Rabbit Cortical Bone Research Articles

1078 Performance of Improved Forms of Demineralized Bone Fiber Allografts in Rabbit Posterolateral Spine Fusion Model

INTRODUCTION: Biologic fusion is often critical for successful spine procedures. Autograft remains the Gold Standard bone graft due to osteogenic properties but has limitations in practice. New bone graft forms that efficiently incorporate autograft or bone marrow aspirate (BMA) and provide osteoinductive and osteoconductive properties were evaluated in the current study. Grafts were made using demineralized bone fibers (DBF) produced by a patented method that provides long fibers that can effectively entangle autograft and be formed into open cylinders (PAK) that contain and prevent autograft migration and absorb BMA. METHODS: DBF was produced from rabbit cortical bone using the same processes as human clinical product. Pak grafts were made with 1.5cc Fibers formed into a cylinder with 1.5cc volume. Implants into the rabbit PLF model were harvested after 8 weeks and evaluated for fusion by manual palpation and x-rays. RESULTS: All graft forms demonstrated robust new bone formation. Each group demonstrated high fusion rates. Across the groups 10/11 levels were 100% fused (91%) with the remaining 1/11 being 95% reduced motion (functionally fused). Note radiolucency of Group 3 at post-op and appearance of new bone at 8 weeks. CONCLUSIONS: The new graft forms demonstrated robust bone formation and fusion in PLF model. The DBF Fibers provided excellent entanglement of autograft to prevent migration. The PAK form eliminated autograft migration, and absorbed BMA providing osteogenesis to the osteoconductive and osteoinductive graft.

Strontium-loaded 3D intramedullary nail titanium implant for critical-sized femoral defect in rabbits.

The treatment of critical-sized bone defects has long been a major problem for surgeons. In this study, an intramedullary nail shaped three-dimensional (3D)-printed porous titanium implant that is capable of releasing strontium ions was developed through a simple and cost-effective surface modification technique. The feasibility of this implant as a stand-alone solution was evaluated using a rabbit's segmental diaphyseal as a defect model. The strontium-loaded implant exhibited a favorable environment for cell adhesion, and mechanical properties that were commensurate with those of a rabbit's cortical bone. Radiographic, biomechanical, and histological analyses revealed a significantly higher amount of bone ingrowth and superior bone-bonding strength in the strontium-loaded implant when compared to an untreated porous titanium implant. Furthermore, one-year histological observations revealed that the strontium-loaded implant preserved the native-like diaphyseal bone structure without failure. These findings suggest that strontium-releasing 3D-printed titanium implants have the clinical potential to induce the early and efficient repair of critical-sized, load-bearing bone defects.

Understanding basic multicellular unit activity in cortical bone through 3D morphological analysis: New methods to define zones of the remodeling space

The activity of basic multicellular units (BMU) in cortical bone is classically described as a sequential order of events— resorption, reversal and formation. This simplified portrayal of the remodeling process is pervasive despite the reported variability in remodeling space morphology. These variations may reflect meaningful nuances in BMU activity but methods to quantify 3D remodeling space morphology within the context of the cellular activity are currently lacking. This study developed new techniques to define zones of BMU activity based on the 3D morphology of remodeling spaces in rabbit cortical bone and integrated morphological data with the BMU longitudinal erosion rate (LER) to elucidate the spatial-temporal coordination of BMUs and estimate mineral apposition rate (MAR). The tibiae of New Zealand white rabbits (n = 5) were imaged in vivo using synchrotron radiation and two weeks later ex vivo with desktop microCT. The in vivo and ex vivo datasets were co-registered, and 27 remodeling spaces were identified at both timepoints. A radial profile representing the 3D morphology was the platform for partitioning the remodeling spaces into resorption, reversal and formation zones. Manual, automated and semi-automated partitioning approaches were compared, and the zone-segmentations were used to calculate the length, change in radius and slope of each zone. The manual approach most accurately defined the zones of idealized remodeling spaces with known dimensions (relative error = 0.9–9.2 %) while the semi-automated method reliably defined the zones in rabbit remodeling spaces (ICC = 0.85–1.00). Combining LER and the manually derived zone dimensions indicated that a BMU passes through a cross-section in approximately 18.8 days with resorption, reversal and formation taking 4.1, 2.2, and 12.5 days, respectively. MAR estimated by the 3D analysis was not significantly different than that determined with classic histomorphometry (p = 0.48). These techniques have the potential to assess dynamic parameters of bone resorption and formation, eliminate the need for fluorochrome labeling and provide a more comprehensive perspective of the remodeling process.

