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1.  Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings 
The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue.
We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells.
Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt.
We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of osteogenic extracellular matrix. The coated nanosprings enhance normal human osteoblasts cellular behaviors needed for improving osseointegration of orthopedic materials. Thus, metal-coated nanosprings represent a novel biomaterial that could be exploited for improving success rates of orthopedic implant procedures.
PMCID: PMC3276422  PMID: 22284364
nanosprings; nanomaterials; osteoblasts; osseointegration; calcification; bone regeneration
2.  Titanium Implant Osseointegration Problems with Alternate Solutions Using Epoxy/Carbon-Fiber-Reinforced Composite 
Metals  2014;4(4):549-569.
The aim of the article is to present recent developments in material research with bisphenyl-polymer/carbon-fiber-reinforced composite that have produced highly influential results toward improving upon current titanium bone implant clinical osseointegration success. Titanium is now the standard intra-oral tooth root/bone implant material with biocompatible interface relationships that confer potential osseointegration. Titanium produces a TiO2 oxide surface layer reactively that can provide chemical bonding through various electron interactions as a possible explanation for biocompatibility. Nevertheless, titanium alloy implants produce corrosion particles and fail by mechanisms generally related to surface interaction on bone to promote an inflammation with fibrous aseptic loosening or infection that can require implant removal. Further, lowered oxygen concentrations from poor vasculature at a foreign metal surface interface promote a build-up of host-cell-related electrons as free radicals and proton acid that can encourage infection and inflammation to greatly influence implant failure. To provide improved osseointegration many different coating processes and alternate polymer matrix composite (PMC) solutions have been considered that supply new designing potential to possibly overcome problems with titanium bone implants. Now for important consideration, PMCs have decisive biofunctional fabrication possibilities while maintaining mechanical properties from addition of high-strengthening varied fiber-reinforcement and complex fillers/additives to include hydroxyapatite or antimicrobial incorporation through thermoset polymers that cure at low temperatures. Topics/issues reviewed in this manuscript include titanium corrosion, implant infection, coatings and the new epoxy/carbon-fiber implant results discussing osseointegration with biocompatibility related to nonpolar molecular attractions with secondary bonding, carbon fiber in vivo properties, electrical semiconductors, stress transfer, additives with low thermal PMC processing and new coating possibilities.
PMCID: PMC4307950  PMID: 25635227
titanium; composite; bisphenol polymer; carbon fiber; osseointegration; corrosion; infection; estrogen; microbiocircuit; semiconductor
3.  Microstructure and mineral composition of dystrophic calcification associated with the idiopathic inflammatory myopathies 
Arthritis Research & Therapy  2009;11(5):R159.
Calcified deposits (CDs) in skin and muscles are common in juvenile dermatomyositis (DM), and less frequent in adult DM. Limited information exists about the microstructure and composition of these deposits, and no information is available on their elemental composition and contents, mineral density (MD) and stiffness. We determined the microstructure, chemical composition, MD and stiffness of CDs obtained from DM patients.
Surgically-removed calcinosis specimens were analyzed with fourier transform infrared microspectroscopy in reflectance mode (FTIR-RM) to map their spatial distribution and composition, and with scanning electron microscopy/silicon drift detector energy dispersive X-ray spectrometry (SEM/SDD-EDS) to obtain elemental maps. X-ray diffraction (XRD) identified their mineral structure, X-ray micro-computed tomography (μCT) mapped their internal structure and 3D distribution, quantitative backscattered electron (qBSE) imaging assessed their morphology and MD, nanoindentation measured their stiffness, and polarized light microscopy (PLM) evaluated the organic matrix composition.
Some specimens were composed of continuous carbonate apatite containing small amounts of proteins with a mineral to protein ratio much higher than in bone, and other specimens contained scattered agglomerates of various sizes with similar composition (FTIR-RM). Continuous or fragmented mineralization was present across the entire specimens (μCT). The apatite was much more crystallized than bone and dentin, and closer to enamel (XRD) and its calcium/phophorous ratios were close to stoichiometric hydroxyapatite (SEM/SDD-EDS). The deposits also contained magnesium and sodium (SEM/SDD-EDS). The MD (qBSE) was closer to enamel than bone and dentin, as was the stiffness (nanoindentation) in the larger dense patches. Large mineralized areas were typically devoid of collagen; however, collagen was noted in some regions within the mineral or margins (PLM). qBSE, FTIR-RM and SEM/SDD-EDS maps suggest that the mineral is deposited first in a fragmented pattern followed by a wave of mineralization that incorporates these particles. Calcinosis masses with shorter duration appeared to have islands of mineralization, whereas longstanding deposits were solidly mineralized.
The properties of the mineral present in the calcinosis masses are closest to that of enamel, while clearly differing from bone. Calcium and phosphate, normally present in affected tissues, may have precipitated as carbonate apatite due to local loss of mineralization inhibitors.
PMCID: PMC2787294  PMID: 19857267
4.  Biomimetic Nanofibrous Gelatin/Apatite Composite Scaffolds for Bone Tissue Engineering 
Biomaterials  2009;30(12):2252-2258.
