Study on injectable and degradable cement of calcium sulphate and calcium phosphate for bone repair

Injectable calcium sulphate/phosphate cement (CSPC) with degradable characteristic was developed by introduction of calcium sulphate (CS) into calcium phosphate cement (CPC). The setting time, compressive strength, composition, degradation, cells and tissue responses to the CSPC were investigated. The results show that the injectable CSPC with optimum L/P ratio exhibited good injectability, and had suitable setting time and mechanical properties. Furthermore, the CSPC had good degradability and its degradation significantly faster than that of CPC in Tris–HCl solution. Cell culture results indicate that CSPC was biocompatible and could support MG63 cell attachment and proliferation. To investigate the in vivo biocompatibility and osteogenesis, the CSPC were implanted in the bone defects of rabbits. Histological evaluation shows that the introduction of CS into CPC enhanced the efficiency of new bone formation, and CSPC exhibited good biocompatibility, degradability and osteoconductivity with host bone in vivo. It can be concluded that the injectable CSPC had a significant clinical advantage over CPC, and might have potential to be applied in orthopedic, reconstructive and maxillofacial surgery, especially for minimally invasive techniques.     

Preparation and properties of an injectable thermo-sensitive double crosslinking hydrogel based on thiolated chitosan/beta-glycerophosphate

An injectable thermo-sensitive double crosslinking hydrogel based on thiolated chitosan (CS-TGA)/beta-glycerophosphate (β-GP) was prepared by combining physical and chemical crosslinking. The effect of the concentrations of CS-TGA and β-GP on gelation temperature of CS-TGA-GP hydrogel was investigated. The gelation and mechanical properties of CS-TGA-GP in situ gel system were studied by oscillatory rheology and unconfined compression testing. By the physical interaction of CS-TGA and β-GP, CS-TGA-GP system undergoes a fast gelation at body temperature within 2 min. In addition, CS-TGA-GP hydrogel contains a low concentration of β-GP, which significantly decreases the toxicity of the gel. Owing to the chemical crosslinking of disulfide bonds, the gel is durable and possesses high-mechanical strength without introducing any potential cytotoxicity. The integrity of CS-TGA-GP hydrogel maintains for more than 30 days both in vitro and in vivo, and the interior morphology visualized by scanning electron microscopy reveals that the gel has interconnected porous network structure. In vitro release behavior of protein from CS-TGA-GP hydrogel was investigated using BSA as model protein. There is a sustained protein release from the gel without any initial burst. In vitro cytotoxicity, hemolysis, and histopathological analysis reveal that the gel is biocompatible. These features indicate that CS-TGA-GP hydrogel is a promising candidate for injectable protein delivery system and biomedical applications.     

The gelation behaviors of the reactive blends of nylon1212 and functional elastomer

Studies on the gelation behaviors of the reactive blends of nylon1212 and functional elastomer were carried out. The results show that the curves of the storage modulus(G′)–frequency (ω) exhibit a gel plateau in the low ω region, and the transition from liquid-like to solid-like viscoelastic behaviors emerges with the concentration of SEBS-g-MA increasing. There exist the gelation behaviors in the blending process similar to those of crosslinking polymer. Based on Winter’s method, the gel point of blends is determined to be, φ_g = 17.45 wt%, and the corresponding value of tanδ is 1.44. The gel index n calculated is 0.61 and the gel strength S _g is 1.08 × 10⁴ Pa s^0.61. However, the non-reactive blends of nylon1212 and elastomer have no emergence of gelation behaviors. The morphology analysis shows that the gel point for the reactive blends is a threshold of cocontinuous morphology, and morphology analysis can also be a method to determine the gel point.     

