Fabrication of an rhBMP-2 loaded porous β-TCP microsphere-hyaluronic acid-based powder gel composite and evaluation of implant osseointegration

Methods to improve osseointegration that include implantation of rhBMP-2 with various kinds of carriers are currently of considerable interest. The present study was conducted to evaluate if the rhBMP-2 loaded β-TCP microsphere-hyaluronic acid-based powder-like hydrogel composite (powder gel) can act as an effective rhBMP-2 carrier for implantation in host bone with a bone defect or poor bone quality. The release pattern for rhBMP-2 was then evaluated against an rhBMP-2-loaded collagen sponge as a control group. Dental implants were also inserted into the tibias of three groups of rabbits: an rhBMP-2 (200 µg) loaded powder gel composite implanted group, an implant only group, and a powder gel implanted group. Micro-CT and histology of the implanted areas were carried out four weeks later. The rhBMP-2 powder gel released less rhBMP-2 than the collagen sponge, but it continued a slow release for more than 7 days. The rhBMP-2 powder gel composite improved osseointegration of the dental implant by increasing the amount of new bone formation in the implant pitch and it improved the bone quality and bone quantity of new bone. The histology results indicated that the rhBMP-2 powder gel composite improved the osseointegration in the cortical bone as well as the marrow space along the fixture. The bone-to-implant contact ratio of the rhBMP-2 (200 µg) loaded powder gel composite implanted group was significantly higher than those of the implant only group and the powder gel implanted group. The powder gel appeared to be a good carrier and could release rhBMP-2 slowly to promote the formation of new bone following implantation in a bone defect, thereby improving implant osseointegration.     

生物可吸收储存式药物控释载体制备研究

针对骨科疾病治疗中传统的用药方式存在的问题,研究制备了一种生物可吸收靶向式药物控释载体。首先在体外模拟天然骨的生物矿化过程,利用材料的自组装机制合成了羟基磷灰石／胶原类骨仿生复合材料,再采用热致分相／非溶剂抽提成孔技术进一步与聚乳酸复合制备了三维多孔储存式药物控释载体。通过对材料的表征和模型化合物控释实验,结果显示羟基磷灰石／胶原复合材料与天然骨类似,羟基磷灰石／胶原／聚乳酸储存式载体能达到控制释放的目的。     

Controlled release of small molecules from silica xerogel with limited nanoporosity

Conventional sol–gel processing requires several distinct steps involving hydrolysis, condensation and drying to obtain a highly porous, glassy solid material. With the goal of achieving controlled release of small molecules, herein we focus on the acceleration of the condensation and drying steps by casting the hydrolyzed sol on a large open surface to achieve a denser 100 % silica xerogel structure. Thus, cast xerogel with a more limited porosity was prepared. The effect of synthesis parameters during sol–gel synthesis on the release kinetics of bupivacaine, vancomycin and cephalexin was investigated. The release kinetics fitted well with the Higuchi model, suggesting a diffusional release mechanism. Combining the release and nanostructure data, the formation mechanism of cast xerogel is described. Without introducing additional precursors or additives into sol–gel systems, sol–gel casting is an easy technique that further expands the applicability of sol–gel materials as excellent carriers for the controlled release of a variety of drugs.     

Heparin-functionalized collagen matrices with controlled release of basic fibroblast growth factor

Tissue engineering scaffolds with controlled long-term release of growth factors are constructed in an attempt to mimic the intelligent ability of the extracellular matrix (ECM) to release endogenous growth factors. In this study, collagen sponges (Collagen group) were modified by N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) crosslinking (EDC/NHS group) and heparin immobilization (EDC/NHS-H group), and subsequently seeded with human umbilical vein endothelial cells (HUVECs). Native and modified sponges were pre-adsorbed with basic fibroblast growth factor (bFGF) to evaluate the sustained release and bioactive maintenance of bFGF from the sponges. We found that modified collagen matrices permitted HUVECs to proliferate and migrate well and to distribute uniformly. The EDC/NHS-H group exhibited an excellent sustained-release profile and bioactive maintenance of the pre-adsorbed bFGF as compared with the Collagen and EDC/NHS groups. These results suggest that heparin-functionalized collagen matrices can support a controlled release of bFGF and thus, have potential as a tissue engineering scaffold.     

