Enhanced in vitro cell activity on silicon-doped vaterite/poly(lactic acid) composites

A biodegradable composite with silicon-species releasability was prepared using poly(l-lactic acid) (PLLA) and silicon-doped vaterite (SiV) particles. SiV with particle diameters of approximately 1 mum was prepared using aminopropyltriethoxysilane (APTES) as the silicon species by a carbonation process and then mixed with PLLA in methylene chloride according to a SiV to PLLA weight ratio of 1:2, resulting in the preparation of composite slurry. A composite film was prepared by dipping a cover glass in the slurry. The composite films were incubated in a culture medium for 7 days and the silicon concentration of the medium was measured to estimate the species releasability of the composites. A trace amount of silicon species was continuously released from the composites for 7 days, the amount depending on the content of APTES in SiV. On the composite releasing silicon species, mouse osteoblast-like cells (MC3T3-E1 cells) were significantly stimulated to proliferate and differentiate in comparison with those on a composite containing no silicon species. The proliferation of the cells on the composites releasing larger amounts of silicon species (0.51mgl(-1)day(-1)) was higher than that on the composites releasing smaller amount of the species (0.21mgl(-1)day(-1)). The silicon species in the composites were effective in enhancing the cellular functions. The composites were expected to be useful as a scaffold material for bone tissue engineering.     

Preparation of poly(L-lactic acid)-polysiloxane-calcium carbonate hybrid membranes for guided bone regeneration

A novel poly(L-lactic acid) (PLLA)/calcium carbonates hybrid membrane containing polysiloxane was prepared using aminopropyltriethoxysilane (APTES) for biodegradable bone-guided regeneration. Carboxy groups in the PLLA made a chemical bond with amino groups in APTES, resulting in the formation of the hybrid membrane. The polysiloxane-hybridized PLLA was an amorphous phase. The membrane formed hydroxycarbonate apatite (HCA) on its surface after 3d of soaking in simulated body fluid (SBF). X-ray energy-dispersive spectroscopy showed that the HCA layer includes Si with Ca and P. After soaking the membrane in SBF, almost no Si was present in SBF. The membrane is expected to be a satisfactory substrate for the formation of the silicon-containing HCA layer using the SBF-soaking method. A result of osteoblast-like cellular proliferation on the membrane and the membrane coated with silicon-containing HCA showed no cell toxicity. The membrane coated with silicon-containing HCA had much higher cell-proliferation ability than the membrane.     

Electrospinning biomedical nanocomposite fibers of hydroxyapatite/poly(lactic acid) for bone regeneration

Development of fibrous matrices of bioceramic-biopolymer nanocomposite offers great potential in the field of bone regeneration and tissue engineering. However, in order to produce electrospun fibers with homogeneous structure, it is essential for the ceramic powder to be fine and to remain stable in suspension. Herein, we developed a novel method whereby the bioceramic hydroxyapatite (HA) was kept in suspension in biopolymer poly(lactic acid) (PLA). The strategy was to introduce a surfactant hydroxysteric acid (HSA) between the hydrophilic HA powder and the hydrophobic chloroform-dissolved PLA. The HA nanopowder was dispersed effectively in HSA and mixed homogeneously with PLA. Continuous and uniform fibers were generated successfully with diameters of approximately 1-2 microm, and featured a well-developed nanocomposite structure of HA nanopowder-dispersed PLA. Initial cellular assays showed excellent cell attachment and proliferation and also enhanced expression of alkaline phosphatase at 7 days of culturing. The HA-PLA nanocomposite fibers may be potentially useful in tissue engineering applications, particularly as three-dimensional substrates for bone growth.     

Preparation of poly(lactic acid) composites containing calcium carbonate (vaterite)

A new type of ceramic-polymer biomaterial having excellent apatite-forming ability in simulated body fluid was prepared by hot-pressing a mixture of poly(-L-lactic acid) (PLA) and calcium carbonate (vaterite). After PLA dissolved in methylene chloride was mixed with calcium carbonate consisting of vaterite, the mixture was dried completely and subsequently hot-pressed uniaxially under a pressure of 40 MPa at 180 degrees C. When 30 wt% vaterite was introduced, the modulus of elasticity was effectively improved by 3.5-6 GPa, which was about twice higher than the modulus of PLA. The composite showed no brittle fracture behavior and a comparably high bending strength of approximately 50 MPa. The composite containing 30 wt% vaterite formed a 5-15-microm-thick bonelike apatite layer on its surface after soaking in SBF at 37 degrees C even for 1-3d.     

Electrospun poly(lactic acid-co-glycolic acid) scaffolds for skin tissue engineering

Electrospun fiber matrices composed of scaffolds of varying fiber diameters were investigated for potential application of severe skin loss. Few systematic studies have been performed to examine the effect of varying fiber diameter electrospun fiber matrices for skin regeneration. The present study reports the fabrication of poly[lactic acid-co-glycolic acid] (PLAGA) matrices with fiber diameters of 150-225, 200-300, 250-467, 500-900, 600-1200, 2500-3000 and 3250-6000nm via electrospinning. All fiber matrices found to have a tensile modulus from 39.23+/-8.15 to 79.21+/-13.71MPa which falls in the range for normal human skin. Further, the porous fiber matrices have porosity between 38 to 60% and average pore diameters between 10 to 14mum. We evaluated the efficacy of these biodegradable fiber matrices as skin substitutes by seeding them with human skin fibroblasts (hSF). Human skin fibroblasts acquired a well spread morphology and showed significant progressive growth on fiber matrices in the 350-1100nm diameter range. Collagen type III gene expression was significantly up-regulated in hSF seeded on matrices with fiber diameters in the range of 350-1100nm. Based on the need, the proposed fiber skin substitutes can be successfully fabricated and optimized for skin fibroblast attachment and growth.     

