Bone growth in rapid prototyped porous titanium implants

Two porous titanium implants with a pore size diameter of 800 and 1200 microm (Ti800 and Ti1200) and an interconnected network were manufactured using rapid prototyping. Their dimensions and structure matched those of the computer assisted design. The porosity of the implants was around 60%. Their compressive strength and Young's modulus were around 80 MPa and 2.7 GPa, respectively. These values are comparable to those of cortical bone. The implants were implanted bilaterally in the femoral epiphysis of 15 New Zealand White rabbits. After 3 and 8 weeks, abundant bone formation was found inside the rapid prototyped porous titanium implants. For the Ti1200 implants, bone ingrowth was (23.9 +/- 3.5)% and (10.3 +/- 2.8)%, respectively. A significant statistical difference (p < 0.05) was found for bone ingrowth in the Ti1200 between the two delays. The percentage of bone directly apposited on titanium was (35.8 +/- 5.4)% and (30.5 +/- 5.0)%. No significant difference was found for bone-implant contact between the different time periods and pore sizes. This work demonstrates that manufacturing macroporous titanium implants with controlled shape and porosity using a rapid prototyping method is possible and that this technique is a good candidate for orthopedic and maxillofacial applications.     

Pore throat size and connectivity determine bone and tissue ingrowth into porous implants: three-dimensional micro-CT based structural analyses of porous bioactive titanium implants

A porous structure comprises pores and pore throats with a complex three-dimensional (3D) network structure, and many investigators have described the relationship between average pore size and the amount of bone ingrowth. However, the influence of network structure or pore throats for tissue ingrowth has rarely been discussed. Four types of bioactive porous titanium implants with different pore sizes and porosities (6mm in diameter and 15 mm long) were analyzed using specific algorithms for 3D analysis of interconnectivity based on a micro focus X-ray computed tomography system. In vivo histomorphometric analysis was performed using the very same implants implanted into the femoral condyles of male rabbits for 6 and 12 weeks. This matching study revealed that more poorly differentiated pores tended to have narrow pore throats, especially in their shorter routes to the outside. In addition, for assessment of the entire implant, we proposed new two indices that represent the degree of bone and tissue ingrowth into an implant by considering the effect of narrow pore throats. Data obtained suggest that this sort of novel analysis is useful for evaluating bone and tissue ingrowth into porous biomaterials.     

In vivo bone response to porous calcium phosphate cement

We conducted an in vivo experiment to evaluate the resorption rate of a calcium phosphate cement (CPC) with macropores larger than 100 microm, using the CPC called Biocement D (Merck Biomaterial, Darmstadt, Germany), which after setting only shows pores smaller than 1 microm. The gas bubble method used during the setting process created macroporosity. Preset nonporous and porous cement implants were inserted into the trabecular bone of the tibial metaphysis of goats. The size of the preset implants was 6 mm and the diameter of the drill hole was 6.3 mm, leaving a gap of 0.3 mm between implant surface and drill wall. After 2 and 10 weeks, the animals were euthanized and cement implants with surrounding bone were retrieved for histologic evaluation. Light microscopy at 2 weeks revealed that the nonporous implants were surrounded by connective tissue. On the cement surface, we observed a monolayer of multinucleated cells. Ten weeks after implantation, the nonporous implants were still surrounded by connective tissue. However, a thin layer of bone now covered the implant surface. No sign of cement resorption was observed. In contrast, the porous cement evoked a completely different bone response. At 2 weeks, bone formation had already occurred inside the implant porosity. Bone formation even appeared to occur as a result of osteoinduction. Also, at their outer surface, the porous implants were completely surrounded by bone. At 2 weeks, about 31% of the initial cement was resorbed. After 10 weeks, 81% of the initial phosphate cement was resorbed and new bone was deposited. On the basis of these observations, we conclude that the creation of macropores can significantly improve the resorption rate of CPC. This increased degradation is associated with almost complete bone replacement.     