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled‐release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine.     

Preparation and characterization of collagen microspheres for sustained release of VEGF

In this study, we prepared injectable collagen microspheres for the sustained delivery of recombinant human vascular endothelial growth factor (rhVEGF) for tissue engineering. Collagen solution was formed into microspheres under a water-in-oil emulsion condition, followed by crosslinking with water-soluble carbodiimide. Various sizes of collagen microspheres in the range of 1–30 μm diameters could be obtained by controlling the surfactant concentration and rotating speed of the emulsified mixture. Particle size proportionally decreased with increasing the rotating speed (1.8 μm per 100 rpm increase in the range of 300–1,200 rpm) and surfactant concentration (3.1 μm per 0.1% increase in the range of 0.1–0.5%). The collagen microspheres showed a slight positive charge of 8.86 and 3.15 mV in phosphate-buffered saline and culture medium, respectively. Release study showed the sustained release of rhVEGF for 4 weeks. Released rhVEGF was able to induce capillary formation of human umbilical vein endothelial cells, indicating the maintenance of rhVEGF bioactivity after release. In conclusion, the results suggest that the collagen microspheres have potential for sustained release of rhVEGF.     

Tamibarotene-loaded citric acid-crosslinked alkali-treated collagen matrix as a coating material for a drug-eluting stent

Abstract

Tamibarotene-loaded biodegradable matrices with antithrombogenic and drug-releasing properties were prepared in a crosslinking reaction between amino groups of alkali-treated collagen (AlCol) and active ester groups of trisuccinimidyl citrate. The resulting matrices were characterized by their residual amino group concentrations, swelling ratios and thermal, antithrombogenic and drug-releasing properties. It was clarified that the addition of tamibarotene does not inhibit matrix formation. After immersion in water, the swelling ratio of a matrix became lower than that prior to immersion. Thermal analysis indicated that AlCol interacted with tamibarotene. The addition of tamibarotene to the matrix did not influence the antithrombogenic property of the resulting matrix. A matrix with a high crosslinking density had a prolonged tamibarotene elution time. These results demonstrate that tamibarotene-loaded matrices have great potential as a coating material for drug-eluting stents.     

Myocardial‐Infarction‐Responsive Smart Hydrogels Targeting Matrix Metalloproteinase for On‐Demand Growth Factor Delivery

Although in situ restoration of blood supply to the infarction region and attenuating pre‐existing extracellular matrix degradation remain potential therapeutic approaches for myocardial infarction (MI), local delivery of therapeutics has been limited by low accumulation (inefficacy) and unnecessary diffusion (toxicity). Here, a dual functional MI‐responsive hydrogel is fabricated for on‐demand drug delivery to promote angiogenesis and inhibit cardiac remodeling by targeting upregulated matrix metalloproteinase‐2/9 (MMP‐2/9) after MI. A glutathione (GSH)‐modified collagen hydrogel (collagen‐GSH) is prepared by conjugating collagen amine groups with GSH sulfhydryl groups and the recombinant protein GST‐TIMP‐bFGF (bFGF: basic fibroblast growth factor) by fusing bFGF with glutathione‐S‐transferase (GST) and MMP‐2/9 cleavable peptide PLGLAG (TIMP). Specific binding between GST and GSH significantly improves the amount of GST‐TIMP‐bFGF loaded in collagen‐GSH hydrogel. The TIMP peptide enclosed between GST and bFGF responds to MMPs for on‐demand release during MI. Additionally, the TIMP peptide is a competitive substrate of MMPs that inhibits the excessive degradation of cardiac matrix by MMPs after MI. GST‐TIMP‐bFGF/collagen‐GSH hydrogels promote the recovery of MI rats by enhancing vascularization and ameliorating myocardium remodeling. The results suggest that on‐demand growth factor delivery by synchronously controlling binding and responsive release to promote angiogenesis and attenuate cardiac remodeling might be promising for the treatment of ischemic heart disease.     