Mimicking certain features (e.g. nanoscale topography and biological cues) of natural extracellular matrix (ECM) is advantageous for the successful regeneration of damaged tissue. In this study, nanofibrous gelatin/apatite (NF-gelatin/apatite) composite scaffolds have been fabricated to mimic both the physical architecture and chemical composition of natural bone ECM. A thermally induced phase separation (TIPS) technique was developed to prepare nanofibrous gelatin (NF-gelatin) matrix. The NF-gelatin matrix mimicked natural collagen fibers and had an average fiber diameter of about 150 nm. By integrating the TIPS method with porogen leaching, three-dimensional NF-gelatin scaffolds with well-defined macropores were fabricated. In comparison to Gelfoam® (a commercial gelatin foam) with similar pore size and porosity, the NF-gelatin scaffolds exhibited a much higher surface area and mechanical strength. The surface area and compressive modulus of NF-gelatin scaffolds were more than 700 times and 10 times higher than that of Gelfoam®, respectively. The NF-gelatin scaffolds also showed excellent biocompatibility and mechanical stability. To further enhance pre-osteoblast cell differentiation as well as improving mechanical strength, bone-like apatite particles (< 2 μm) were incorporated onto the surface of NF-gelatin scaffolds via a simulated body fluid (SBF) incubation process. The NF-gelatin/apatite scaffolds showed significantly higher mechanical strength than NF-gelatin scaffolds 5 days after SBF treatment. Furthermore, the incorporated apatite in the NF-gelatin/apatite composite scaffold enhanced the ostgeogenic differentiation. The expression of BSP and OCN in the osteoblast-(NF-gelatin/apatite composite) constructs was about 5 times and 2 times higher than in the osteoblast-(NF-gelatin) constructs 4 week after cell culture. The biomimetic NF-gelatin/apatite scaffolds are, therefore, excellent for bone tissue engineering.
PMCID: PMC2679864  PMID: 19152974
5.  Evaluation of Biological Properties of Electron Beam Melted Ti6Al4V Implant with Biomimetic Coating In Vitro and In Vivo 
PLoS ONE  2012;7(12):e52049.
High strength porous titanium implants are widely used for the reconstruction of craniofacial defects because of their similar mechanical properties to those of bone. The recent introduction of electron beam melting (EBM) technique allows a direct digitally enabled fabrication of patient specific porous titanium implants, whereas both their in vitro and in vivo biological performance need further investigation.
In the present study, we fabricated porous Ti6Al4V implants with controlled porous structure by EBM process, analyzed their mechanical properties, and conducted the surface modification with biomimetic approach. The bioactivities of EBM porous titanium in vitro and in vivo were evaluated between implants with and without biomimetic apatite coating.
The physical property of the porous implants, containing the compressive strength being 163 - 286 MPa and the Young’s modulus being 14.5–38.5 GPa, is similar to cortical bone. The in vitro culture of osteoblasts on the porous Ti6Al4V implants has shown a favorable circumstance for cell attachment and proliferation as well as cell morphology and spreading, which were comparable with the implants coating with bone-like apatite. In vivo, histological analysis has obtained a rapid ingrowth of bone tissue from calvarial margins toward the center of bone defect in 12 weeks. We observed similar increasing rate of bone ingrowth and percentage of bone formation within coated and uncoated implants, all of which achieved a successful bridging of the defect in 12 weeks after the implantation.
This study demonstrated that the EBM porous Ti6Al4V implant not only reduced the stress-shielding but also exerted appropriate osteoconductive properties, as well as the apatite coated group. The results opened up the possibility of using purely porous titanium alloy scaffolds to reconstruct specific bone defects in the maxillofacial and orthopedic fields.
PMCID: PMC3525565  PMID: 23272208
6.  UV- Killed Staphylococcus aureus Enhances Adhesion and Differentiation of Osteoblasts on Bone-associated Biomaterials 
Titanium alloys (Ti) are the preferred material for orthopaedic applications. However, very often, these metallic implants loosen over a long period and mandate revision surgery. For implant success, osteoblasts must adhere to the implant surface and deposit a mineralized extracellular matrix. Here, we utilized UV-killed Staphylococcus aureus as a novel osteoconductive coating for Ti surfaces. S. aureus expresses surface adhesins capable of binding to bone and biomaterials directly. Furthermore, interaction of S. aureus with osteoblasts activates growth factor-related pathways that potentiate osteogenesis. While UV-killed S. aureus cells retain their bone-adhesive ability, they do not stimulate significant immune modulator expression. All of the above properties were utilized for a novel implant coating so as to promote osteoblast recruitment and subsequent cell functions on the bone-implant interface. In the present study, osteoblast adhesion, proliferation, and mineralized extracellular matrix synthesis were measured on Ti surfaces coated with fibronectin with and without UV-killed bacteria. Osteoblast adhesion was enhanced on Ti alloy surfaces coated with bacteria compared to uncoated surfaces while cell proliferation was sustained comparably on both surfaces. Osteoblast markers such as collagen, osteocalcin, alkaline phosphatase activity and mineralized nodule formation were increased on Ti alloy coated with bacteria compared to uncoated surfaces.
PMCID: PMC2943998  PMID: 20725968
Titanium implant surfaces; Osseointegration; Staphylococcus aureus; Calvarial osteoblasts; Osteoblast differentiation
7.  Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions 
Biomaterials  2010;31(25):6618-6627.
The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the nonclassical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 hours after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes.
PMCID: PMC2904357  PMID: 20621767
extrafibrillar mineralisation; intrafibrillar mineralisation; matrix protein analogues; reconstituted collagen fibrils; tissue engineering materials
8.  In Vitro Mineralization of Dense Collagen Substrates: A Biomimetic Approach Toward the Development of Bone-Graft Materials 
Acta Biomaterialia  2011;7(8):3158-3169.