Peri- and intra-implant bone response to microporous Ti coatings with surface modification

Bone growth on and into implants exhibiting substantial surface porosity is a promising strategy in order to improve the long-term stable fixation of bone implants. However, the reliability in clinical applications remains a point of discussion. Most attention has been dedicated to the role of macroporosity, leading to the general consensus of a minimal pore size of 50–100 μm in order to allow bone ingrowth. In this in vivo study, we assessed the feasibility of early bone ingrowth into a predominantly microporous Ti coating with an average thickness of 150 μm and the hypothesis of improving the bone response through surface modification of the porous coating. Implants were placed in the cortical bone of rabbit tibiae for periods of 2 and 4 weeks and evaluated histologically and histomorphometrically using light microscopy and scanning electron microscopy. Bone with osteocytes encased in the mineralized matrix was found throughout the porous Ti coating up to the coating/substrate interface, highlighting that osseointegration of microporosities (<10 μm) was achievable. The bone trabeculae interweaved with the pore struts, establishing a large contact area which might enable an improved load transfer and stronger implant/bone interface. Furthermore, there was a clear interconnection with the surrounding cortical bone, suggesting that mechanical interlocking of the coating in the host bone in the long term is possible. When surface modifications inside the porous structure further reduced the interconnective pore size to the submicrometer level, bone ingrowth was impaired. On the other hand, application of a sol–gel-derived bioactive glass–ceramic coating without altering the pore characteristics was found to significantly improve bone regeneration around the coating, while still supporting bone ingrowth.     

Investigation as to the osteoinductivity of macroporous calcium phosphate cement in goats

For bone formation in critical-sized or poor healing defects, osteoinductive behavior of synthetic bone grafts is crucial. Although the osteoconductive behavior of calcium phosphate (CaP) cement is generally accepted, its osteoinductive potential is less reported. In this study, osteoinduction of porous CaP cement was investigated. Four goats received each six subcutaneous placed prehardened porous CaP cement implants. Implantation time was 3 and 6 months. After explantation, histological evaluation and scoring with a histological grading scale for soft-tissue implants were performed. The histological sections revealed that the implants degraded for more than 50% over time. The implants had lost their macroporous structure from 3 months on. A medium-thick fibrous capsule with a few inflammatory cells surrounded the implants after 3 months. This capsule significantly decreased in thickness after 6 months. Throughout the implant ingrowth of fibrous tissue was seen with scattered foci of inflammatory cells. Cement particles were surrounded by a layer of inflammatory cells. The massive inflammatory response in the interstice was seen after 3 months, which disappeared after 6 months implantation. No bone formation was detected in any of the specimens. The fast degradation and thereby collapsing of the porous structure of our CaP cement implant might have prevented osteoinduction.     

Osteoinduction of porous bioactive titanium metal

This is the first report of bone induction in a non-osseous site by titanium metal, which has long been recognized as a non-bioactive material. After undergoing specific chemical and thermal treatments, porous bioactive titanium induced bone formation without the need of additional osteogenic cells or osteoinductive agents. Four types of titanium implants were implanted in the dorsal muscles of mature beagle dogs, and were examined histologically after periods of 3 and 12 months. Chemically and thermally treated titanium, as well as pure titanium, was implanted either as porous blocks or as fibre mesh cylinders. Bone formation was found only in the chemically and thermally treated porous block implants removed after 12 months. The present study shows that even a non-soluble metal that contains no calcium or phosphorus can be an osteoinductive material when treated to form an appropriate macrostructure and microstructure. This finding may elucidate the nature of osteoinduction, and lead to the advent of epochal osteoinductive biomaterials for tissue regeneration.     

Bone induction by porous glass ceramic made from Bioglass (45S5)

Porous glass ceramic, which was prepared from Bioglass powder (45S5, U.S. Biomaterials) by foaming with diluted H(2)O(2) solution and sintering at 1000 degrees C for 2 h, was implanted as cylinders (5 mm in diameter and 6 mm in length) in thigh muscles of dogs for 3 months. Histological observation was made on thin un-decalcified sections. Bone formation was histologically found in pores of all implants (X16) retrieved from 16 dogs. The bone tissue was also identified with backscattered scanning electron microscopy observation (BSE) and energy dispersive X-ray microanalysis (EDX). This is the first report of bone induction in soft tissues of animals by glass ceramic that has long been recognized as a bioactive (osteoconductive) biomaterial. The present results justify the impetus to investigate the osteoinductivity of calcium phosphate-based biomaterials, to study the mechanism of bone induction (osteoinduction) by calcium phosphate-based biomaterials, to develop osteoinductive calcium phosphate-based biomaterials, and to examine the relation between osteoinduction and osteoconduction.     