Self-assembly of mineralized collagen composites

This paper presents a review of the current understanding of the structure, self-assembly mechanisms, and properties of mineralized collagen fibril composites in connective tissues, such as in lamellar bones, woven bones, zebrafish skeletal bone, and ivory. Recent work involving biomimetic synthesis of new materials with the structure of mineralized collagen is described. The focus in the paper is mainly on materials containing type I collagen, with mineralization by Ca–P crystals although some other systems are also described. Investigation and simulation of naturally occurring fibril structures can offer some new ideas in the design and fabrication of new functional materials, for applications such as bone grafts or for use as scaffolds in tissue engineering and biomimetic engineering materials. The development of bone grafts based on the mineralization of self-assembled collagen fibrils in vivo and in vitro is an active area of research. This kind of bone graft composite has already shown great promise and success in clinical applications, on account of its compositional and structural similarity to autologous bone. It is suggested that future work in this should focus on both basic theoretical aspects as well as the development of applications. In particular issues including control of morphology, incorporation of foreign ions, interaction with biomolecules, and the assembly of organic and inorganic phases are all still not well understood. In the area of applications, the design of composite materials with a hierarchical structure closer to that of natural hard tissues, and the synthesis of bone grafts and tooth regenerative materials, as well as biomimetic functional materials, are areas currently being examined by many research groups.     

Growth and phenotypic expression of human endothelial cells cultured on a glass-reinforced hydroxyapatite

Glass-reinforced hydroxyapatite composites (GR-HA) are bone regenerative materials that are characterized by their increased mechanical properties, when compared to synthetic hydroxyapatite. Bonelike is a GR-HA that is a result of the addition of a CaO-P(2)O(5) based glass to a HA matrix. This biomaterial has been successfully applied in clinical bone regenerative applications. This work aims to evaluate the ability of Bonelike to support the adhesion, proliferation and phenotypic expression of human endothelial cells, aiming to establish new bone tissue engineering pre-endothelialization strategies. Bonelike discs, regardless of being submitted to a pre-immersion treatment with culture medium, were seeded with first passage human umbilical vein endothelial cells, and characterized regarding proliferation and differentiation events. Pre-immersed Bonelike allowed the adhesion, proliferation and phenotype expression of endothelial cells. Seeded materials presented positive immunofluorescent staining for PECAM-1 and a tendency for the formation of cord-like arrangements under angiogenesis-stimulating conditions, although, compared to standard culture plates, a slight decreased cell growth was observed. In this way, Bonelike may be a suitable candidate for pre-endothelialization approaches in bone tissue engineering applications.     

Bone regeneration potential of a soybean-based filler: experimental study in a rabbit cancellous bone defects

Autologous and allogenic bone grafts are considered as materials of choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of alternative biomaterials. The present work compares the bone regeneration potential of a soybean based bone filler (SB bone filler) in comparison to a commercial 50:50 poly(d,l lactide–glycolide)-based bone graft (Fisiograft^® gel) when implanted into a critical size defect (6-mm diameter, 10-mm length) in rabbit distal femurs. The histomorphometric and microhardness analyses of femoral condyles 4, 8, 16 and 24 weeks after surgery showed that no significant difference was found in the percentage of both bone repair and bone in-growth in the external, medium and inner defect areas. The SB filler-treated defects showed significantly higher outer bone formation and microhardness results at 24 weeks than Fisiograft^® gel (P < 0.05). Soybean-based biomaterials clearly promoted bone repair through a mechanism of action that is likely to involve both the scaffolding role of the biomaterial for osteoblasts and the induction of their differentiation.     