Bone is an organic-inorganic composite which has hierarchical structuring that leads to high strength and toughness. The nanostructure of bone consists of nanocrystals of hydroxyapatite embedded and aligned within the interstices of collagen fibrils. This unique nanostructure leads to exceptional properties, both mechanical and biological, making it difficult to emulate bone properties without having a bone-like nanostructured material. A primary goal of our group’s work is to use biomimetic processing techniques that lead to bone-like structures.
In our prior studies, we demonstrated that intrafibrillar mineralization of porous collagen sponges, leading to a bone-like nanostructure, can be achieved using a polymer-induced liquid-precursor (PILP) mineralization process. The objective of this study was to investigate the use of this polymer-directed crystallization process to mineralize dense collagen substrates. To examine collagen scaffolds that truly represent the dense-packed matrix of bone, manatee bone was demineralized to isolate its collagen matrix, consisting of a dense, lamellar osteonal microstructure. This biogenic collagen scaffold was then remineralized using polyaspartate to direct the mineralization process through an amorphous precursor pathway.
Various conditions investigated included polymer molecular weight, substrate dimension and mineralization time. Mineral penetration depths of up to 100 μms were achieved using this PILP process, compared to no penetration with only surface precipitates observed for the conventional crystallization process. Electron microscopy, wide-angle X-ray diffraction, and thermal analysis were used to characterize the resulting hydroxyapatite/collagen composites. These studies demonstrate that the original interpenetrating bone nanostructure and osteonal microstructure could be recovered in a biogenic matrix using the PILP process.
PMCID: PMC3261505  PMID: 21550424
Biomineralization; bone; hydroxyapatite; amorphous calcium phosphate; biomimetic; collagen
9.  Modular titanium alloy neck adapter failures in hip replacement - failure mode analysis and influence of implant material 
Modular neck adapters for hip arthroplasty stems allow the surgeon to modify CCD angle, offset and femoral anteversion intraoperatively. Fretting or crevice corrosion may lead to failure of such a modular device due to high loads or surface contamination inside the modular coupling. Unfortunately we have experienced such a failure of implants and now report our clinical experience with the failures in order to advance orthopaedic material research and joint replacement surgery.
The failed neck adapters were implanted between August 2004 and November 2006 a total of about 5000 devices. After this period, the titanium neck adapters were replaced by adapters out of cobalt-chromium. Until the end of 2008 in total 1.4% (n = 68) of the implanted titanium alloy neck adapters failed with an average time of 2.0 years (0.7 to 4.0 years) postoperatively. All, but one, patients were male, their average age being 57.4 years (36 to 75 years) and the average weight 102.3 kg (75 to 130 kg). The failures of neck adapters were divided into 66% with small CCD of 130° and 60% with head lengths of L or larger. Assuming an average time to failure of 2.8 years, the cumulative failure rate was calculated with 2.4%.
A series of adapter failures of titanium alloy modular neck adapters in combination with a titanium alloy modular short hip stem was investigated. For patients having received this particular implant combination risk factors were identified which were associated with the occurence of implant failure. A Kaplan-Meier survival-failure-analysis was conducted. The retrieved implants were analysed using microscopic and chemical methods. Modes of failure were simulated in biomechanical tests. Comparative tests included modular neck adapters made of titanium alloy and cobalt chrome alloy material.
Retrieval examinations and biomechanical simulation revealed that primary micromotions initiated fretting within the modular tapered neck connection. A continuous abrasion and repassivation process with a subsequent cold welding at the titanium alloy modular interface. Surface layers of 10 - 30 μm titanium oxide were observed. Surface cracks caused by fretting or fretting corrosion finally lead to fatigue fracture of the titanium alloy modular neck adapters. Neck adapters made of cobalt chrome alloy show significantly reduced micromotions especially in case of contaminated cone connection. With a cobalt-chromium neck the micromotions can be reduced by a factor of 3 compared to the titanium neck. The incidence of fretting corrosion was also substantially lower with the cobalt-chromium neck configuration.
Failure of modular titanium alloy neck adapters can be initiated by surface micromotions due to surface contamination or highly loaded implant components. In the present study, the patients at risk were men with an average weight over 100 kg. Modular cobalt chrome neck adapters provide higher safety compared to titanium alloy material.
PMCID: PMC2824687  PMID: 20047653
10.  Effects of electrospun submicron fibers in calcium phosphate cement scaffold on mechanical properties and osteogenic differentiation of umbilical cord stem cells 
Acta biomaterialia  2011;7(11):4037-4044.
Fibrous scaffolds are promising for tissue engineering because of the high surface area and fibrous features mimicking the extracellular matrix in vivo. Calcium phosphate cements (CPCs) can be injected and self-set in the bone defect. A literature search revealed no report on stem cell seeding on CPC containing electrospun submicron fibers. The objective of this study was to investigate the effects of electrospun fibers in CPC on mechanical properties and human umbilical cord mesenchymal stem cell (hUCMSC) proliferation, osteogenic differentiation and mineralization for the first time. Poly(d,l-lactide-co-glycolide) (PLGA) fibers were made via an electrospinning technique to yield an average fiber diameter of 650 nm. The fibers were incorporated into CPC consisting of tetracalcium phosphate, dicalcium phosphate anhydrous and chitosan lactate. Fiber volume fractions were 0%, 2.5%, 5% and 10%. CPC with 10% fibers had a flexural strength that was twice, and work-of-fracture (toughness) an order of magnitude, those of CPC without fibers. hUCMSCs proliferated rapidly and synthesized bone minerals while attaching to the electrospun fiber-CPC scaffolds. Alkaline phosphatase, osteocalcin, and collagen I expressions of hUCMSCs were doubled, while mineralization was increased by 40%, when fiber volume fraction in CPC was increased from 0% to 10%. The enhanced cell function was attributed to the high surface area and biomimetic features of the fiber-CPC scaffold. In conclusion, incorporating submicron fibers into CPC greatly improved the strength and toughness of CPC. Creating submicron fibrous features in CPC was a useful method for enhancing the osteogenic differentiation and mineralization of stem cells. The novel electrospun fiber-CPC-hUCMSC construct is promising for stem cell delivery and bone tissue engineering.
