Natural Cancellous Bone Research Articles

J0320505 骨組織工学用複合系多孔質材料の創製と評価([J032-05]ポリマー・セラミックス・メタル多孔質材料の開発と応用(5),材料力学部門)

In bone tissue engineering, porous composite materials of biopolymers and bioactive ceramics have been planned to be utilized as scaffolds for tissue regeneration with stem cells. Continuous porous structure is one of the key factors for scaffolds in order to ensure cell growth and formation of extra-cellular matrix (ECM) inside the scaffolds. In the present study, different kinds of porous composite scaffolds were developed for bone tissue engineering applications and their mechanical and biological properties were examined. For example, β-TCP particle filled collagen sponge exhibited an excellent biocompatibility with mesenchymal stem cells although its compressive properties were quite low compared to natural cancellous bone. On the contrary, sintered porous bioceramic structures with introduction of secondary polymer phase possessed better mechanical properties with proper biocompatibility. A novel porous scaffold with drug release function was also successfully developed to activate regeneration of bone. In this paper, the fabrication methods and the fundamental properties of those porous composite materials developed for bone regeneration are summarized.

Anabolic potential of bone mineral in human periosteal fibroblasts using steroid markers of healing

A deproteinized natural cancellous bone mineral (B) was studied in a cell culture model for its anabolic potential using two radiolabelled steroid substrates, 14C-testosterone (14C-T) and 14C-4-androstenedione (14C-4-A) independently; in the presence or absence of the anti-androgen finasteride (F) and minocycline (M). Culture medium was assayed for the biologically active metabolite 5alpha-dihydrotestosterone (DHT) a marker of regenerative potential and wound healing. Confluent monolayer cultures of human periosteal fibroblasts were incubated in Eagle’s minimum essential medium with each of the substrates 14C-T and 14C-4-A. Incubations were performed with previously established optimal concentrations of B5 (milligrams/ml), M25 (μg/ml) and F5 (μg/ml) alone and in combination (n=6) for 24h. The eluent was solvent extracted with ethyl acetate (2mlx2) and subjected to TLC in a benzene/acetone solvent system (4:1 v/v) for separation of metabolites; they were quantified using a radioisotope scanner. The yield of DHT was increased over controls in response to B and M with both substrates 14C-T and 14C-4-A by 1.7, 1.8-fold and 1.7, 1.6-fold respectively (n=6; p<0.001; one way ANOVA). Combined incubations of B and M resulted in similar yields. F inhibited DHT yields with both radiolabelled substrates by 2–3-fold (n=6; p<0.001) which was overcome by a combined incubation of F+B to values similar to those of controls (p<0.01). Documented pro-anabolic effects of minocycline were applicable as a standard for confirmation of responses to B. Significant increases in yields of DHT in response to B and M with both substrates indicate their anabolic potential in periosteal fibroblasts with implications for wound healing.

Effects of Sodium Hydroxide, Sodium Hypochlorite, and Gaseous Hydrogen Peroxide on the Natural Properties of Cancellous Bone

Processed xenegeneic cancellous bone represents an alternative to bone autograft. In order to observe the effects of present prion inactivation treatments on the natural properties of xenogeneic cancellous bones, we treated bovine bone granules with sodium hydroxide (NaOH), sodium hypochlorite (NaOCl), and gaseous hydrogen peroxide (gH2 O2 ) respectively in this study. The microstructure, composition, and mineral content of the granules were evaluated by scanning electron micrograph, energy dispersive X-ray spectroscopy, ash analysis, and micro-computed tomography. The biomechanical property was analyzed by a materials testing machine. The cytocompatibility was evaluated by using a mouse fibroblast cell line (3T3). The microstructure, organic content, and mechanical strength were dramatically altered at the surface of bone in both NaOH- and NaOCl-treated groups, but not in the gH2 O2 -treated group. Compared with the gH2 O2 -treated group, attachment and proliferation of 3T3 were reduced in either NaOH- or NaOCl-treated groups. As the consequence, gH2 O2 treatment may be a useful approach of disinfection for the preparation of natural cancellous bone with well-preserved structural, mechanical, and biological properties.