Three-Dimensional Printing of Polycaprolactone/Nano-Hydroxyapatite Composite Scaffolds with a Pore Size of 300/500 µm is Histocompatible and Promotes Osteogenesis Using Rabbit Cortical Bone Marrow Stem Cells.

BACKGROUND Many patients have bone defects that exceed the healing size. This study aimed to construct polycaprolactone/nano-hydroxyapatite (PCL/nHA) composite scaffolds with different pore sizes and investigate the osteogenesis and histocompatibility of cortical bone mesenchymal stem cells (BMSCs-C) seeded on it after inoculation. MATERIAL AND METHODS After mixing PCL and nHA proportionally, three-dimensional (3D) printing was used to print scaffolds. Porosity, compressive strength, and elastic modulus of PCL/nHA scaffolds were tested. The proliferation of BMSCs-C cells was examined and osteogenesis, chondrogenesis, and adipogenesis were evaluated. BMSCs-C cells were inoculated into 3D printing scaffolds, and histocompatibility between BMSCs-C cells and scaffolds was observed by the cell count kit (CCK-8) assay and LIVE/DEAD staining. After inoculating BMSCs-C cells into scaffolds, alkaline phosphatase (ALP) activity and calcium content were measured. RESULTS There was no obvious difference in characteristics between the 3 PCL/nHA composite scaffolds. The porosity, compressive strength, and elastic modulus of the 300/500-μm scaffold were between those of the 300-μm and 500-μm scaffolds. With increasing pore size, the mechanical properties of the scaffold decrease. BMSCs-C cells demonstrated faster growth and better osteogenic, adipogenic, and chondrogenic differentiation; therefore, BMSCs-C cells were selected as seed cells. PCL/nHA composite scaffolds with different pore sizes had no obvious toxicity and demonstrated good biocompatibility. All scaffolds showed higher ALP activity and calcium content. CONCLUSIONS The 300/500 μm mixed pore size scaffold took into account the mechanical properties of the 300 μm scaffold and the cell culture area of the 500 μm scaffold, therefore, 300/500 μm scaffold is a better model for the construction of tissue engineering scaffolds.

Growth Factor Comparison in Cortical Demineralized Bone Matrix that Demineralized Using Chloric and Acetic Acid

Background: Demineralized Bone Matrix (DBM) is an alternative biomaterial that needs particular acid and a specific time to optimize the growth factors preservation. The best demineralization protocol for preserving growth factors in DBM is currently unavailable. This study aimed to investigate DBM extraction methods using different acids and times to maintain the optimum growth factors.Methods: This in vitro experimental laboratory study used a randomized controlled post-test-only group design. We characterized the IGF-1, BMP2, and TGF-β content of 1 gram of New Zealand White Rabbit cortical bone immersed in 0.6 M hydrochloric and 0.5 M acetic acid at 3, 6, and 9 days. We then analyzed the differences in growth factor levels in each acid and analyzed them statistically.Results: IGF-1 levels were higher in DBM demineralized acetic acid than hydrochloric acid. BMP-2 and TGF-β levels were higher in DBM demineralized using hydrochloric acid. The concentration of growth factors decreased with time in DBM demineralized using acetic acid. The highest growth factor level was obtained after six days of immersion in hydrochloric acid.Conclusion: The average value of IGF-1 in DBM demineralized with acetic acid was higher than in hydrochloric acid. Otherwise, BMP 2 and TGF-β were better in hydrochloric acid. We also found that the growth factor level in hydrochloric acid reached its peak level at six days and then decreased. These results suggest the importance of not over-demineralizing the bone to maintain growth factors, and further research should be conducted to improve DBM processing.

Demineralized Cortical Bone Matrix Augmented With Peripheral Blood-Derived Mesenchymal Stem Cells for Rabbit Medial Meniscal Reconstruction.