Comparison of an experimental bone cement with a commercial control, Hydroset™

Glass polyalkenoate cements based on strontium calcium zinc silicate glasses (Zn-GPCs) and high molecular weight polyacrylic acids (PAA) (MW; 52,000–210,000) have been shown to exhibit mechanical properties and in vitro bioactivity suitable for arthroplasty applications. Unfortunately, these formulations exhibit working times and setting times which are too short for invasive surgical applications such as bone void filling and fracture fixation. In this study, Zn-GPCs were formulated using a low molecular weight PAA (MW; 12,700) and a modifying agent, trisodium citrate dihydrate (TSC), with the aim of improving the rheological properties of Zn-GPCs. These novel formulations were then compared with commercial self-setting calcium phosphate cement, Hydroset™, in terms of compressive strength, biaxial flexural strength and Young’s modulus, as well as working time, setting time and injectability. The novel Zn-GPC formulations performed well, with prolonged mechanical strength (39 MPa, compression) greater than both vertebral bone (18.4 MPa) and the commercial control (14 MPa). However, working times (2 min) and rheological properties of Zn-GPCs, though improved, require further modifications prior to their use in minimally invasive surgical techniques.     

Fabrication of calcium phosphate-calcium sulfate injectable bone substitute using chitosan and citric acid

In this study, an injectable bone substitute (IBS) consisting of citric acid, chitosan solution as the liquid phase and tetra calcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA) and calcium sulfate hemihydrate (CSH) powders as the solid phase was prepared. Four groups containing different percentages (0–30%) of calcium sulfate hemihydrate (CSH, CaSO₄ · 0.5H₂O) were investigated. Initial setting times for IBS with CSH were longer than those without CSH. The setting times for all compositions were in the range of 25–45 min. The injectability was improved by the addition of CSH in the present system. Scanning electron microscopy images showed that fiber-like crystallization appeared in the cements. The enhancement of crystallinity was confirmed by XRD profiles where the peak intensity of HAp increased with incubation time and the addition of CSH. Also, the compressive strength increased with the addition of CSH. The maximum compressive strength obtained for IBS was with 20% CSH after 28-day incubation in 100% humidity at 37°C.     

Evaluation of Injectable Composite Material Comprising Biphasic Bone Substitutes and Crosslinked Collagen

Bone tissue engineering has emerged as a promising approach to regenerate bone tissue, and injectable biomaterials have shown potential for bone regeneration applications due to their ease of administration and ability to fill irregularly shaped defects. This study aims to develop and characterize an injectable composite material comprising biphasic bone substitutes (BBS) and crosslinked porcine collagen type I for bone regeneration applications. The collagen is crosslinked via a UVA-riboflavin crosslinking strategy and evaluated by testing the physicochemical properties, including the rheological behavior, dynamic storage modulus (G′) and loss modulus (G″), and in vitro degradation process. The results show that the crosslinked collagen (xCol) exhibits suitable physicochemical properties for injectability and improved viscoelasticity and degradation resistance. Furthermore, xCol is then combined with BBS in a predetermined ratio, obtaining the injectable composite material. The biocompatibility of the materials is evaluated in vitro by XTT and BrdU assays on fibroblasts and preosteoblasts. The results demonstrate that the composite material is biocompatible and supporting pre-osteoblasts proliferation. In conclusion, the injectable composite material BBS-xCol has promising physiochemical and biological properties for bone regeneration applications. Further studies are warranted to evaluate its efficacy in vivo and optimize its composition for clinical translation.     

Evaluation of two novel aluminum-free,zinc-based glass polyalkenoate cements as alternatives to PMMA bone cement for use in vertebroplasty and balloon kyphoplasty

Vertebroplasty (VP) and balloon kyphoplasty (BKP) are now widely used for treating patients in whom the pain due to vertebral compression fractures is severe and has proved to be refractory to conservative treatment. These procedures involve percutaneous delivery of a bolus of an injectable bone cement either directly to the fractured vertebral body, VB (VP) or to a void created in it by an inflatable bone tamp (BKP). Thus, the cement is a vital component of both procedures. In the vast majority of VPs and BKPs, a poly(methyl methacrylate) (PMMA) bone cement is used. This material has many shortcomings, notably lack of bioactivity and very limited resorbability. Thus, there is room for alternative cements. We report here on two variants of a novel, bioactive, Al-free, Zn-based glass polyalkenoate cement (Zn-GPC), and how their properties compare to those of an injectable PMMA bone cement (SIMPL) that is widely used in VP and BKP. The properties determined were injectability, radiopacity, uniaxial compressive strength, and biaxial flexural modulus. In addition, we compared the compression fatigue lives of a validated synthetic osteoporotic VB model (a polyurethane foam cube with an 8 mm-diameter through-thickness cylindrical hole), at 0–2300 N and 3 Hz, when the hole was filled with each of the three cements. A critical review of the results suggests that the performance of each of the Zn-GPCs is comparable to that of SIMPL; thus, the former cements merit further study with a view to being alternatives to an injectable PMMA cement for use in VP and BKP.     