不同温度煅烧骨作骨形态发生蛋白载体的试验研究

采用不同温度天然煅烧骨与骨形态发生蛋白复合物进行兔桡骨骨缺损修复的动物试验。发现600℃的煅烧骨与骨形态发生蛋白的复合材料（BMP／SB）与900℃及1200℃的BMP／SB材料在诱导成骨及修复骨缺损中作用不如后两者。900℃及1200℃的BMP／SB材料在新骨形成量上要高于600℃的BMP／SB材料（P〈0．05），而后两者间无明显差别（P〉0.05）。但900℃的煅烧骨材料力学强度大，制备温度相对低，故经济效益明显。研究提示900℃的的煅烧骨与600℃、1200℃的煅烧骨相比，更适于用作骨形态发生蛋白的载体。     

磷酸钙生物材料固有骨诱导性的研究现状与展望

磷酸钙陶瓷因与骨的无机组成相似,具有良好生物相容性和骨引导性,无抗原性,被广泛应用于骨缺损修复,但一直认为磷酸钙陶瓷是一类仅具有骨引导性,而无骨诱导性的生物活性材料。大量研究表明通过材料自身组成和结构优化,可赋予磷酸钙陶瓷骨诱导性。本文对磷酸钙骨诱导现象发现和确证、骨诱导过程和机制、影响骨诱导性的材料学因素和骨诱导与动物种属关系、骨诱导相关的间充质细胞来源、以及骨诱导性材料的应用研究现状进行综述,并对磷酸钙生物材料骨诱导性及相关的研究方向予以展望。     

Osteoinduction of hydroxyapatite/β-tricalcium phosphate bioceramics in mice with a fractured fibula

Many studies have shown that calcium phosphate ceramics can induce bone formation in non-osseous sites without the application of any osteoinductive biomolecules, but the mechanisms of this phenomenon (intrinsic osteoinduction of bioceramics) remain unclear. In this study, we compared the intrinsic osteoinduction of porous hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) implanted in mice at different sites. In 30 mice the left fibula was fractured and the right fibula was kept intact. A porous HA/β-TCP cylinder was implanted into both the left (group 1) and right (group 2) leg muscles of each animal. In addition, two HA/β-TCP cylinders were bilaterally implanted into leg subcutaneous pockets (group 3) in each of the remaining 15 mice. New bone formation was studied in the three groups by histology, histomorphometry and immunostaining. In group 1 new bone was observed at week 6 and bone marrow appeared at week 12. In group 2 new bone was observed at week 8 and bone marrow appeared at week 12. The new bone area percentage in group 1 was significantly higher than in group 2 at both weeks 8 and 12. In contrast, group 3 did not show any new bone within the period studied. These differences were explained based on the location of the implants and thus their proximity to the osteogenic environment of fracture healing. The results support the hypothesis that intrinsic osteoinduction by calcium phosphate ceramics is the result of adsorption of osteoinductive substances on the surface.     

Qualitative and quantitative observations of bone tissue reactions to anodised implants

Research projects focusing on biomaterials related factors; the bulk implant material, the macro-design of the implant and the microsurface roughness are routinely being conducted at our laboratories. In this study, we have investigated the bone tissue reactions to turned commercially pure (c.p.) titanium implants with various thicknesses of the oxide films after 6 weeks of insertion in rabbit bone. The control c.p. titanium implants had an oxide thickness of 17-200 nm while the test implants revealed an oxide thickness between 600 and 1000 nm. Routine histological investigations of the tissue reactions around the implants and enzyme histochemical detections of alkaline and acid phosphatase activities demonstrated similar findings around both the control and test implants. In general, the histomorphometrical parameters (bone to implant contact and newly formed bone) revealed significant quantitative differences between the control and test implants. The test implants demonstrated a greater bone response histomorphometrically than control implants and the osteoconductivity was more pronounced around the test implant surfaces. The parameters that differed between the implant surfaces, i.e. the oxide thickness, the pore size distribution, the porosity and the crystallinity of the surface oxides may represent factors that have an influence on the histomorphometrical results indicated by a stronger bone tissue response to the test implant surfaces, with an oxide thickness of more than 600 nm.     