Tissue engineering is a promising treatment strategy for meniscal regeneration after meniscal injury. However, existing scaffold materials and seed cells still have many disadvantages. The objective of the present study is to explore the feasibility of peripheral blood-derived mesenchymal stem cells (PBMSCs) augmented with demineralized cortical bone matrix (DCBM) pretreated with TGF-β3 as a tissue-engineered meniscus graft and the repair effect. PBMSCs were collected from rabbit peripheral blood and subjected to three-lineage differentiation and flow cytometry identification. DCBM was prepared by decalcification, decellularization, and cross-linking rabbit cortical bone. Various characteristics such as biomechanical properties, histological characteristics, microstructure and DNA content were characterized. The cytotoxicity and the effects of DCBM on the adhesion and migration of PBMSCs were evaluated separately. The meniscus-forming ability of PBMSCs/DCBM complex in vitro induced by TGF-β3 was also evaluated at the molecular and genetic levels, respectively. Eventually, the present study evaluated the repair effect and cartilage protection effect of PBMSCs/DCBM as a meniscal graft in a rabbit model of medial meniscal reconstruction in 3 and 6 months. The results showed PBMSCs positively express CD29 and CD44, negatively express CD34 and CD45, and have three-lineage differentiation ability, thus can be used as tissue engineering meniscus seed cells. After the sample procedure, the cell and DNA contents of DCBM decreased, the tensile modulus did not decrease significantly, and the DCBM had a pore structure and no obvious cytotoxicity. PBMSCs could adhere and grow on the scaffold. Under induction of TGF-β3, PBMSCs/DCBM composites expressed glycosaminoglycan (GAG), and the related gene expression also increased. The results of the in vivo experiments that the PBMSCs/DCBM group had a better repair effect than the DCBM group and the control group at both 12 and 24 weeks, and the protective effect on cartilage was also better. Therefore, the application of DCBM augmented with PBMSCs for meniscus injury treatment is a preferred option for tissue-engineered meniscus.

An alternative ex vivo method to evaluate the osseointegration of Ti–6Al–4V alloy also combined with collagen

Due to the increasing number of orthopedic implantation surgery and advancements in biomaterial manufacturing, chemistry and topography, there is an increasing need of reliable and rapid methods for the preclinical investigation of osseointegration and bone ingrowth. Implant surface composition and topography increase osteogenicity, osteoinductivity, osteoconductivity and osseointegration of a prosthesis. Among the biomaterials used to manufacture an orthopedic prosthesis, titanium alloy (Ti–6Al–4V) is the most used. Type I collagen (COLL I) induces cell function, adhesion, differentiation and bone extracellular matrix component secretion and it is reported to improve osseointegration if immobilized on the alloy surface. The aim of the present study was to evaluate the feasibility of an alternative ex vivo model, developed by culturing rabbit cortical bone segments with Ti–6Al–4V alloy cylinders (Ti-POR), fabricated through the process of electron beam melting (EBM), to evaluate osseointegration. In addition, a comparison was made with Ti-POR coated with COLL I (Ti-POR-COLL) to evaluate osseointegration in terms of bone-to-implant contact (BIC) and new bone formation (nBAr/TAr) at 30, 60 and 90 d of culture. After 30 and 60 d of culture, BIC and nBAr/TAr resulted significantly higher in Ti-POR-COLL implants than in Ti-POR. No differences have been found at 90 d of culture. With the developed model it was possible to distinguish the biomaterial properties and behavior. This study defined and confirmed for the first time the validity of the alternative ex vivo method to evaluate osseointegration and that COLL I improves osseointegration and bone growth of Ti–6Al–4V fabricated through EBM.

Direct Assessment of Rabbit Cortical Bone Basic Multicellular Unit Longitudinal Erosion Rate: A 4D Synchrotron-Based Approach.

Cortical bone remodeling is carried out by basic multicellular units (BMUs), which couple resorption to formation. Although fluorochrome labeling has facilitated study of BMU formative parameters since the 1960s, some resorptive parameters, including the longitudinal erosion rate (LER), have remained beyond reach of direct measurement. Indeed, our only insights into this spatiotemporal parameter of BMU behavior come from classical studies that indirectly inferred LER. Here, we demonstrate a 4D in vivo method to directly measure LER through in-line phase contrast synchrotron imaging. The tibias of rabbits (n=15) dosed daily with parathyroid hormone were first imaged in vivo (synchrotron micro-CT; day 15) and then ex vivo 14 days later (conventional micro-CT; day 29). Mean LER assessed by landmarking the co-registered scans was 23.69 ± 1.73 μm/d. This novel approach holds great promise for the direct study of the spatiotemporal coordination of bone remodeling, its role in diseases such as osteoporosis, as well as related treatments. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Collagen Membrane for Guided Bone Regeneration in Dental and Orthopedic Applications.