Injectable thermosensitive hydrogel based on chitosan and quaternized chitosan and the biomedical properties

A novel injectable thermosensitive hydrogel (CS–HTCC/α β-GP) was successfully designed and prepared using chitosan (CS), quaternized chitosan (HTCC) and α,β-glycerophosphate (α,β-GP) without any additional chemical stimulus. The gelation point of CS–HTCC/α β-GP can be set at a temperature close to normal body temperature or other temperature above 25°C. The transition process can be controlled by adjusting the weight ratio of CS to HTCC, or different final concentration of α,β-GP. The optimum formulation is (CS + HTCC) (2% w/v), CS/HTCC (5/1 w/w) and α,β-GP 8.33% or 9.09% (w/v), where the sol–gel transition time was 3 min at 37°C. The drug released over 3 h from the CS–HTCC/α,β-GP thermosensitive hydrogel in artificial saliva pH 6.8. In addition, CS–HTCC/α,β-GP thermosensitive hydrogel exhibited stronger antibacterial activity towards two periodontal pathogens (Porphyromonas gingivalis, P.g and Prevotella intermedia, P.i). CS–HTCC/α, β-GP thermosensitive hydrogel was a considerable candidate as a local drug delivery system for periodontal treatment.     

A novel sheep vertebral bone defect model for injectable bioactive vertebral augmentation materials

New injectable bone substitutes have been developed that are, unlike polymethylmethacrylate, biologically active and have an osteogenic effect leading to osteogenesis and bone remodeling for vertebroplasty or kyphoplasty. In this study, we developed a sheep vertebral bone defect model to evaluate the new bioactive materials and assessed the feasibility of the model in vivo. Bone voids were experimentally created on lumbar vertebrae L2–L5 with L1 and L6 left intact as a normal control in mature sheep. The defect vertebrae L2–L5 in each sheep were randomized to receive augmentation with calcium phosphate cement (CPC) or sham. Vertebrae (L1–L6) were collected after 2 and 24 weeks of the cement augmentation and their strength and stiffness, as well as osseointegration activity and biodegradability, were evaluated. Finally, CPC significantly improved the strength and stiffness of vertebrae but did not yet restore it to the normal level at 24 weeks. Osteogenesis occurred at a substantially high level after 24 weeks of CPC augmentation or sham. Therefore, the sheep vertebral model with one void, 6.0 mm in diameter and 15.0 mm in depth, is replicable and can be used for evaluating the new injectable bioactive materials in vertebral augmentation or reconstruction.     

Injectability of brushite-forming Mg-substituted and Sr-substituted α-TCP bone cements

The influence of magnesium- and strontium-substitutions on injectability and mechanical performance of brushite-forming α-TCP cements has been evaluated in the present work. The effects of Mg- and Sr-substitutions on crystalline phase composition and lattice parameters were determined through quantitative X-ray phase analysis and structural Rietveld refinement of the starting calcium phosphate powders and of the hardened cements. A noticeable dependence of injectability on the liquid-to-powder ratio (LPR), smooth plots of extrusion force versus syringe plunger displacement and the absence of filter pressing effects were observed. For LPR values up to 0.36 ml g⁻¹, the percentage of injectability was always higher and lower for Mg-containing cements and for Sr-containing cements, respectively, while all the pastes could be fully injected for LPR > 0.36 ml g⁻¹. The hardened cements exhibited relatively high wet compressive strength values (~17–25 MPa) being the Sr- and Mg-containing cements the strongest and the weakest, respectively, holding an interesting promise for uses in trauma surgery such as for filling bone defects and in minimally invasive techniques such as percutaneous vertebroplasty to fill lesions and strengthen the osteoporotic bone.     