In situ ring-opening polymerization of hydroxyapatite/poly(ethylene adipate)-co-(ethylene terephthalate) biomimetic composites

Hydroxyapatite/poly(ethylene adipate)-co-poly(ethylene terephthalate) biomaterials (HAp/PEA-co-PET) have been prepared by ring opening polymerization (ROP) of cyclic oligo(ethylene adipate)-co-oligo(ethylene terephthalate) (C-OEA-co-C-OET) in the porous hydroxyapatite (HAp) scaffolds at 250 °C for 24 h under vacuum. The content of ROP-PEA-co-PET in the HAp/PEA-co-PET composite was about 20 wt% with the values of number average molecular weight $({\overline{M}_{{\rm n}}})$ and weight average molecular weight $({\overline{M}_{{\rm W}}})$ of 3380 and 7160 g/mol, respectively. Compressive strength and modulus of the HAp/PEA-co-PET composites were about 29 and 246 MPa, respectively. These mechanical properties were higher than those of the porous HAp templates and natural cancellous bone. In vitro bioactivity of the HAp/PEA-co-PET composites was studied by soaking in simulated body fluid (SBF) under the flowing system at the rate of 130 mL/day for 7, 14, 21 and 28 days. The formation of hydroxyapatite nanocrystals was observed on the composite surfaces through the consumption of calcium and phosphorus from the SBF solution, indicating the bioactivity of these HAp/PEA-co-PET composites. These results indicated the competency of HAp/PEA-co-PET composites for biomedical applications.

Preparation and Properties of Porous β–Tricalcium Phosphate Bone Graft

The need for bone repair has increased as the population ages. However, currently, the bone grafts still have some disadvantages, such as low compressive strength and porosity, which limit their use. In order to solve these disadvantages, in this study, the porous beta-tricalcium phosphate (β-TCP) anorganic bone graft were prepared from healthy bovine cancellous bone by cell-free, defat and twice calcinations. X-ray diffraction (XRD) was used to investigate the chemical composition of the bone graft. And the morphology, porosity and mechanical strength of the bone graft were also evaluated. The results showed that most constituent of the bone graft was β-TCP. In addition, the bone graft scaffold exhibited the macro and micro porous structure and the porosity was 57.63%, just as the nature cancellous bone. The compressive strength was 4.47±0.63MPa. Above all, the porous β-TCP anorganic bone graft not only has similar chemical composites as the nature cancellous bone, but also it can effectively retain the porous structure of natural cancellous bone and provides optimal channels for the ingrowth of new bone and blood vessels.Therefore, the porous β-TCP anorganic bone graft is a potential biomaterial in bone tissue engineering.

Permeability studies of artificial and natural cancellous bone structures

In the development of artificial cancellous bones, two major factors need to be considered: the integrity of the overall structure and its permeability. Whilst there have been many studies analysing the mechanical properties of artificial and natural cancellous bones, permeability studies, especially those using numerical simulation, are scarce. In this study, idealised cancellous bones were simulated from the morphological indices of natural cancellous bone. Three different orientations were also simulated to compare the anisotropic nature of the structure. Computational fluid dynamics methods were used to analyse fluid flow through the cancellous structures. A constant mass flow rate was used to determine the intrinsic permeability of the virtual specimens. The results showed similar permeability of the prismatic plate-and-rod model to the natural cancellous bone. The tetrakaidecahedral rod model had the highest permeability under simulated blood flow conditions, but the plate counterpart had the lowest. Analyses on the anisotropy of the virtual specimens showed the highest permeability for the horizontal orientation. Linear relationships were found between permeability and the two physical properties, porosity and bone surface area.