Treatment of cortical bone defects is a clinical challenge. Guided bone regeneration (GBR), commonly used in oral and maxillofacial dental surgery, may show promise for orthopedic applications in repair of cortical bone defects. However, a limitation in the use of GBR for cortical bone defects is the lack of an ideal scaffold that provides sufficient mechanical support to bridge the cortical bone with minimal interference in the repair process. We have developed a new collagen membrane, CelGro™, for use in GBR. We report the material characterization of CelGro and evaluate the performance of CelGro in translational preclinical and clinical studies. The results show CelGro has a bilayer structure of different fiber alignment and is composed almost exclusively of type I collagen. CelGro was found to be completely acellular and free from xenoantigen, α-gal (galactose-alpha-1,3-galactose). In the preclinical study of a rabbit cortical bone defect model, CelGro demonstrated enhanced bone-remodeling activity and cortical bone healing. Microcomputed tomography evaluation showed early bony bridging over the defect area 30 days postoperatively, and nearly complete restoration of mature cortical bone at the bone defect site 60 days postoperatively. Histological analysis 60 days after surgery further confirmed that CelGro enables bridging of the cortical bone defect by induction of newly formed cortical bone. Compared to a commercially available collagen membrane, Bio-Gide®, CelGro showed much better cortical alignment and reduced porosity at the defect interface. As selection of orthopedic patients with cortical bone defects is complex, we conducted a clinical study evaluating the performance of CelGro in guided bone regeneration around dental implants. CelGro was used in GBR procedures in a total of 16 implants placed in 10 participants. Cone-beam computed tomography images show significantly increased bone formation both horizontally and vertically, which provides sufficient support to stabilize implants within 4 months. Together, the findings of our study demonstrate that CelGro is an ideal membrane for GBR not only in oral and maxillofacial reconstructive surgery but also in orthopedic applications (Clinical Trial ID ACTRN12615000027516).

Estudo comparativo da resistência mecânica da força de compressão entre biomateriais naturais, sintéticos e mistos

RESUMO: As afecções ortopédicas com perda de tecido ósseo são um desafio tanto na medicina veterinária quanto na medicina humana. Portanto, não é raro ortopedistas se depararem com fraturas cominutivas irredutíveis de ossos longos, neoplasias ósseas ou não-uniões, que necessitam de procedimentos cirúrgicos reparadores, por meio da substituição de segmento ou preenchimento de falha óssea com o uso de biomateriais. Pretende-se com esta pesquisa avaliar e comparar a resistência mecânica entre biomateriais naturais, sintéticos e mistos. Foram utilizados sete grupos experimentais compostos por seis corpos de provas cada: Grupo 1 , tecido ósseo cortical de coelho (OSSO - controle); Grupo 2, cimento ósseo (CO); Grupo 3, cimento ósseo autoclavado (COA); Grupo 4, cimento ósseo e macrofragmento ósseo cortical de cão (COMaFO); Grupo 5, cimento ósseo e macrofragmento ósseo autoclavado de cortical de cão (COMaFOA); Grupo 6, cimento ósseo e microfragmento ósseo cortical de cão (COMiFO) e Grupo 7, cimento ósseo e microfragmento ósseo cortical de cão (COMiFOA). Os corpos de prova foram submetidos a ensaios mecânicos de compressão axial controlada em máquina universal de ensaio Emic®. O teste era interrompido quando ocorria queda brusca na curva do gráfico indicando falência da amostra. Em relação à Força máxima, os grupos COA, COMaFOA e COMiFOA não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COMaFO e COMiFO diferiram estatisticamente do grupo controle (OSSO; p<0,01). Quanto a rigidez relativa, os grupos COMaFOA e COMiFOA não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COA, COMaFO e COMiFO diferiram estatisticamente do grupo controle (OSSO; p<0,01). Comparando a deformação, os grupos COMaFo, COMaFOA e COMiFO não diferiram estatisticamente do grupo controle (OSSO; p>0,01). Já os grupos CO, COA e COMiFOA diferiram estatisticamente do grupo controle (OSSO; p<0,01). Conclui-se que apenas os grupos COMaFOA e COMiFOA apresentaram propriedades mecânicas muito semelhantes às do grupo controle (OSSO). Por isso, devido a essas características, esses dois biomateriais (COMaFOA e COMiFOA) seriam os mais indicados como substitutos na reparação de falhas ósseas.