PMCID: PMC3185192  PMID: 21763791
Electrospun Fibers; Calcium Phosphate Cement; Human Cord Stem Cells; Osteogenic Differentiation; Strength and Toughness; Bone Tissue Engineering
11.  Differences between top-down and bottom-up approaches in mineralizing thick, partially-demineralized collagen scaffolds 
Acta biomaterialia  2010;7(4):1742-1751.
Biominerals exhibit complex hierarchical structures derived from bottom-up self-assembly mechanisms. Type I collagen serves as the building block for mineralized tissues such as bone and dentin. In the present study, 250–300 μm thick, partially-demineralized collagen scaffolds exhibiting a gradient of demineralization from the base to surface were mineralized using a classical top-down approach and a non-classical bottom-up approach. The top-down approach involved epitaxial growth over seed crystallites. The bottom-up approach utilized biomimetic analogs of matrix proteins to stabilize amorphous calcium phosphate nanoprecursors and template apatite nucleation and growth within the collagen matrix. Micro-computed tomography and transmission electron microscopy were employed to examine mineral uptake and apatite arrangement within the mineralized collagen matrix. The top-down approach could only mineralize the base of the partially-demineralized scaffold where remnant seed crystallites were abundant. Minimal mineralization was observed along the surface of the scaffold; extrafibrillar mineralization was predominantly observed. Conversely, the entire partially-demineralized scaffold including apatite-depleted collagen fibrils was mineralized by the bottom-up approach, with evidence of both intrafibrillar and extrafibrillar mineralization. Understanding the different mechanisms involved in these two mineralization approaches is pivotal in adopting the optimum strategy for fabricating novel nanostructured materials in bioengineering research.
PMCID: PMC3050119  PMID: 21111071
biomimetics; bottom-up; collagen; ion-mediated; particle-mediated; mineralization; top-down
12.  Calcium phosphate deposition rate, structure and osteoconductivity on electrospun poly(l-lactic acid) matrix using electrodeposition or simulated body fluid incubation 
Acta biomaterialia  2013;10(1):10.1016/j.actbio.2013.08.041.
Mineralized nanofibrous scaffolds have been proposed as promising scaffolds for bone regeneration due to their ability to mimic both nanoscale architecture and chemical composition of natural bone extracellular matrix (ECM). In this study, a novel electrodeposition method was compared with an extensively explored simulated body fluid (SBF) incubation method in terms of the deposition rate, chemical composition, and morphology of calcium phosphate formed on electrospun fibrous thin matrices with a fiber diameter in the range from about 200 nm to about 1400 nm prepared using 6, 8, 10 and 12 wt% poly(l-lactic acid) (PLLA) solutions in a mixture of dichloromethane and acetone (2:1 in volume). The effects of the surface modification using the two mineralization techniques on osteoblastic cell (MC3T3-E1) proliferation and differentiation were also examined. It was found that electrodeposition was two to three orders of magnitude faster than the SBF method in mineralizing the fibrous matrices, reducing the mineralization time from about two weeks to an hour to achieve the same amounts of mineralization. The mineralization rate also varied with the fiber diameter but in opposite directions between the two mineralization methods. As a general trend, the increase of fiber diameter resulted in a faster mineralization rate for the electrodeposition method but a slower mineralization rate for the SBF incubation method. Using the electrodeposition method, one can control the chemical composition and morphology of the calcium phosphate by varying the electric deposition potential and electrolyte temperature to tune the mixture of dicalcium phosphate dihydrate (DCPD) and hydroxy apatite (HAp). Using the SBF method, one can only obtain a low crystallinity HAp. The mineralized electrospun PLLA fibrous matrices from either method similarly facilitate the proliferation and osteogenic differentiation of preosteoblastic MC3T3-E1 cells as compared to neat PLLA matrices. Therefore, the electrodeposition method can be utilized as a fast and versatile technique to fabricate mineralized nanofibrous scaffolds for bone tissue engineering.
PMCID: PMC3840094  PMID: 24012605
Electrodeposition; SBF incubation; Calcium phosphate; Nanofiber; Bone tissue engineering
13.  In Vivo Ectopic Bone Formation by Devitalized Mineralized Stem Cell Carriers Produced Under Mineralizing Culture Condition 
BioResearch Open Access  2014;3(6):265-277.