Bone formation in transforming growth factor beta-1-coated porous poly(propylene fumarate) scaffolds

This study determined the bone growth into pretreated poly(propylene fumarate) (PPF) scaffolds implanted into a subcritical size, rabbit cranial defect. PPF scaffolds were constructed by using a photocrosslinking-porogen leaching technique. These scaffolds were then either prewetted (PPF-Pw), treated with RF glow-discharge (PPF-Gd), coated with fibronectin (PPF-Fn), or coated with rhTGF-beta1 (PPF-TGF-beta1). One of each scaffold type was then placed into the cranium of nine rabbits. The rabbits were sacrificed after 8 weeks, and the scaffolds were retrieved for histological analysis. The most bone formation was present in the PPF-TGF-beta1 implants; the newly formed bone had a trabecular appearance together with bone marrow-like tissue. Little or no bone formation was observed in implants without rhTGF-beta1. These histological findings were confirmed by image analysis. Bone surface area, bone area percentage, pore fill percentage, and pore area percentage were significantly higher in the rhTGF-beta1-coated implants than in the noncoated implants. No statistical difference was seen between the PPF-Fn, PPF-Pw, or PPF-Gd scaffolds for these parameters. Quadruple fluorochrome labeling showed that in PPF-TGF-beta1 implants bone formation mainly started in the interior of a pore and proceeded toward the scaffold. We conclude that (a) PPF-TGF-beta1 scaffolds can indeed adequately induce bone formation in porous PPF, and (b) PPF scaffolds prepared by the photocrosslinking-porogen leaching technique are good candidates for the creation of bone graft substitutes.     

Biological performance in goats of a porous titanium alloy-biphasic calcium phosphate composite

In this study, porous 3D fiber deposition titanium (3DFT) and 3DFT combined with porous biphasic calcium phosphate ceramic (3DFT+BCP) implants, both bare and 1 week cultured with autologous bone marrow stromal cells (BMSCs), were implanted intramuscularly and orthotopically in 10 goats. To assess the dynamics of bone formation over time, fluorochrome markers were administered at 3, 6 and 9 weeks and the animals were sacrificed at 12 weeks after implantation. New bone in the implants was investigated by histology and histomorphometry of non-decalcified sections. Intramuscularly, no bone formation was found in any of the 3DFT implants, while a very limited amount of bone was observed in 2 BMSC 3DFT implants. 3DFT+BCP and BMSC 3DFT+BCP implants showed ectopic bone formation, in 8 and 10 animals, respectively. The amount of formed bone was significantly higher in BMSC 3DFT+BCP as compared to 3DFT+BCP implants. Implantation on transverse processes resulted in significantly more bone formation in composite structure as compared to titanium alloy alone, both with and without cells. Unlike intramuscularly, the presence of BMSC did not have a significant effect on the amount of new bone either in metallic or in composite structure. Although the 3DFT is inferior to BCP for bone growth, the reinforcement of the brittle BCP with a 3DFT cage did not negatively influence osteogenesis, osteoinduction and osteoconduction as previously shown for the BCP alone. The positive effect of BMSCs was observed ectopically, while it was not significant orthotopically.     

The effect of direct electrical current stimulation on the bone/porous metallic implant interface

Cylindrical porous plugs (6.35 mm dia. 11 mm long, average pore size of 190 μ dia.) made of electrically conductive Co-Cr-Mo surgical alloy powders were implanted in the canine femur. An electrical stimulation device (mercury battery, 1.35 V, connected in series with a 150 kΩ resistor) was attached to all implants directly. The in vivo current was about 8 μA for the stimulated implants while no current was delivered for the control ones.

After predetermined implant periods, tensile test specimens were made to measure the interfacial strength between bone and implants. Some samples were used for histological observations.