The Effect of Plasma Electrolytic Oxidation Parameters on Coating Morphology and Corrosion Behavior

Zirconium (Zr) and Zr-based materials have been widely used in biomedical and dental applications. Although these materials have better chemical and mechanical properties compared to titanium-based implants for biomedical applications, their poor bioactivity is still a challenge that needs to be overcome in order to increase the cell attachment properties [1-3]. In this study, the surface of Zr substrates was modified by plasma electrolytic oxidation (PEO) to induce a significant changing the surface chemistry and topology and thus increase bioactivity. The PEO processes were conducted at different current frequencies ranging from 80 to 420 Hz in a 30 g/l calcium acetate monohydrate (CA), 8 g/l β-glycerophosphate disodium salt pentahydrate (β-GP) and 2 g/l KOH containing electrolyte solution. The phase and chemical composition, as well as the morphology of the coatings were investigated by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and optical surface profilometry (OSP) techniques. The corrosion performance of the fabricated coatings, which is also crucial for biomedical applications, were evaluated. The results revealed that the current frequency had a profound influence on the microstructure and the chemical composition of the fabricated coatings. The SEM and OSP experiments revealed that the increase in the current frequency resulted in rougher surface coatings with bigger pores. The corrosion behaviour of each samples conducted in simulated body fluid (SBF) at the temperature of 36.5 °C will be presented. References 1- V. Sollazzo, F. Pezzetti, A. Scarano, A. Piattelli, C.A. Bignozzi, L. Massari, G. Brunelli, F. Carinci, Zirconium oxide coating improves implant osseointegration in vivo, Dent Mater, 24 (2008) 357-361. 2- P. Thomsen, C. Larsson, L.E. Ericson, L. Sennerby, J. Lausmaa, B. Kasemo, Structure of the interface between rabbit cortical bone and implants of gold, zirconium and titanium, J Mater Sci-Mater M, 8 (1997) 653-665.3- Y.Y. Yan, Y. Han, Structure and bioactivity of micro-arc oxidized zirconia films, Surf Coat Tech, 201 (2007) 5692-5695.

Evaluation of the 3D spatial distribution of the Calcium/Phosphorus ratio in bone using computed-tomography dual-energy analysis

PurposeThe Calcium/Phosphorus (Ca/P) ratio was shown to vary between healthy bones and bones with osteoporotic symptoms. The relation of the Ca/P ratio to bone quality remains under investigation. To study this relation and determine if the ratio can be used to predict bone fractures, a non-invasive 3D imaging technique is required. The first aim of this study was to test the effectiveness of a computed-tomography dual-energy analysis (CT-DEA) technique developed to assess the Ca/P ratio in bone apatite (collagen-free bone) in identifying differences between healthy and inflammation-mediated osteoporotic (IMO) bones. The second aim was to extend the above technique for its application to a more complex structure, intact bone, that could potentially lead to clinical use. MethodsFor the first aim, healthy and IMO rabbit cortical bone apatite samples were assessed. For the second aim, some changes were made to the technique, which was applied to healthy and IMO intact bone samples. ResultsStatistically significant differences between healthy and IMO bone apatite were found for the bulk Ca/P ratio, low Ca/P ratio proportion and interconnected low Ca/P ratio proportion. For the intact bone samples, the bulk Ca/P ratio was found to be significantly different between healthy and IMO. ConclusionsResults show that the CT-DEA technique can be used to identify differences in the Ca/P ratio between healthy and osteoporotic, in both bone apatite and intact bone. With quantitative imaging becoming an increasingly important advancement in medical imaging, CT-DEA for bone decomposition could potentially have several applications.

Which is the best method of sterilization for recycled bone autograft in limb salvage surgery: a radiological, biomechanical and histopathological study in rabbit.