Functionalization of tissue engineering scaffolds with in vitro–generated bone-like extracellular matrix (ECM) represents an effective biomimetic approach to promote osteogenic differentiation of stem cells in vitro. However, the bone-forming capacity of these constructs (seeded with or without cells) is so far not apparent. In this study, we aimed at developing a mineralizing culture condition to biofunctionalize three-dimensional (3D) porous scaffolds with highly mineralized ECM in order to produce devitalized, osteoinductive mineralized carriers for human periosteal-derived progenitors (hPDCs). For this, three medium formulations [i.e., growth medium only (BM1), with ascorbic acid (BM2), and with ascorbic acid and dexamethasone (BM3)] supplemented with calcium (Ca2+) and phosphate (PO43−) ions simultaneously as mineralizing source were investigated. The results showed that, besides the significant impacts on enhancing cell proliferation (the highest in BM3 condition), the formulated mineralizing media differentially regulated the osteochondro-related gene markers in a medium-dependent manner (e.g., significant upregulation of BMP2, bone sialoprotein, osteocalcin, and Wnt5a in BM2 condition). This has resulted in distinguished cell populations that were identifiable by specific gene signatures as demonstrated by the principle component analysis. Through devitalization, mineralized carriers with apatite crystal structures unique to each medium condition (by X-ray diffraction and SEM analysis) were obtained. Quantitatively, BM3 condition produced carriers with the highest mineral and collagen contents as well as human-specific VEGF proteins, followed by BM2 and BM1 conditions. Encouragingly, all mineralized carriers (after reseeded with hPDCs) induced bone formation after 8 weeks of subcutaneous implantation in nude mice models, with BM2-carriers inducing the highest bone volume, and the lowest in the BM3 condition (as quantitated by nano-computed tomography [nano-CT]). Histological analysis revealed different bone formation patterns, either bone ossicles containing bone marrow surrounding the scaffold struts (in BM2) or bone apposition directly on the struts' surface (in BM1 and BM3). In conclusion, we have presented experimental data on the feasibility to produce devitalized osteoinductive mineralized carriers by functionalizing 3D porous scaffolds with an in vitro cell-made mineralized matrix under the mineralizing culture conditions.
PMCID: PMC4245878  PMID: 25469312
biomineralization; bone regeneration; devitalization; mineralized extracellular matrix; osteoinductive carriers
14.  Ultra-structural defects cause low bone matrix stiffness despite high mineralization in osteogenesis imperfecta mice☆ 
Bone  2012;50(6):1317-1323.
Bone is a complex material with a hierarchical multi-scale organization from the molecule to the organ scale. The genetic bone disease, osteogenesis imperfecta, is primarily caused by mutations in the collagen type I genes, resulting in bone fragility. Because the basis of the disease is molecular with ramifications at the whole bone level, it provides a platform for investigating the relationship between structure, composition, and mechanics throughout the hierarchy. Prior studies have individually shown that OI leads to: 1. increased bone mineralization, 2. decreased elastic modulus, and 3. smaller apatite crystal size. However, these have not been studied together and the mechanism for how mineral structure influences tissue mechanics has not been identified. This lack of understanding inhibits the development of more accurate models and therapies. To address this research gap, we used a mouse model of the disease (oim) to measure these outcomes together in order to propose an underlying mechanism for the changes in properties. Our main finding was that despite increased mineralization, oim bones have lower stiffness that may result from the poorly organized mineral matrix with significantly smaller, highly packed and disoriented apatite crystals. Using a composite framework, we interpret the lower oim bone matrix elasticity observed as the result of a change in the aspect ratio of apatite crystals and a disruption of the crystal connectivity.
► We have investigated osteogenesis imperfecta mice (oim) bone matrix. ► oim mice have lower matrix stiffness and higher mineralization than wild type mice. ► Poor correlation found between stiffness and mineralization. ► Changes in apatite crystal structure and connectivity may play a role the degradation of material properties despite higher density.
PMCID: PMC3407875  PMID: 22449447
Osteogenesis imperfecta; Bone matrix; Stiffness; Mineralization; Mouse model
15.  Biological assemblies provide novel templates for the synthesis of hierarchical structures and facilitate cell adhesion** 
Advanced functional materials  2008;18(24):3972-3980.
Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the self-assembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic “bottom-up” construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.
Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the self-assembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic “bottom-up” construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.
PMCID: PMC2746078  PMID: 19768126
dentin matrix protein 1; leucine zipper; self-assembly; cell adhesion; mineralized matrix
16.  Rapid prototyped porous nickel–titanium scaffolds as bone substitutes 
Journal of Tissue Engineering  2014;5:2041731414540674.
While calcium phosphate–based ceramics are currently the most widely used materials in bone repair, they generally lack tensile strength for initial load bearing. Bulk titanium is the gold standard of metallic implant materials, but does not match the mechanical properties of the surrounding bone, potentially leading to problems of fixation and bone resorption. As an alternative, nickel–titanium alloys possess a unique combination of mechanical properties including a relatively low elastic modulus, pseudoelasticity, and high damping capacity, matching the properties of bone better than any other metallic material. With the ultimate goal of fabricating porous implants for spinal, orthopedic and dental applications, nickel–titanium substrates were fabricated by means of selective laser melting. The response of human mesenchymal stromal cells to the nickel–titanium substrates was compared to mesenchymal stromal cells cultured on clinically used titanium. Selective laser melted titanium as well as surface-treated nickel–titanium and titanium served as controls. Mesenchymal stromal cells had similar proliferation rates when cultured on selective laser melted nickel–titanium, clinically used titanium, or controls. Osteogenic differentiation was similar for mesenchymal stromal cells cultured on the selected materials, as indicated by similar gene expression levels of bone sialoprotein and osteocalcin. Mesenchymal stromal cells seeded and cultured on porous three-dimensional selective laser melted nickel–titanium scaffolds homogeneously colonized the scaffold, and following osteogenic induction, filled the scaffold’s pore volume with extracellular matrix. The combination of bone-related mechanical properties of selective laser melted nickel–titanium with its cytocompatibility and support of osteogenic differentiation of mesenchymal stromal cells highlights its potential as a superior bone substitute as compared to clinically used titanium.