The present results show that in vivo electrical stimulation substantially increased the strength of the union between porous implants and bone when compared to the controls up to 12 weeks. Histological observations show that the increased strength is mainly due to the increased new bone formation in the pores of implants.

It was also observed that the fractional callus volume in the intramedullary canal for the stimulated samples retained more than the controls after reaching maximum at 3 weeks.     

Impact of pore size on the vascularization and osseointegration of ceramic bone substitutes in vivo

The repair of bone defects with biomaterials depends on a sufficient vascularization of the implantation site. We analyzed the effect of pore size on the vascularization and osseointegration of biphasic calcium phosphate particles, which were implanted into critical-sized cranial defects in Balb/c mice. Dense particles and particles with pore sizes in the ranges 40-70, 70-140, 140-210, and 210-280 microm were tested (n = 6 animals per group). Angiogenesis, vascularization, and leukocyte-endothelium interactions were monitored for 28 days by intravital microscopy. The formation of new bone and the bone-interface contact (BIC) were determined histomorphometrically. Twenty-eight days after implantation, the functional capillary density was significantly higher with ceramic particles whose pore sizes exceeded 140 microm [140-210 microm: 6.6 (+/-0.8) mm/mm(2); 210-280 microm: 7.3 (+/-0.6) mm/mm(2)] than with those whose pore sizes were lesser than 140 microm [40-70 microm: 5.3 (+/-0.4) mm/mm(2); 70-140 microm: 5.6 (+/-0.3) mm/mm(2)] or with dense particles [5.7 (+/-0.8) mm/mm(2)]. The volume of newly-formed bone deposited within the implants increased as the pore size increased [40-70 microm: 0.07 (+/-0.02) mm(3); 70-140 microm: 0.10 (+/-0.06) mm(3); 140-210 microm: 0.13 (+/-0.05) mm(3); 210-280 microm: 0.15 (+/-0.06) mm(3)]. Similar results were observed for the BIC. The data demonstrates pore size to be a critical parameter governing the dynamic processes of vascularization and osseointegration of bone substitutes.     

Osteoconduction at porous hydroxyapatite with various pore configurations

To assess the histological response and the reinforcing effects of bone ingrowth within porous hydroxyapatite (HA) implants depending on pore geometry, four kinds of cylindrical-type with parallel linear pores phi50, 100, 300, 500 microm), one kind of sponge-type with irregular interconnecting pores (phi250 microm) and one cross-type with crossing linear pores (phi100 x 120 microm) of porous HA were prepared. Eighty-four rabbits were divided into six groups, and a 5 x 5 x 7 mm sized porous HA block was inserted through the medial cortical window of the proximal tibia. Histomorphological changes were examined using light and scanning electron microscopy. A biomechanical compression test was performed using material test machines. After implantation, the implants showed different histological changes depending on pore geometry. Active osteoconduction was also found in the phi50 microm sized cylindrical-type porous HA. Evidence of remodeling of new bone and bone marrow formation within porous HA was found in the larger cylindrical-types (phi300, 500 microm), and the sponge- and cross-types. The biomechanical test showed that the ultimate compressive strength increased significantly in the phi300 microm sized cylindrical-type, and in the sponge- and cross-types eight weeks after implantation. Porous HA with cylindrical pores could be a useful graft material due to its strength, osteoconductivity and the ease with which its pore geometry can be controlled.     

Mechanical and biological properties of hydroxyapatite/tricalcium phosphate scaffolds coated with poly(lactic-co-glycolic acid)