BackgroundLimb salvage surgery is a treatment of choice for sarcomas of the extremities. One of the options in skeletal reconstruction after tumour resection is by using a recycled bone autograft. The present accepted methods of recycling bone autografts include autoclaving, pasteurization and irradiation. At the moment there is lack of studies that compare the effectiveness of various sterilization methods used for recycling bone autografts and their effects in terms of bone incorporation. This study was performed to determine the effects of different methods of sterilization on bone autografts in rabbit by radiological, biomechanical and histopathological evaluations.MethodsFresh rabbit cortical bone is harvested from the tibial diaphysis and sterilized extracorporeally by pasteurization (n = 6), autoclaving (n = 6), irradiation (n = 6) and normal saline as control group (n = 6). The cortical bones were immediately reimplanted after the sterilization process. The subsequent process of graft incorporation was examined over a period of 12 weeks by serial radiographs, biomechanical and histopathological evaluations. Statistical analysis (ANOVA) was performed on these results. Significance level (α) and power (β) were set to 0.05 and 0.90, respectively.ResultsRadiographic analysis showed that irradiation group has higher score in bony union compared to other sterilization groups (p = 0.041). ANOVA analysis of ‘failure stress’, ‘modulus’ and ‘strain to failure’ demonstrated no significant differences (p = 0.389) between treated and untreated specimens under mechanical loading. In macroscopic histopathological analysis, the irradiated group has the highest percentage of bony union (91.7 percent). However in microscopic analysis of union, the pasteurization group has significantly higher score (p = 0.041) in callus formation, osteocytes percentage and bone marrow cellularity at the end of the study indicating good union potential.ConclusionsThis experimental study shown that both irradiation and pasteurization techniques have more favourable outcome in terms of bony union based on radiographic and histopathological evaluations. Autoclaving has the worst outcome. These results indicate that extracorporeal irradiation or pasteurization of bone autografts, are viable option for recycling bone autografts. However, pasteurization has the best overall outcomes because of its osteocytes preservation and bone marrow cellularity.

The effect of sterilization on the dynamic mechanical properties of paired rabbit cortical bone

The optimal sterilization method for load bearing allografts remains a clinical concern. Recently, supercritical carbon dioxide (SCCO2) treatments have been shown to be capable of terminally sterilizing a range of bacteria and viruses, while preserving the static mechanical properties of cortical bone. This study evaluated the effect of SCCO2 treatment compared with two doses of gamma irradiation, on clinically relevant dynamic mechanical properties of cortical bone. Quasi-static testing was also performed to compare the impairment of treatment. Whole paired adult rabbit humeri were dissected and randomly assigned into either SCCO2 Control, SCCO2 Additive or gamma irradiation at 10 or 25kGy treatment groups. The bones were treated and mechanically tested in three-point bending, with the lefts acting as controls for the treated rights. Maximum load, energy to failure and stiffness were evaluated from static tests. The number of cycles to failure was determined for fatigue at 6–60% of the ultimate load. This study found that SCCO2 treatment with or without additive did not alter static or dynamic mechanical properties. Gamma irradiation had a deleterious dose dependent effect, with statistically significant (p<0.05) reductions in all static mechanical parameters at 25kGy. This effect was increased in fatigue with statistically significant decreases in both the 10 and 25kGy dose groups. This study highlights the expediency of SCCO2 treatment for load bearing bone allograft processing as terminal sterilization can be achieved while maintaining both the quasi-static and dynamic mechanical properties of the graft.

Validation of a finite element model of a unilateral external fixator in a rabbit tibia defect model

In case of large segmental defects in load-bearing bones, an external fixator is used to provide mechanical stability to the defect site. The overall stiffness of the bone–fixator system is determined not only by the fixator design but also by the way the fixator is mounted to the bone. This stiffness is an important factor as it will influence the biomechanical environment to which tissue engineering scaffolds and regenerating tissues are exposed. A finite element (FE) model can be used to predict the system stiffness. The goal of this study is to develop and validate a 3D anatomical FE model of a bone–fixator system which includes a previously developed unilateral external fixator for a large segmental defect model in the rabbit tibia. It was hypothesized that the contact interfaces between bone and fixator screws play a major role for the prediction of the stiffness. In vitro mechanical testing was performed in order to measure the axial stiffness of cortical bone from mid-shaft rabbit tibiae and of the tibia–fixator system, as well as the bending stiffness of individual fixator screws, inserted in bone. μCT-based case-specific FE models of cortical bone and SCREW–BONE specimens were created to simulate the corresponding mechanical test set-ups. The Young's modulus of rabbit cortical bone as well as appropriate screw–bone contact settings were derived from those FE models. We then used the derived settings in an FE model of the tibia–fixator system. The difference between the FE predicted and measured axial stiffness of the tibia–fixator system was reduced from 117.93% to 7.85% by applying appropriate screw–bone contact settings. In conclusion, this study shows the importance of screw–bone contact settings for an accurate fixator stiffness prediction. The validated FE model can further be used as a tool for virtual mechanical testing in the design phase of new tissue engineering scaffolds and/or novel patient-specific external fixation devices.