Design and fabrication of a novel porous implant with pre-set channels based on ceramic stereolithography for vascular implantation

Being a multi-etiological factors disease, osteonecrosis of the femoral head affects many young people, leading to the collapse of the femur head; eventually the hip arthroplasty is needed if not treated in time. Unfortunately, as yet, no satisfactory therapy to repair necrotic bone at an early stage is present. Novel implants with pre-set channels were designed for the treatment of early femoral head necrosis. Ceramic stereolithography was applied to fabricate the green part from β-TCP powder. Other processes, such as dehydration, rinsing, drying and sintering, were processed successively. The final ceramic part remains the same as the engineered part in both shape and internal structure. No significant deformation or crack occurred. X-ray diffraction showed that no facies changed or chemical reaction occurred during the fabrication process. The chemical composition remains the same as that of the original β-TCP powder. The compressive strength is 23.54 MPa, close to that of natural cancellous bone. Novel implants with a pre-set channel were designed and fabricated for blood vessel implantation. Bioceramic stereolithography technology based directly on the CAD model in this research shows advantages in accurate design, optimization of 3D scaffold and critical control of the fabrication process. This proposed implant shows promising clinical application in the restoration of early femoral head necrosis.

Bioactive glass scaffold with similar structure and mechanical properties of cancellous bone

Natural bone is an amazing material because of its structure and mechanics. It is a challenge to find a bone substituted material mimicking natural bone intended to suffice for the need for bone repair and regeneration. In this study, a biomorphic material with similar structure and mechanical properties of cancellous bone has been fabricated by using demineralized cancellous bone as a template. Well-oriented particles in the pore walls could be observed clearly. The compressive strength of this scaffold was up to about 4.9 ± 0.2 MPa, close to the lower limit of the natural bone and 30 times of bioactive glass scaffolds, even though the porosity was up to 90%. This kind of bioactive glass composite scaffolds presented high resistance to deformation and showed an excellent shape restoration property. It is very close to a natural cancellous bone with a similar macro- and micro-structure and force-extension behavior. These results suggested this material would have a good potential in bone tissue engineering.

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a "biomimetic niche." The CD34(+) cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2-5weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2weeks in culture, the CD34(+) cells from umbilical cord blood in the 3D system increased 3.3-4.8 folds when compared with the initial CD34(+) cells. CD34(+)/CD38(-) cells accounted for 82-90% of CD34(+) cells. After 5weeks, CD34(+)/CD38(-) cells in the 3D system increased when compared with initial (1.3±0.3×10(3) vs. 1.0±0.5×10(4), p<0.05), but were decreased in the 2D system (1.3±0.3×10(3) vs. 2.5±0.7×10(2), p<0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6-9.3 folds vs. 1.0-1.5 folds) after 2weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2-7.2 folds increase over input at 2weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.

Fabrication and Characterization of Ag-coated BCP Scaffold Derived from Sponge Replica Process

As a starting material, BCP (biphasic calcium phosphate) nano powder was synthesized by a hydrothermal microwave-assisted process. A highly porous BCP scaffold was fabricated by the sponge replica method using 60 ppi (pore per inch) of polyurethane sponge. The BCP scaffold had interconnected pores ranging from to , which were similar to natural cancellous bone. To realize the antibacterial property, a microwave-assisted nano Ag spot coating process was used. The morphology and distribution of nano Ag particles were different depending on the coating conditions, such as concentration of the solution, microwave irradiation times, etc. With an increased microwave irradiation time, the amount of coated nano Ag particles increased. The surface of the BCP scaffold was totally covered with nano Ag particles homogeneously at 20 seconds of microwave irradiation time when 0.6 g of was used. With an increased amount of and irradiation time, the size of the coated particles increased. Antibacterial activities of the solution extracted from the Ag-coated BCP scaffold were examined against gram-negative (Escherichia coli) and gram-positive bacteria (Staphylococcus aureus). When 0.6 g of was used for coating the Ag-coated scaffold, it showed higher antibacterial activities than that of the Ag-coated scaffold using 0.8 g of .

