilayers

The biomechanical stability of osseointegrated implants is of particular importance, especially the stability which is achieved from structural manipulation at the interface between the implant surface and the bone tissues. Nanoscale β-tricalcium phosphate-immobilized titanium was prepared by discharge into a physiological buffered saline solution. Compared with hydroxyapatite, it has been shown to be effective in generating a bone-like chemical structure on the surface by cooperative interaction between osteoblastic cells and the β-tricalcium phosphate. The present study, after cell cultivation, investigates the nanostructures and biomechanical property differences of a mineralized layer formed on two samples of nano-calcium phosphate-immobilized titanium. A scanning probe microscope study revealed that the mineralized tissue formed on the β-tricalcium phosphate samples after 1 week of cell culture showed significantly higher roughness, compared with hydroxyapatite samples. Nanoindentation micromechanical evaluation of the in vitro generated multilayered structures exhibited thicker bone-like mineralized layers on the β-tricalcium phosphate samples. A successful modification of titanium implants through the cooperative interaction between osteoblastic cells and nano β-tricalcium phosphate is anticipated.