The cast imaging of the osteon lacunar‐canalicular system and the implications with functional models of intracanalicular flow

A casting technique with methyl-methacrylate (MMA) was applied to the study of the osteon lacunar-canalicular network of human and rabbit cortical bone. The MMA monomer infiltration inside the vascular canals and from these into the lacunar-canalicular system was driven by capillarity, helped by evaporation and the resulting negative pressure in a system of small pipes. There was uniform, centrifugal penetration of the resin inside some osteons, but this was limited to a depth of four to five layers of lacunae. Moreover, not all of the osteon population was infiltrated. This failure can be the result of one of two factors: the incomplete removal of organic debris from the canal and canalicular systems, and lack of drainage at the osteon external border. These data suggest that each secondary osteon is a closed system with a peripheral barrier (represented by the reversal line). As the resin advances into the osteon, the air contained inside the canalicula is compressed and its pressure increases until infiltration is stopped. The casts gave a reliable visualization of the lacunar shape, position and connections between the lacunae without the need for manipulations such as cutting or sawing. Two systems of canalicula could be distinguished, the equatorial, which connected the lacunae (therefore the osteocytes) lying on the same concentric level, and the radial, which established connections between different levels. The equatorial canalicula radiated from the lacunar border forming ramifications on a planar surface around the lacuna, whereas the radial canalicula had a predominantly straight direction perpendicular to the equatorial plane. The mean length of the radial canalicula was 40.12 ± 10.26 μm in rabbits and 38.4 ± 7.35 μm in human osteons; their mean diameter was 174.4 ± 71.12 nm and 195.7 ± 79.58 nm, respectively. The mean equatorial canalicula diameter was 237 ± 66.04 nm in rabbit and 249.7 ± 73.78 nm in human bones, both significantly larger (P < 0.001) than the radial. There were no significant differences between the two species. The lacunar surface measured on the equatorial plane was higher in rabbit than in man, but the difference was not statistically significant. The cast of the lacunar-canalicular network obtained with the reported technique allows a direct, 3-D representation of the system architecture and illustrates how the connections between osteocytes are organized. The comparison with models derived by the assumption of the role of hydraulic conductance and other mechanistic functions provides descriptive, morphological data to the ongoing discussion on the Haversian system biology.

Age‐related changes in organization and content of the collagen matrix in rabbit cortical bone

The organization and composition of the collagen matrix of cortical bone changes as the bone matures due to growth and mechanical loading. We aimed to investigate the composition and organization of the collagen matrix in rabbit cortical bone during maturation using Fourier transform infrared (FTIR) microspectroscopy and polarized light microscopy (PLM). FTIR and PLM findings were compared to biochemical analysis from an earlier study. Mid-diaphyseal samples from left femora of female New Zealand White rabbits were used. The animal age ranged from newborn to 18-month old (5 age groups, n = 10 per group). The bones had earlier been decalcified and evaluated with biochemistry. In this study, collagen content, orientation, collagen cross-linking and spatial heterogeneity of all parameters was evaluated. Similar results were obtained when collagen content was evaluated with FTIR and PLM compared to the collagen content assessed with BA. Collagen content, orientation and collagen maturity increased significantly until the age of 3 months and remained similar thereafter. Simultaneously, spatial heterogeneity of the measured parameters decreased. Based on these findings, it seems that the collagen matrix of rabbit bone attains its mature state around 3 months of age, which is before the overall skeletal maturity is reached.