PMCID: PMC4221926  PMID: 25383165
Bone tissue engineering; osteogenic differentiation; nickel–titanium; scaffold; selective laser melting
17.  Bisphenyl-Polymer/Carbon-Fiber-Reinforced Composite Compared to Titanium Alloy Bone Implant 
Aerospace/aeronautical thermoset bisphenyl-polymer/carbon-fiber-reinforced composites are considered as new advanced materials to replace metal bone implants. In addition to well-recognized nonpolar chemistry with related bisphenol-polymer estrogenic factors, carbon-fiber-reinforced composites can offer densities and electrical conductivity/resistivity properties close to bone with strengths much higher than metals on a per-weight basis. In vivo bone-marrow tests with Sprague-Dawley rats revealed far-reaching significant osseoconductivity increases from bisphenyl-polymer/carbon-fiber composites when compared to state-of-the-art titanium-6-4 alloy controls. Midtibial percent bone area measured from the implant surface increased when comparing the titanium alloy to the polymer composite from 10.5% to 41.6% at 0.8 mm, P < 10−4, and 19.3% to 77.7% at 0.1 mm, P < 10−8. Carbon-fiber fragments planned to occur in the test designs, instead of producing an inflammation, stimulated bone formation and increased bone integration to the implant. In addition, low-thermal polymer processing allows incorporation of minerals and pharmaceuticals for future major tissue-engineering potential.
PMCID: PMC4278381  PMID: 25553057
18.  Sciatic nerve regeneration in rats by a promising electrospun collagen/poly(ε-caprolactone) nerve conduit with tailored degradation rate 
BMC Neuroscience  2011;12:68.
To cope with the limitations faced by autograft acquisitions particularly for multiple nerve injuries, artificial nerve conduit has been introduced by researchers as a substitute for autologous nerve graft for the easy specification and availability for mass production. In order to best mimic the structures and components of autologous nerve, great efforts have been made to improve the designation of nerve conduits either from materials or fabrication techniques. Electrospinning is an easy and versatile technique that has recently been used to fabricate fibrous tissue-engineered scaffolds which have great similarity to the extracellular matrix on fiber structure.
In this study we fabricated a collagen/poly(ε-caprolactone) (collagen/PCL) fibrous scaffold by electrospinning and explored its application as nerve guide substrate or conduit in vitro and in vivo. Material characterizations showed this electrospun composite material which was made of submicron fibers possessed good hydrophilicity and flexibility. In vitro study indicated electrospun collagen/PCL fibrous meshes promoted Schwann cell adhesion, elongation and proliferation. In vivo test showed electrospun collagen/PCL porous nerve conduits successfully supported nerve regeneration through an 8 mm sciatic nerve gap in adult rats, achieving similar electrophysiological and muscle reinnervation results as autografts. Although regenerated nerve fibers were still in a pre-mature stage 4 months postoperatively, the implanted collagen/PCL nerve conduits facilitated more axons regenerating through the conduit lumen and gradually degraded which well matched the nerve regeneration rate.
All the results demonstrated this collagen/PCL nerve conduit with tailored degradation rate fabricated by electrospinning could be an efficient alternative to autograft for peripheral nerve regeneration research. Due to its advantage of high surface area for cell attachment, it is believed that this electrospun nerve conduit could find more application in cell therapy for nerve regeneration in future, to further improve functional regeneration outcome especially for longer nerve defect restoration.
PMCID: PMC3148572  PMID: 21756368
19.  Surface Oxide Net Charge of a Titanium Alloy; Comparison Between Effects of Treatment With Heat or Radiofrequency Plasma Glow Discharge 
In the current study, we have compared the effects of heat and radiofrequency plasma glow discharge (RFGD) treatment of a Ti6Al4V alloy on the physico-chemical properties of the alloy’s surface oxide. Titanium alloy (Ti6Al4V) disks were passivated alone, heated to 600 °C, or RFGD plasma treated in pure oxygen. RFGD treatment did not alter the roughness, topography, elemental composition or thickness of the alloy’s surface oxide layer. In contrast, heat treatment altered oxide topography by creating a pattern of oxide elevations approximately 50–100 nm in diameter. These nanostructures exhibited a three-fold increase in roughness compared to untreated surfaces when RMS roughness was calculated after applying a spatial high-pass filter with a 200 nm cutoff wavelength. Heat treatment also produced a surface enrichment in aluminum and vanadium oxides. Both RFGD and heat treatment produced similar increases in oxide wettability. Atomic force microscopy (AFM) measurements of metal surface oxide net charge signified by a long range force of attraction to or repulsion from a (negatively charged) silicon nitride AFM probe were also obtained for all three experimental groups. Force measurements showed that the RFGD-treated Ti6Al4V samples demonstrated a higher net positive surface charge at pH values below 6 and a higher net negative surface charge at physiological pH (pH values between 7 and 8) compared to control and heat-treated samples These findings suggest that RFGD treatment of metallic implant materials can be used to study the role of negatively charged surface oxide functional groups in protein bioactivity, osteogenic cell behavior and osseointegration independently of oxide topography.