体外壳聚糖-明胶-生长因子缓释支架对骨髓间充质干细胞增殖的影响

制备具有缓释功能的壳聚糖-明胶-碱性成纤维细胞生长因子(bFGF)-肝细胞生长因子(HGF)三维大孔支架,探讨两种生长因子在体外对骨髓间充质干细胞(BMSCs)增殖的协同作用。方法:采用冷冻干燥法,将不同比例的壳聚糖、明胶、bFGF和HGF依次混合,使其成为具有一定孔径的三维缓释支架。取SD大鼠乳鼠股骨和胫骨骨髓,分离、培养BMSCs。取生长状态良好的第三代BMSCs,将其接种于96孔板后,加入支架混合培养,5d后进行MTT细胞增殖测定。结果:在与含1μg/mL的bFGF支架混合培养,以及与同时含1μg/mL的bFGF、1μg/mL的HGF支架混合培养后,BMSCs增殖明显(P<0.05),但这二组间无统计学差异(P>0.05)。分别与含0.1μg/mL的bFGF支架、0.01μg/mL的bFGF支架、1μg/mL的HGF支架混合培养后,细胞增殖无统计学意义(P>0.05)。结论:壳聚糖-明胶可作为生长因子缓释支架材料;bFGF具有促进BMSCs增殖的作用,促进作用的大小与加入bFGF的量有关;HGF对BMSCs不具有增殖作用;在实验浓度范围内bFGF和HGF体外促进BMSCs增殖上不具有协同性。     

New Composite Bone Cement Based on Hydroxyapatite and Nanosilver

Although total joint replacement surgery has become common in recent years, problems due to bacterial infection remain a significant complication following this procedure. One approach in our study was to obtain a self-cured bone cement based on hydroxyapatite with nanosilver (Hap-Ag) and ZrO₂ and polymer matrix based on 2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)-phenyl]propane/triethyleneglycol dimethacrylate. New materials were tested for: in vitro silver release, compressive strength (CS), compressive modulus (CM), and radiopacity. In vitro silver release increased in time and depended of silver content in cement. The highest silver release was registered for the cement with 1.26 wt% silver content. The results reveal that the CS for bone cement was between 133.37 and 146.70 MPa and CM was between 1.68 and 1.82 GPa (p > 0.05). A slow increasing of CM was registered for samples with 1.5/1 powder/liquid ratio. Addition of nanosilver and ZrO₂ increased radiopacity of experimental bone cement.     

The effect of ionic dissolution products of Ca–Sr–Na–Zn–Si bioactive glass on in vitro cytocompatibility

Many commercial bone grafts cannot regenerate healthy bone in place of diseased bone. Bioactive glasses have received much attention in this regard due to the ability of their ionic dissolution products to promote cell proliferation, cell differentiation and activate gene expression. Through the incorporation of certain ions, bioactive glasses can become therapeutic for specific pathological situations. Calcium–strontium–sodium–zinc–silicate glass bone grafts have been shown to release therapeutic levels of zinc and strontium, however the in vitro compatibility of these materials is yet to be reported. In this study, the in vitro cytocompatibility of three different calcium–strontium–sodium–zinc–silicate glasses was examined as a function of their ion release profiles, using Novabone® bioglass as a commercial comparison. Experimental compositions were shown to release Si⁴⁺ ranging from 1 to 81 ppm over 30 days; comparable or enhanced release in comparison to Novabone. The maximum Ca²⁺ release detected for experimental compositions was 9.1 ppm, below that reported to stimulate osteoblasts. Sr²⁺ release was within known therapeutic ranges, and Zn²⁺ release ranged from 0.5 to 1.4 ppm, below reported cytotoxic levels. All examined glass compositions show equivalent or enhanced in vitro compatibility in comparison to Novabone. Cells exposed to BT112 ionic products showed enhanced cell viabilities indicating cell proliferation was induced. The ion release profiles suggest this effect was due to a synergistic interaction between certain combinations and concentrations of ions. Overall, results indicate that the calcium–strontium–sodium–zinc–silicate glass compositions show equivalent or even enhanced in vitro compatibility compared to Novabone®.     