Comparison between Infrared and Raman Spectroscopic Analysis of Maturing Rabbit Cortical Bone

The molecular composition of the organic and inorganic matrices of bone undergoes alterations during maturation. The aim of this study was to compare Fourier transform infrared (FT-IR) and near-infrared (NIR) Raman microspectroscopy techniques for characterization of the composition of growing and developing bone from young to skeletally mature rabbits. Moreover, the specificity and differences of the techniques for determining bone composition were clarified. The humeri of female New Zealand White rabbits, with age range from young to skeletally mature animals (four age groups, n = 7 per group), were studied. Spectral peak areas, intensities, and ratios related to organic and inorganic matrices of bone were analyzed and compared between the age groups and between FT-IR and Raman microspectroscopic techniques. Specifically, the degree of mineralization, type-B carbonate substitution, crystallinity of hydroxyapatite (HA), mineral content, and collagen maturity were examined. Significant changes during maturation were observed in various compositional parameters with one or both techniques. Overall, the compositional parameters calculated from the Raman spectra correlated with analogous parameters calculated from the IR spectra. Collagen cross-linking (XLR), as determined through peak fitting and directly from the IR spectra, were highly correlated. The mineral/matrix ratio in the Raman spectra was evaluated with multiple different peaks representing the organic matrix. The results showed high correlation with each other. After comparison with the bone mineral density (BMD) values from micro-computed tomography (micro-CT) imaging measurements and crystal size from XRD measurements, it is suggested that Raman microspectroscopy is more sensitive than FT-IR microspectroscopy for the inorganic matrix of the bone. In the literature, similar spectroscopic parameters obtained with FT-IR and NIR Raman microspectroscopic techniques are often compared. According to the present results, however, caution is required when performing this kind of comparison.

Increased bone apposition on a titanium oxide surface incorporating phosphate and strontium

This study investigated the osteoconductivity of titanium (Ti) implants with a phosphate (P)- and strontium (Sr) ion-incorporated oxide surface, produced by hydrothermal treatment in the rabbit cortical and cancellous bone, for future biomedical applications as a biocompatible endosseous implant surface. The P- and Sr ion-incorporated Ti implants (P/Sr implant) were produced by hydrothermal treatment using a P- and Sr-containing solution. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy and optical profilometry. Forty screw implants (20 control and 20 experimental) were placed in the tibiae and femoral condyles of 10 New Zealand White rabbits. The surface in vivo osteoconductivity of the P/Sr implants was compared with micro-arc oxidized (TO) implants with surface calcium and P chemistry by histomorphometric analysis in the cortical and cancellous bone after 4 weeks of implantation. The P/Sr implants showed moderately rough surface features and had lower R(a) values than the TO implants. Histologically, more direct bone apposition was observed on the surface of the P/Sr implants. The P/Sr implants displayed significantly higher bone-to-implant contact percentages compared with the TO implant in both the tibiae and the femoral condyles (P<0.01). The results indicate that the hydrothermally produced P- and Sr ion-incorporated Ti oxide surface may be effective in improving implant osseointegration in both cortical and cancellous bone by increasing bone apposition, due to its surface properties combining micro-topography, P/Sr chemistry and superior wettability.

Collagen and mineral deposition in rabbit cortical bone during maturation and growth: Effects on tissue properties

We characterized the composition and mechanical properties of cortical bone during maturation and growth and in adult life in the rabbit. We hypothesized that the collagen network develops earlier than the mineralized matrix. Growth was monitored, and the rabbits were euthanized at birth (newborn), and at 1, 3, 6, 9, and 18 months of age. The collagen network was assessed biochemically (collagen content, enzymatic and non-enzymatic cross-links) in specimens from the mid-diaphysis of the tibia and femur and biomechanically (tensile testing) from decalcified whole tibia specimens. The mineralized matrix was analyzed using pQCT and 3-point bend tests from intact femur specimens. The collagen content and the Young's modulus of the collagen matrix increased significantly until the rabbits were 3 months old, and thereafter remained stable. The amount of HP and LP collagen cross-links increased continuously from newborn to 18 months of age, whereas PEN cross-links increased after 6 months of age. Bone mineral density and the Young's modulus of the mineralized bone increased until the rabbits were at least 6 months old. We concluded that substantial changes take place during the normal process of development in both the biochemical and biomechanical properties of rabbit cortical bone. In cortical bone, the collagen network reaches its mature composition and mechanical strength prior to the mineralized matrix.