The influence of approaches for the purification of natural cancellous bone grafts: Morphology, microstructure, composition, strength and biocompatibility study

To find an optimal approach for preparation of porous scaffold for tissue engineering, natural cancellous bone were processed with several techniques: calcination, lyophilization, treatment of chemical reagents and supercritical CO 2 fluid extraction, respectively. Scanning electron micrograph, energy dispersive X-ray microanalysis, X-ray diffraction, Micro-CT, mechanical test and T-cell proliferation assay were used to observe and compare morphology, microstructure, composition, mechanical strength and biocompatibility of the different scaffolds. After processing all the scaffolds exhibited natural porous three-dimension microstructure, and the composition and properties of natural bone, such as Ca/P ratio and hydroxyapatite crystal were still preserved in all of them. Except scaffolds processed with lyophilization, all the other scaffolds showed no influence on the proliferation of T-cell. And scaffolds processed with supercritical CO 2 fluid performed higher compressive strength than that of the other scaffolds, which was similar to natural bones. As the consequence, supercritical CO 2 fluid extraction may be a useful approach for preparation of natural scaffolds for tissue engineering.

PCL Infiltration into a BCP Scaffold Strut to Improve the Mechanical Strength while Retaining Other Properties

A highly porous Biphasic Calcium Phosphate (BCP) scaffold was fabricated by the sponge replica method with a microwave sintering technique. The BCP scaffold had interconnected pores ranging from 80 μm to 1000 μm, which were similar to natural cancellous bone. To enhance the mechanical properties of the porous scaffold, infiltration of polycaprolactone (PCL) was employed. The microstructure of the BCP scaffold was optimized using various volume percentages of polymethylmethacrylate (PMMA) for the infiltration process. PCL successfully infiltrated into the hollow space of the strut formed after the removal of the polymer sponge throughout the degassing and high pressure steps. The microstructure and material properties of the BCP scaffold (i.e., pore size, morphology of infiltrated and coated PCL, compressive strength, and porosity) were evaluated. When a 30 vol% of PMMA was used, the PCL-BCP scaffold showed the highest compressive strength. The compressive strength values of the BCP and PCL-BCP scaffolds were approximately 1.3 and 2MPa, respectively. After the PCL infiltration process, the porosity of the PCL-BCP scaffold decreased slightly to 86%, whereas that of the BCP scaffold was 86%. The number of pores in the 10 μm to 20 μm rage, which represent the pore channel inside of the strut, significantly decreased. The in-vitro study confirmed that the PCL-infiltrated BCP scaffold showed comparable cell viability without any cytotoxic behavior.

A novel, low temperature method for the preparation of ß-TCP/HAP biphasic nanostructured ceramic scaffold from natural cancellous bone

In this study, a novel method was used to synthesize ß-TCP/HAP biphasic ceramic scaffold from natural cancellous bone. Bovine bone was calcined to remove the organic content. The remaining material was then soaked in P 2O 5 solution for different periods. P ions were doped into cancellous bone and reacted with hydroxyapatite (HAP) to produce ß-tricalcium phosphate (ß-TCP) at room temperature without any subsequent high-temperature heat treatment. By increasing the soaking time, the crystalline phase composition of the calcined bone gradually changed from HAP into a HAP/ß-TCP biphasic structure with different HAP/TCP ratios. The crystallite size of HAP and ß-TCP was smaller than 100 nm.

In vitro testing of the osteoinductive potential of different bony allograft preparations

Bony allografts are used frequently in the clinic for bone defect filling, however, less comparative data concerning their osteoinductive potential are available. The purpose of the present study was the comparative analysis of different allograft preparations. From five donors, we investigated fresh-frozen cancellous bone (native), peracetic acid–ethanol sterilized (PES) cancellous bone, cortical bone and demineralised bone matrix (DBM). In addition, two commercially available DBM products from five different donors were analyzed: Allomatrix® (Wright Medical Technology Inc.) and DBX putty® (Synthes GmbH). For positive control and as a clinically used growth factor, BMP-2 was chosen. To investigate the osteoinductivity C2C12 cells were cultured with the different materials and the effect on cell proliferation and alkaline phosphatase activity were measured. Proliferation was significantly enhanced by the native cancellous bone, Allomatrix, and BMP-2 and decreased by the PES-processed cancellous bone. The osteogenic differentiation was significantly enhanced by BMP-2 and the two commercial DBM products and decreased by PES-sterilized cancellous bone. All tested materials revealed a high donor-dependent variability. This is the first comparative study on the osteoinductivity of bony allografts frequently used in clinic.