Bioglass as a carrier for reindeer bone protein extract in the healing of rat femur defect

Bioactive glasses have been developed as scaffolds for bone tissue engineering but combination with reindeer bone protein extract has not been evaluated. We investigated the effects of bone protein extract implants (5–40 mg dosages) with bioglass (BG) carrier on the healing of rat femur defects. Bioglass implants and untreated defects served as controls. All doses of extract increased bone formation compared with the control groups, and bone union was enhanced with doses of 10 mg or more. In comparison with untreated defect, mean cross-sectional bone area at the defect site was greater when implants with BG + 15 mg of extract or bioglass alone were used, bone density at the defect site was higher in all bioglass groups with and without bone extract, and the BG + 15 mg extract dosage marginally increased bone torsional stiffness in mechanical testing. Bioglass performed well as a carrier candidate for reindeer bone protein extract.     

Synthesis and characterization of mechanically strong carboxymethyl cellulose–gelatin–hydroxyapatite nanocomposite for load-bearing orthopedic application

Novel three-dimensional hybrid polymer–hydroxyapatite nanocomposites have been developed as load-bearing synthetic bone graft through in situ mineralization process, using natural polymers carboxymethyl cellulose (CMC) and gelatin (Gel) as matrix. This process is simple and does not involve any chemical cross-linker. Detailed structural and physicochemical characterization of the samples disclosed that incorporation of gelatin with CMC assists the formation of CMC-Gel polymeric network of new conformational structure through non-covalent interactions (H-bond). The formation of hydroxyapatite (HA) in this polymeric network was occurred in such a fashion that the HA serves as bridging molecule which strengthen the polymeric network more and formed a mechanically strong three-dimensional CMC-Gel-HA nanocomposite. The synthesized CMC-Gel-HA nanocomposites have compressive strength and modulus in the range of 40–86 MPa and 0.4–1.2 GPa, respectively, analogous to human cancellous as well as cortical bone. In vitro cell interaction of the synthesized nanocomposites with osteoblast-like MG-63 cells has been evaluated. Results showed that synthesized CMC-Gel-HA nanocomposite promote cells for high alkaline phosphatase activity and extracellular mineralization. Extracellular mineralization ability of nanocomposite was investigated by alizarin red staining and von Kossa staining. Biodegradable nature and bone apatite formation ability of CMC-Gel-HA nanocomposite under simulated physiological environment were investigated by different characterization processes. Results indicated that the synthesized CMC-Gel-HA nanocomposite has great potential to be used as regenerative bone graft in major load-bearing region.     

In vitro evaluation of bioactive strontium-based ceramic with rabbit adipose-derived stem cells for bone tissue regeneration

The development of bone replacement materials is an important objective in the field of orthopaedic surgery. Due to the drawbacks of treating bone defects with autografts, synthetic bone graft materials have become optional. So in this work, a bone tissue engineering approach with radiopaque bioactive strontium incorporated calcium phosphate was proposed for the preliminary cytocompatibility studies for bone substitutes. Accumulating evidence indicates that strontium containing biomaterials promote enhanced bone repair and radiopacity for easy imaging. Hence, strontium calcium phosphate (SrCaPO₄) and hydroxyapatite scaffolds have been investigated for its ability to support and sustain the growth of rabbit adipose-derived mesenchymal stem cells (RADMSCs) in vitro. They were characterized via Micro-CT for pore size distribution. Cells used were isolated from New Zealand White rabbit adipose tissue, characterized by FACS and via differentiation into the osteogenic lineage by alkaline phosphatase, Masson’s trichome, Alizarin Red and von Kossa staining on day 28. Material-cell interaction was observed by SEM imaging of cell morphology on contact with material. Live–Dead analysis was done by confocal laser scanning microscopy and cell cluster analysis via μCT. The in vitro biodegradation, elution and nucleation of apatite formation of the material was evaluated using simulated body fluid and phosphate buffered saline in static regime up to 28 days at 37 °C. These results demonstrated that SrCaPO₄ is a good candidate for bone tissue engineering applications and with osteogenically-induced RADMSCs, they may serve as potential implants for the repair of critical-sized bone defects.     