Hyaluronan-based heparin-incorporated hydrogels for generation of axially vascularized bioartificial bone tissues: in vitro and in vivo evaluation in a PLDLLA–TCP–PCL-composite system

Smart matrices are required in bone tissue-engineered grafts that provide an optimal environment for cells and retain osteo-inductive factors for sustained biological activity. We hypothesized that a slow-degrading heparin-incorporated hyaluronan (HA) hydrogel can preserve BMP-2; while an arterio–venous (A–V) loop can support axial vascularization to provide nutrition for a bio-artificial bone graft. HA was evaluated for osteoblast growth and BMP-2 release. Porous PLDLLA–TCP–PCL scaffolds were produced by rapid prototyping technology and applied in vivo along with HA-hydrogel, loaded with either primary osteoblasts or BMP-2. A microsurgically created A–V loop was placed around the scaffold, encased in an isolation chamber in Lewis rats. HA-hydrogel supported growth of osteoblasts over 8 weeks and allowed sustained release of BMP-2 over 35 days. The A–V loop provided an angiogenic stimulus with the formation of vascularized tissue in the scaffolds. Bone-specific genes were detected by real time RT-PCR after 8 weeks. However, no significant amount of bone was observed histologically. The heterotopic isolation chamber in combination with absent biomechanical stimulation might explain the insufficient bone formation despite adequate expression of bone-related genes. Optimization of the interplay of osteogenic cells and osteo-inductive factors might eventually generate sufficient amounts of axially vascularized bone grafts for reconstructive surgery.     

PEG-grafted chitosan nanoparticles as an injectable carrier for sustained protein release

The development of injectable nanoparticulate “stealth” carriers for protein delivery is a major challenge. The objective of this work was to investigate the possibility of achieving the controlled release of a model protein, insulin, from PEG-grafted chitosan (PEG-g-chitosan) nanoparticles (mean diameter 150–300 nm) prepared by the ion gelation method. Insulin was efficiently incorporated into the nanoparticles, and reached as high as 38%. In vitro release showed that it could control the insulin release by choosing the composition, loading and release temperature. We observed that the composition of the nanoparticle surface (C/O ratio) increased from 2.40 to 3.23, with an increase in the incubation time. Therefore, we concluded that during this time, insulin release from PEG-g-chitosan nanoparticles followed a diffusion mechanism in which erosion was negligible. The experiments also demonstrated that PEG-g-chitosan helped to maintain the natural structure of the protein entrapped in the nanoparticles.     

Click synthesis of the thermo- and pH-sensitive hydrogels containing β-cyclodextrins

Novel intelligent hydrogels containing β-cyclodextrins were prepared by tandem physical and chemical crosslinking method based on Diels–Alder reaction. First, dienophile-functionalized cyclodextrins (HCD–AMI) were synthesized by the coupling reaction of hydroxyethyl-β-cyclodextrins and N-maleoyl alanine (AMI); diene-functionalized polymers (PFMIPA) were synthesized by free radical copolymerization of N-isopropylacrylamide and furfuryl amine maleic acid monoamide, a novel monomer synthesized in our lab. Then, the LCSTs of the PFMIPA were estimated by transmittance measurements of copolymer solutions. After the as-synthesized PFMIPA and HCD–AMI were dissolved separately in water and mixed, the hydrogels with physical crosslinks formed quickly within 10 s at 37 °C. Subsequently, chemical crosslinks came into being gradually due to Diels–Alder reaction. Therefore, there are both physical crosslinks and chemical crosslinks in as-prepared hydrogels, resulting in the improvement of the mechanical strength of the hydrogels. And the in vitro degradation behaviors of the resultant hydrogels were given a pilot study. A general gravimetric method was used to study the swelling behavior of the hydrogels. It was found that the hydrogels showed good pH/temperature-sensitivity. The strategy described here has several advantages for preparing intelligent hydrogels including tunable gelation rate, mild reaction conditions, no initiator or catalyzer, and no organic solvent. We believe that this novel, potentially biocompatible hydrogels could have biomedical applications, especially in the area of tissue engineering and drug-controlled release carriers.     