Modification of bone graft by blending with lecithin to improve hydrophilicity and biocompatibility

Lecithin was blended to improve the hydrophilicity and biocompatibility of bone graft containing poly(l-lactic acid) (PLLA). Solution blending and freeze drying were used to fabricate symmetrical scaffolds containing different percentages of lecithin (lecithin: PLLA = 0, 5, 10 wt%). Scanning electron microscopy showed that the scaffolds maintained the three-dimensional porous structure. A water uptake experiment proved the significant improvement of hydrophilicity of the blend scaffold. With the addition of lecithin, the compressive strength and compressive modulus decreased. When the weight ratio of lecithin to PLLA was up to 10%, the compressive strength was still more than the lower limit of natural cancellous bone. To test the biocompatibility of the scaffolds, cell culture in vitro and subcutaneous implantation in vivo were performed. MC3T3-E1 preosteoblastic cells were cultured on the scaffolds for 7 days. Methylthiazol tetrazolium assay and laser scanning confocal microscopy were used to exhibit proliferation and morphology of the cells. The subcutaneous implantation in rats tested inflammatory response to the scaffolds. The results proved the better biocompatibility and milder inflammatory reactions of the blend scaffold (lecithin: PLLA = 5%) compared with the scaffold without lecithin. The modified scaffold containing lecithin is promising for bone tissue engineering.

Mechanical properties of open-cell foam synthetic thoracic vertebrae

This study presents comprehensive morphological and mechanical properties (static, dynamic) of open-cell rigid foams (Pacific Research Laboratories Inc. Vashon, WA) and a synthetic vertebral body derived from each of the foams. Synthetic vertebrae were comprised of a cylindrical open-cell foam core enclosed by a fiberglass resin cortex. The open-cell rigid foam was shown to have similar morphology and porosity as human vertebral cancellous bone, and exhibited a crush or fracture consolidation band typical of open-celled materials and cancellous bone. However, the foam material density was 40% lower than natural cancellous bone resulting in a lower compressive apparent strength and apparent modulus in comparison to human bone. During cyclic, mean compression fatigue tests, the synthetic vertebrae exhibited an initial apparent modulus, progressive modulus reduction, strain accumulation and S-N curve behaviour similar to human and animal vertebral cancellous bone. Synthetic open-cell foam vertebrae offer researchers an alternative to human vertebral bone for static and dynamic biomechanical experiments, including studies examining the effects of cement injection.

Cancellous bone adaptation to in vivo loading in a rabbit model

Biophysical stimuli are important to the development and maintenance of cancellous bone, but the regulatory mechanisms need to be understood. We investigated the effects of mechanical loading applied in vivo to native cancellous bone in the rabbit on bone formation and trabecular realignment. A novel device was developed to apply controlled compressive loads to cancellous bone in situ. The effect of loading on cancellous bone volume fraction and architecture was quantified. A 4-week experiment was performed in rabbits with devices implanted bilaterally. Cyclic 1 MPa pressures were applied daily to the right limb for 10, 25, or 50 cycles at 0.5 Hz, and the left limb served as the control without any applied loading. Microcomputed tomography and histomorphometry were used to characterize the cancellous tissue within a 4-mm spherical volume located below the loading core. In vivo cyclic loading significantly increased the bone volume fraction, direct trabecular thickness, mean intercept length, and mineral apposition rate in the loaded limbs compared with contralateral limbs. Insufficient evidence was found to demonstrate an effect of number of cycles on the cancellous adaptation between loaded and control limbs. Using a rabbit model, we demonstrated that mechanical loading applied to cancellous bone in situ increased bone formation and altered trabecular morphology. This in vivo model will allow further investigation of cancellous functional adaptation to controlled mechanical stimuli and the influence of mechanical loading parameters, metabolic status, and therapeutic agents.