PMCID: PMC2966390  PMID: 20880672
titanium alloy; oxides; surface treatments; net surface charge; wettability
20.  Self-setting collagen-calcium phosphate bone cement: Mechanical and cellular properties 
Calcium phosphate cement (CPC) can conform to complex bone cavities and set in-situ to form bioresorbable hydroxyapatite. The aim of this study was to develop a CPC-collagen composite with improved fracture resistance, and to investigate the effects of collagen on mechanical and cellular properties. A type-I bovine-collagen was incorporated into CPC. MC3T3-E1 osteoblasts were cultured. At CPC powder/liquid mass ratio of 3, the work-of-fracture (mean±sd; n=6) was increased from (22±4) J/m2 at 0% collagen, to (381±119) J/m2 at 5% collagen (p≤0.05). At 2.5–5% of collagen, the flexural strength at powder/liquid ratios of 3 and 3.5 was 8–10 MPa. They matched the previously-reported 2–11 MPa of sintered porous hydroxyapatite implants. SEM revealed that the collagen fibers were covered with nano-apatite crystals and bonded to the CPC matrix. Higher collagen content increased the osteoblast cell attachment (p≤0.05). The number of live cells per specimen area was (382±99) cells/mm2 on CPC containing 5% collagen, higher than (173±42) cells/mm2 at 0% collagen (p≤0.05). The cytoplasmic extensions of the cells anchored to the nano-apatite crystals of the CPC matrix. In summary, collagen was incorporated into in situ-setting, nano-apatitic CPC, achieving a 10-fold increase in work-of-fracture (toughness) and 2-fold increase in osteoblast cell attachment. This moldable/injectable, mechanically-strong, nano-apatite-collagen composite may enhance bone regeneration in moderate stress-bearing applications.
PMCID: PMC2749899  PMID: 18985758
calcium phosphate bone cement; collagen; cell attachment; cell density; strength; work-of-fracture; bone tissue engineering
21.  Material Properties and Osteogenic Differentiation of Marrow Stromal Cells on Fiber-Reinforced Laminated Hydrogel Nanocomposites 
Acta biomaterialia  2009;6(6):1992-2002.
The fibrils in the bone matrix are glued together by ECM proteins to form laminated structures (osteons) to provide elasticity and a supportive substrate for osteogenesis. The objective of this work was to investigate material properties and osteogenic differentiation of bone marrow stromal (BMS) cells seeded on osteon-mimetic fiber-reinforced hydrogel/apatite composites. Layers of electrospun poly(L-lactide) (L-PLA) fiber mesh coated with a poly(lactide-co-ethylene oxide fumarate) (PLEOF) hydrogel precursor solution were stacked and pressed together, and crosslinked to produce a laminated fiber-reinforced composite. Hydroxyapatite (HA) nanocrystals were added to the precursor solution to produce an osteoconductive matrix for BMS cells. Acrylamide-terminated RGD peptide (Ac-GRGD) was conjugated to the PLEOF/HA hydrogel phase to promote focal point adhesion of BMS cells. Laminates were characterized with respect to Young’s modulus, degradation kinetics, and osteogenic differentiation of BMS cells. The moduli of the laminates under dry and wet conditions were significantly higher than those of the fiber mesh and PLEOF/HA hydrogel, and within the range of values reported for wet human cancellous bone. At days 14 and 21, ALPase activity of the laminates was significantly higher than those of the fiber mesh and hydrogel. Lamination significantly increased the extent of mineralization of BMS cells and laminates with HA and conjugated with RGD (Lam-RGD-HA) had 2.7-, 3.5-, and 2.8-fold higher calcium content (compared to laminates without HA or RGD) after 7, 14, and 21 days, respectively. The Lam-RGD-HA group had significantly higher expression of osteopontin (OP) and osteocalcin (OC) compared to the hydrogel or laminates without HA or RGD, consistent with the higher ALPase activity and calcium content of Lam-RGD-HA. Laminated osteon-mimetic structures have the potential to provide mechanical strength to the regenerating region as well as supporting the differentiation of progenitor cells to the osteogenic lineage.
PMCID: PMC2862832  PMID: 19995620
22.  Enzyme Directed Templating of Artificial Bone Mineral** 
Bone is one of Nature’s most remarkable materials, not only for its mechanical properties but also for its ability to repair fractures and remodel its microstructure in response to stress. At the nanoscale bone is a supramolecular matrix of collagen fibers reinforced by hydroxyapatite crystals with a high degree of order. Emulating elements of the biological synthesis of this composite could help develop strategies for advanced materials. Previous work has demonstrated the use of functionalized peptide amphiphile nanofibers in a two-dimensional system to emulate hydroxyapatite mineralization in natural bone. We describe here an artificial, in vitro biomineralization process that allows a similar process to occur in three dimensions. The system employs the natural enzyme alkaline phosphatase and a phosphorylated, anionic nanofiber gel matrix to template hydroxyapatite nanocrystals with size, shape, and crystallographic orientation resembling natural bone mineral. The formation of this biomimetic mineral in three dimensions results from the synergy of fiber-induced nucleation and the temporal control of phosphate ion harvesting by the enzyme. Gradual enzymatic harvesting of ions for crystal growth and the strong nucleating ability of the phosphorylated fibers suppresses uncontrolled precipitation of mineral. The strategy could lead to biomimetic materials to promote bone regeneration or the synthesis of hybrid materials with crystallographically defined structures.
PMCID: PMC3075609  PMID: 22068437
Biomineralization; Hydroxyapatite Nanocrystals; Supramolecular Materials; Synthetic Biology; Gels
23.  Hypermineralized whale rostrum as the exemplar for bone mineral 
Connective tissue research  2013;54(3):167-175.