Physico-chemical and in vitro biological evaluation of strontium/calcium silicophosphate glass

Strontium is known to reduce bone resorption and stimulate bone formation. Incorporation of strontium into calcium phosphate bioceramics has been widely reported. In this work, calcium and calcium/strontium silicophosphate glasses were synthesized from the sol–gel process and their rheological, thermal, and in vitro biological properties were studied and compared to each other. The results showed that the gel viscosity and thus the rate of gel formation increased by using strontium in glass composition and by increasing aging temperature. In strontium-containing glass, the crystallization temperature increased and the type of the crystallized phase was different to that of strontium-free glass. Both glasses favored precipitation of calcium phosphate layer when they were soaked in simulated body fluid; however strontium seemed to retard the rate of precipitation slightly. The in vitro biodegradation rate of the strontium/calcium silicophosphate glass was higher than that of strontium-free one. The cell culture experiments carried out using rat calvaria osteoblasts showed that the incorporation of strontium into the glass composition stimulated proliferation of the cells and enhanced their alkaline phosphatase activity, depending on cell culture period.     

Effect of the La<Subscript>2</Subscript>O<Subscript>3</Subscript> sol–gel coating on the alumina scale adherence on a model Fe–20Cr–5Al alloy at 1100 °C

The effect of lanthanum sol–gel coatings was studied in order to improve the alumina scale adherence during the model Fe–20Cr–5Al alloy oxidation, at 1100 °C, in air. Various sol–gel coating procedures were applied. Argon annealing of the lanthanum sol–gel coating was tested at temperatures ranging between 600 and 1000 °C. The coating crystallographic nature was characterized by X-ray diffraction (XRD) depending on the annealing temperature. The oxidation process has been examined at 1100 °C by in situ XRD on blank Fe–20Cr–5Al, sol–gel coated and argon-annealed specimens. This study shows that the coating argon annealing at 1000 °C leads to the preferential formation of LaAlO₃ instead of La₂O₃. This coating procedure leads to an alumina scale formation showing the best adherence under thermal cycling conditions at 1100 °C.     

A gel-forming poly-l-guluronic acid produced from no guluronate-rich marine algae using new hydrolysis method: test for endovascular embolization

To prepare a gel-forming poly-l-guluronic acid (Poly-G) from no guluronate-rich Laminaria japonica, a new hydrolysis method was employed with a lower HCl concentration (0.025–0.15 M) and a shorter treatment time (5 min). The Poly-Gs were set to measure purity, presence of poly-l-guluronic block, molecular weight distribution, polymer yield, viscosity, and compressive gel strength. Finally, the Poly-G was tested to embolize the renal vascular system by using a rabbit model and angiography. Optimized Poly-G could be selected with respect to wt% concentration, polymer yield, gel-forming stability, viscosity, and gel strength as an endovascular embolizing agent. Overall, 0.4–0.6% of 0.03 M-Poly-G obtained from acid treatment with 0.03 M of HCl had molecular weights greater than 80 kDa, and the best gelling capacity with an injectable viscosity (30–120 cP). It was successfully delivered into the vascular bed of a rabbit kidney and was shown angiographically to embolize the renal vascular system.     