Material Changes in Osteoporotic Human Cancellous Bone Following Infiltration with Acrylic Bone Cement for a Vertebral Cement Augmentation

Bone cement infiltration can be effective at mechanically augmenting osteoporotic vertebrae. While most published literature describes the gain in mechanical strength of augmented vertebrae, we report the first measurements of viscoelastic material changes of cancellous bone due to cement infiltration. We infiltrated cancellous core specimen harvested from osteoporotic cadaveric spines with acrylic bone cement. Bone specimen before and after cement infiltration were subjected to identical quasi-static and relaxation loading in confined and free compression. Testing data were fitted to a linear viscoelastic model of compressible material and the model parameters for cement, native cancellous bone, and cancellous bone infiltrated (composite) with cement were identified. The fitting demonstrated that the linear viscoelastic model presented in this paper accurately describes the mechanical behaviour of cement and bone, before and after infiltration. Although the composite specimen did not completely adopt the properties of bulk bone cement, the stiffening of cancellous bone due to cement infiltration is considerable. The composite was, for example, 8.5 times stiffer than native bone. The local stiffening of cancellous bone in patients may alter the load transfer of the augmented motion segment and may be the cause of subsequent fractures in the vertebrae adjacent to the ones infiltrated with cement. The material model and parameters in this paper, together with an adequate finite-element model, can be helpful to investigate the load shift, the mechanism for subsequent fractures, and filling patterns for ideal cement infiltration.

Platelet concentration and its effect on bone formation in calvarial defects: An experimental study in rabbits

Statement of problem. The use of the platelet concentration technique is widespread in dental implant surgery. However, its effect or mechanism is not clearly understood. Purpose. This study introduced an animal model for the platelet concentration technique and evaluated its effect on bone formation with natural cancellous bovine bone mineral. Material and methods. Adult New Zealand white rabbits were used as the animal model. A density gradient medium was used to obtain a constant platelet count for the preparation of platelet concentrates. In the experimental group, natural cancellous bovine bone mineral with added platelet concentrates was grafted onto critically sized bony defects of the rabbit calvarium. Bone formation in the tissue sections was evaluated with soft x-ray imaging and computer tomography. Results. The average platelet count of the rabbit platelet concentrates was 1487 × 103/μL (287% concentrated). In all the tested parameters, greater bone densities were obtained in grafts that were combined with platelet concentrates. Conclusion. This study showed that the rabbit is a useful animal model for studying the platelet concentration technique. When combined with grafts of natural cancellous bovine bone mineral, the technique increased bone formation. (J Prosthet Dent 2001;86:428-33.)

An Investigation into the Feasibility of Implementing Fractal Paradigms to Simulate Cancellous Bone Structure

Cancellous bone consists of a framework of solid trabeculae interspersed with bone marrow. The structure of the bone tissue framework is highly convoluted and complex, being fractal and statistically self-similar over a limited range of magnifications. To date, the structure of natural cancellous bone tissue has been defined using 2D and 3D imaging, with no facility to modify and control the structure. The potential of four computer-generated paradigms has been reviewed based upon knowledge of other fractal structures and chaotic systems, namely Diffusion Limited Aggregation (DLA)perolation and Epidemics, Cellular Automata, and a regular Grid with randomly relocated nodes. The resulting structures were compared for their ability to create realistic structures of cancellous bone rather than reflecting growth and form processes. Although the creation of realistic computer-generated cancellous bone structures is difficult, it should not be impossible. Future work considering the combination of fractal and chaotic paradigms is underway.