Although bone is a nanocomposite of mineral and collagen, mineral has been the more elusive component to study. A standard for bone mineral clearly is needed. We hypothesized that the most natural, least-processed bone mineral could be retrieved from the most highly mineralized bone. We therefore studied the rostrum of the toothed whale Mesoplodon densirostris, which has the densest recognized bone. Essential to establishment of a standard for bone mineral is documentation that the proposed tissue is bone-like in all properties except for its remarkably high concentration of mineral. Transmitted-light microscopy of unstained sections of rostral material shows normal bone morphology in osteon geometry, lacunae concentration, and vasculature development. Stained sections reveal extremely low density of thin collagen fibers throughout most of the bone, but enrichment in and thicker collagen fibers around vascular holes and in a minority of osteons. FE-SEM shows the rostrum to consist mostly of dense mineral prisms. Most rostral areas have the same chemical-structural features, Raman spectroscopically dominated by strong bands at ~962 Δcm−1 and weak bands at ~2940 Δcm−1. Spectral features indicate that the rostrum is composed mainly of the calcium phosphate mineral apatite and has only about 4 wt.% organic content. The degree of carbonate substitution (~8.5 wt.% carbonate) in the apatite is in the upper range found in most types of bone. We conclude that, despite its enamel-like extraordinarily high degree of mineralization, the rostrum is in all other features bone-like. Its mineral component is the long-sought uncontaminated, unaltered exemplar of bone mineral.
PMCID: PMC3789591  PMID: 23586370
bone mineral; rostrum; apatite; histology; scanning electron microscopy; Raman spectroscopy
24.  Gelatin-apatite bone mimetic co-precipitates incorporated within biopolymer matrix to improve mechanical and biological properties useful for hard tissue repair 
Journal of Biomaterials Applications  2014;28(8):1213-1225.
Synthetic biopolymers are commonly used for the repair and regeneration of damaged tissues. Specifically targeting bone, the composite approach of utilizing inorganic components is considered promising in terms of improving mechanical and biological properties. We developed gelatin-apatite co-precipitates which mimic the native bone matrix composition within poly(lactide-co-caprolactone) (PLCL). Ionic reaction of calcium and phosphate with gelatin molecules enabled the co-precipitate formation of gelatin-apatite nanocrystals at varying ratios. The gelatin-apatite precipitates formed were carbonated apatite in nature, and were homogeneously distributed within the gelatin matrix. The incorporation of gelatin-apatite significantly improved the mechanical properties, including tensile strength, elastic modulus and elongation at break, and the improvement was more pronounced as the apatite content increased. Of note, the tensile strength increased to as high as 45 MPa (a four-fold increase vs. PLCL), the elastic modulus was increased up to 1500 MPa (a five-fold increase vs. PLCL), and the elongation rate was ∼240% (twice vs. PLCL). These results support the strengthening role of the gelatin-apatite precipitates within PLCL. The gelatin-apatite addition considerably enhanced the water affinity and the acellular mineral-forming ability in vitro in simulated body fluid; moreover, it stimulated cell proliferation and osteogenic differentiation. Taken together, the GAp-PLCL nanocomposite composition is considered to have excellent mechanical and biological properties, which hold great potential for use as bone regenerative matrices.
PMCID: PMC4107824  PMID: 23985536
Composite materials; bone mimetic; bioactive ceramics; polymer matrix; mechanical properties; bone regeneration
25.  Novel bioactive Co-based alloy/FA nanocomposite for dental applications 
Dental Research Journal  2012;9(2):173-179.
Dental cobalt base alloys are biocompatible dental materials and have been widely used in dentistry. However, metals are bioinert and may not present bioactivity in human body. Bioactivity is the especial ability to interact with human body and make a bonding to soft and hard tissues. The aim of the present research was fabrication and bioactivity evaluation of novel cobalt alloy/Fluorapatite nanocomposite (CoA/FaNC) with different amounts of Fluorapatite (FA) nanopowder.
Materials and Methods:
Co-Cr-Mo alloy (ASTM F75) powder was prepared and mixed in a planetary ball mill with different amounts of FA nanopowders (10, 15, 20% wt). Prepared composite powders were cold pressed and sintered at 1100°C for 4 h. X-ray diffraction (XRD), scanning electron microscopy and transition electron microscopy techniques were used for phase analysis, crystallite size determination of FA and also for phase analysis and evaluation of particle distribution of composites. Bioactivity behavior of prepared nanocomposites was evaluated in simulated body fluid (SBF) for 1 up to 28 days.
Materials and Methods:
Co-Cr-Mo alloy (ASTM F75) powder was prepared and mixed in a planetary ball mill with different amounts of FA nanopowders (10, 15, 20% wt). Prepared composite powders were cold pressed and sintered at 1100°C for 4 h. X-ray diffraction (XRD), scanning electron microscopy and transition electron microscopy techniques were used for phase analysis, crystallite size determination of FA and also for phase analysis and evaluation of particle distribution of composites. Bioactivity behavior of prepared nanocomposites was evaluated in simulated body fluid (SBF) for 1 up to 28 days.
Results showed that nucleus of apatite were formed on the surface of the prepared CoA/FaNC during 1 up to 28 days immersion in the SBF solution. On the other hand, CoA/FaNC unlike Co-base alloy possessed bone-like apatite-formation ability.
It was concluded that bioinert Co-Cr-Mo alloy could be successfully converted into bioactive nanocomposite by adding 10, 15, 20 wt% of FA nano particles.
PMCID: PMC3353694  PMID: 22623934
Bioactivity; Co-Cr-Mo alloy; FA nanopowder; nanocomposite

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