Regeneration of bone, cartilage and osteochondral tissues by tissue engineering has attracted intense attention due to its potential advantages over the traditional replacement of tissues with synthetic implants. Nevertheless, there is still a dearth of ideal or suitable scaffolds based on porous biomaterials, and the present study was undertaken to develop and evaluate a useful porous composite scaffold system. Here, hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds (average pore size: 500 μm; porosity: 87%) were prepared by a polyurethane foam replica method, followed by modification with infiltration and coating of poly(lactic-co-glycolic acid) (PLGA). The thermal shock resistance of the composite scaffolds was evaluated by measuring the compressive strength before and after quenching or freezing treatment. The porous structure (in terms of pore size, porosity and pore interconnectivity) of the composite scaffolds was examined. The penetration of the bone marrow stromal stem cells into the scaffolds and the attachment of the cells onto the scaffolds were also investigated. It was shown that the PLGA incorporation in the HA/TCP scaffolds significantly increased the compressive strength up to 660 kPa and the residual compressive strength after the freezing treatment decreased to 160 kPa, which was, however, sufficient for the scaffolds to withstand subsequent cell culture procedures and a freeze–drying process. On the other hand, the PLGA coating on the strut surfaces of the scaffolds was rather thin (<5 μm) and apparently porous, maintaining the high open porosity of the HA/TCP scaffolds, resulting in desirable migration and attachment of the bone marrow stromal stem cells, although a thicker PLGA coating would have imparted a higher compressive strength of the PLGA-coated porous HA/TCP composite scaffolds.     

Osteogenecity of octacalcium phosphate coatings applied on porous metal implants

The biomimetic route allows the homogeneous deposition of calcium phosphate (Ca-P) coatings on porous implants by immersion in simulated physiologic solution. In addition, various Ca-P phases, such as octacalcium phosphate (OCP) or bone-like carbonated apatite (BCA), which are stable only at low temperatures, can be deposited. In this pilot study, experiments were designed with a twofold-purpose: (1) to investigate the osteoinduction of OCP-coated and noncoated porous tantalum cylinders and of dense titanium alloy cylinders (5 mm in diameter and 10 mm in length) in the back muscle of goats at 12 and 24 weeks (n = 4); and (2) to compare the osteogenic potentials of BCA-coated, OCP-coated, and bare porous tantalum cylinders in a gap of 1 mm created in the femoral condyle of a goat at 12 weeks (n = 2). In the goat muscle, after 12 weeks the OCP-coated porous cylinder had induced ectopic bone as well as bone within the cavity of the OCP-coated dense titanium cylinder. In the femoral condyle, bone did not fill the gap in any of the porous implants. In contrast with the two other groups, OCP-coated porous cylinders exhibited bone formation in the center of the implant. The nature of the Ca-P coating, via its microstructure, its dissolution rate, and its specific interactions with body fluids, may influence the osteogenecity of the Ca-P biomaterial.     

Bone formation process in porous calcium carbonate and hydroxyapatite.

This study determined the bone formation in porous calcium carbonate (CC) and porous hydroxyapatite (HA) in ectopic sites. The bone formation stimulus was derived from bone marrow cells. CC and HA in the shape of disks were implanted with or without rat marrow cells into subcutaneous sites of syngeneic rats. The CC and HA had identical microstructure: pore size was 190-230 microns, porosity was 50-60% and they were fully interconnected. Bone did not form in any implants without marrow cells (disks themselves), whereas bone consistently formed in the pores of all implants with marrow cells after 4 weeks. The bone formation of both CC and HA occurred initially on surface of the pore regions and progressed toward the center of the pore. Scanning electron microscopy and electron-probe microanalysis revealed a continuum of calcium at the interfaces of both bone/CC and bone/HA implants. These results indicate that the bone formation in calcium carbonate derived from marine corals is comparable to the bioactive hydroxyapatite.     

Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants

Rapid prototyping is a valuable implant production tool that enables the investigation of individual geometric parameters, such as shape, porosity, pore size and permeability, on the biological performance of synthetic bone graft substitutes. In the present study, we have employed low-temperature direct 3D printing to produce brushite and monetite implants with different geometries. Blocks predominantly consisting of brushite with channels either open or closed to the exterior were implanted on the decorticated lumbar transverse processes of goats for 12 weeks. In addition, similar blocks with closed channel geometry, consisting of either brushite or monetite were implanted intramuscularly. The design of the channels allowed investigation of the effect of macropore geometry (open and closed pores) and osteoinduction on bone formation orthotopically. Intramuscular implantation resulted in bone formation within the channels of both monetite and brushite, indicating osteoinductivity of these resorbable materials. Inside the blocks mounted on the transverse processes, initial channel shape did not seem to significantly influence the final amount of formed bone and osteoinduction was suggested to contribute to bone formation.