The effect of PLGA sphere diameter on rabbit mesenchymal stem cells in adipose tissue engineering

To investigate the effect of injectable PLGA sphere’s diameter on adipose tissue engineering, rabbit mesenchymal stem cells were attached to various diameters of injectable PLGA spheres (<75; 75–100; 100–150; 150–200; and 200–250 μm). These five groups were cultured in adipogenic media for 2 weeks in vitro and injected into necks of nude mice. Prior to in vivo study, cell proliferation and adipogenic differentiation were determined by hexosaminidase assay and Oil red O staining after 2 weeks. Group C (100–150 μm) showed the highest adipogenic differentiation and the proliferation capacity of Group B (75–100 μm) was significantly higher than that of any other group. We harvested newly formed tissues from necks of nude mice after 1 and 4 weeks. Although PLGA spheres have not been degraded and there was no significant histological difference among various sizes of spheres after 1 week, well-organized fat pads (PLGA spheres were completely degraded) could be observed, and the histology of the 100–150 μm groups resembled that of native tissue after 4 weeks. Based on these experiments, we could conclude that the optimal size of PLGA spheres for adipogenesis was 100–150 μm.     

In vitro macrophage uptake and in vivo biodistribution of PLA–PEG nanoparticles loaded with hemoglobin as blood substitutes: effect of PEG content

The aim of the present work is to investigate the effect of PEG content in copolymer on physicochemical properties, in vitro macrophage uptake, in vivo pharmacokinetics and biodistribution of poly(lactic acid) (PLA)–poly(ethylene glycol) (PEG) hemoglobin (Hb)-loaded nanoparticles (HbP) used as blood substitutes. The HbP were prepared from PLA and PLA–PEG copolymer of varying PEG contents (5, 10, and 20 wt%) by a modified w/o/w method and characterized with regard to their morphology, size, surface charge, drug loading, surface hydrophilicity, and PEG coating efficiency. The in vitro macrophage uptake, in vivo pharmacokinetics, and biodistribution following intravenous administration in mice of HbP labeled with 6-coumarin, were evaluated. The HbP prepared were all in the range of 100–200 nm with highest encapsulation efficiency 87.89%, surface charge −10 to −33 mV, static contact angle from 54.25° to 68.27°, and PEG coating efficiency higher than 80%. Compared with PLA HbP, PEGylation could notably avoid the macrophage uptake of HbP, in particular when the PEG content was 10 wt%, a minimum uptake (6.76%) was achieved after 1 h cultivation. In vivo, besides plasma, the major cumulative organ was the liver. All PLA–PEG HbP exhibited dramatically prolonged blood circulation and reduced liver accumulation, compared with the corresponding PLA HbP. The PEG content in copolymer affected significantly the survival time in blood. Optimum PEG coating (10 wt%) appeared to exist leading to the most prolonged blood circulation of PLA–PEG HbP, with a half-life of 34.3 h, much longer than that obtained by others (24.2 h). These results demonstrated that PEG 10 wt% modified PLA HbP with suitable size, surface charge, and surface hydrophilicity, has a promising potential as long-circulating oxygen carriers with desirable biocompatibility and biofunctionality.     

Evaluation of alginate hydrogels under in vivo–like bioreactor conditions for cartilage tissue engineering

Alginate hydrogels in forms of discs and packed beds of microbeads (~800 μm) were tested in a novel bioreactor at 10% strain using two regimes: at a loading rate of 337.5 μm/s and at sequential increments of 50 μm displacement every 30 min. Compressive strength increased with the increase in alginate concentration (1.5 vs. 2% w/w) and the content of guluronic residues (38.5 vs. 67%). Packed beds of microbeads exhibited significantly higher (~1.5–3.4 fold) compression moduli than the respective discs indicating the effects of gel form and entrapped water. Short-term cultivation of microbeads with immobilized bovine calf chondrocytes (1.5% w/w, 33 × 10⁶ cells/ml) under biomimetic conditions (dynamic compression: 1 h on/1 h off, 0.42 Hz, 10% strain) resulted in cell proliferation and bed compaction, so that the compression modulus slightly increased. Thus, the novel bioreactor demonstrated advantages in evaluation of biomaterial properties and cell-biomaterial interactions under in vivo–like settings.     

Injectable and Crosslinkable PLGA‐Based Microribbons as 3D Macroporous Stem Cell Niche

Poly(lactide‐co‐glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon‐based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon‐shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen‐coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock‐absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA μRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC‐seeded PLGA μRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA μRBs as a promising injectable, macroporous, non‐hydrogel‐based scaffold for cell delivery and tissue regeneration applications.