Comparative investigation of hydroxyapatite/collagen composites prepared by CaCl<Subscript>2</Subscript> addition at different time points in collagen self-assembly process

Mineralization of collagen is a common combination process mainly involving collagen self-assembly and hydroxyapatite formation. Previous measurement of turbidity showed that collagen self-assembly follows the nucleation–growth model. In the present study, 1.0 M CaCl₂ was mixed with 0.6 mg/mL collagen solution in PBS (10 mM phosphate, 80 mM NaCl, pH 7.2, and T = 35 °C) at the beginning of the lag period, at the beginning of the growth period, at t_1/2 (the time to reach half of the total turbidity change), and during the plateau period. Four different hydroxyapatite/collagen (HAp/COL) composites were prepared [COL (1), COL (2), COL (3), and COL (4)]. The optical densities increased with CaCl₂ addition, and a wave trough appeared in the mineralization kinetic curves because of amorphous/crystalline conversion. HAp formation was confirmed by Fourier transform infrared spectroscopy and X-ray diffraction measurements. Energy-dispersive spectroscopy results showed that the calcium/phosphorus ratio of COL (2) is close to that of human bone. Images obtained by scanning electron microscopy revealed that nanosized plate-like HAp of COL (2) formed and became uniformly embedded in collagen, whereas HAp formed large clusters in COL (1), COL (3), and COL (4). Quantitation analyses of collagen and HAp incorporated into composites showed that mineralization at different time points promote collagen fibril generation and have little impact on the HAp content. These results suggest that the composite fabricated by addition of CaCl₂ at the beginning of the growth period is a promising material for bone repair and implantation.     

In vitro studies of calcium phosphate silicate bone cements

A novel calcium phosphate silicate bone cement (CPSC) was synthesized in a process, in which nanocomposite forms in situ between calcium silicate hydrate (C–S–H) gel and hydroxyapatite (HAP). The cement powder consists of tricalcium silicate (C₃S) and calcium phosphate monobasic (CPM). During cement setting, C₃S hydrates to produce C–S–H and calcium hydroxide (CH); CPM reacts with the CH to precipitate HAP in situ within C–S–H. This process, largely removing CH from the set cement, enhances its biocompatibility and bioactivity. The testing results of cell culture confirmed that the biocompatibility of CPSC was improved as compared to pure C₃S. The results of XRD and SEM characterizations showed that CPSC paste induced formation of HAP layer after immersion in simulated body fluid for 7 days, suggesting that CPSC was bioactive in vitro. CPSC cement, which has good biocompatibility and low/no cytotoxicity, could be a promising candidate as biomedical cement.     

Mesoporous bioactive glass as a drug delivery system: fabrication, bactericidal properties and biocompatibility

Implant-associated infection remains a difficult medical problem in orthopaedic surgery. Here, we report on the fabrication of gentamicin-loaded mesoporous bioactive glass (Gent-MBG) for use as a controlled antibiotic delivery system to achieve the sustained release of antibiotics in the local sites of bone defects. The high surface area and mesoporous structure of MBG enable higher drug loading efficiency (79–83 %) than non-mesoporous biological glass (NBG) (18–19 %). Gent-MBG exhibits sustained drug release for more than 6 days, and this controlled release of gentamicin significantly inhibits bacterial adhesion and prevents biofilm formation by S. aureus (ATCC25923) and S. epidermidis (ATCC35984). Biocompatibility tests with human bone marrow stromal cells (hBMSCs) indicate that MBG has better biocompatibility than NBG. Therefore, Gent-MBG can be used as a controlled drug delivery system to prevent and/or treat orthopedic peri-implant infections.