Hydrophilized polycaprolactone nanofiber mesh-embedded poly(glycolic-co-lactic acid) membrane for effective guided bone regeneration

A novel guided bone regeneration (GBR) membrane was fabricated by an immersion precipitation of poly (glycolic-co-lactic acid) (PLGA)/Pluronic F127 solution impregnated in an electrospun polycaprolactone (PCL)/Tween 80 nanofiber mesh. The prepared PCL/Tween 80 nanofiber mesh-embedded PLGA/Pluronic F127 membrane (hydrophilized PCL/PLGA hybrid membrane) had nano-size pores on the top side (which can prevent from fibrous connective tissue infiltration but allow permeation of oxygen and nutrients) and micro-size pores on the bottom side (which can improve adhesiveness with bone). From the comparisons of mechanical properties (tensile and suture pullout strengths), model nutrient (FITC-labeled bovine serum albumin) permeability, and bone regeneration behavior using a rat model (skull bone defect) of the hybrid membrane with those of PLGA/Pluronic F127 membrane (asymmetrically porous, hydrophilized PLGA membrane), PCL/Tween 80 nanofiber mesh (electrospun, hydrophilized PCL nanofiber mesh), and a commercialized GBR membrane, Bio-Gide (collagen type I/III membrane), it was observed that the PCL/PLGA hybrid membrane seems to be highly desirable as a GBR membrane for the selective permeability caused by its unique morphology and osteoconductivity provided by several tens micro-size pores of the bottom side as well as the excellent mechanical strengths by the hybridization of porous PLGA membrane and PCL nanofiber mesh.     

3D-printed poly(lactic acid) scaffolds for trabecular bone repair and regeneration: scaffold and native bone characterization

Purpose: Study objectives were set to (i) fabricate 3D-printed scaffolds/grafts with varying pore sizes, (ii) characterize surface and mechanical properties of scaffolds, (iii) characterize biomechanical properties of bovine trabecular bone, and (iv) evaluate attachment and proliferation of human bone marrow mesenchymal stem cells on 3D-printed scaffolds.

Materials and Methods: Poly(lactic acid) scaffolds were fabricated using 3D-printing technology, and characterized in terms of their surface as well as compressive mechanical properties. Trabecular bone specimens were obtained from bovine and characterized biomechanically under compression. Human bone marrow mesenchymal stem cells were seeded on the scaffolds, and their attachment capacity and proliferation were evaluated.

Results: Contact angles and compressive moduli of scaffolds decreased with increasing pore dimensions of 0.5 mm, 1.0 mm, and 1.25 mm. Biomechanical characterization of trabecular bone yielded higher modulus values as compared to scaffolds with all pore sizes studied. Human bone marrow mesenchymal stem cells attached to the surfaces of all scaffolds yet proliferated more on scaffolds with 1.25 mm pore size.

Conclusions: Collectively, given the similarity between 3D-printed scaffolds and native bone in terms of pore size, porosity, and appropriate mechanical properties of scaffolds, the 3D-printed poly(lactic acid) (PLA) scaffolds of this study appear as candidate substitutes for bone repair and regeneration.     

Poly-L-lactic acid/hydroxyapatite hybrid membrane for bone tissue regeneration

Poly-L-lactic acid (PLLA)/hydroxyapatite (HA) hybrid membranes were fabricated via electrospinning of the PLLA/HA dispersion for use in bone tissue regeneration. The structural properties and morphologies of PLLA and PLLA/HA hybrid membrane were investigated by measuring the Brunauer-Emmett-Teller specific surface area, observations of SEM, and TEM. The dispersion and integrating of HA nanoparticles in the hybrid membrane were studied by energy dispersion X-ray analysis and FTIR. The mechanical properties of PLLA/HA membrane were also measured by tensile tests. For exploring biological behaviors of the hybrid membrane, in vitro degradation tests were carried out. The osteoblast cell (MG-63) was cultured in PLLA/HA hybrid membrane extract containing medium; the cell adhesion and growth capability were investigated by SEM observation and MTT assay. HA nanoparticles were not only dispersed in the PLLA but also reacted with the functional group of PLLA, resulting in strong surface bonding and high tensile strength of hybrid membrane. The cell adhesion and growth on the PLLA/HA hybrid membrane were far better than those on the pure PLLA membrane, which proves that the PLLA/HA hybrid membrane can be one of the promising biomaterials for bone tissue regeneration.     

Control of silicon species released from poly(lactic acid)-polysiloxane hybrid membranes

Novel hybrid membranes consisting of poly(L-lactic acid) (PLLA), aminopropyltriethoxysilane (APTES), and calcium carbonates were prepared for bioresorbable guided bone regeneration. A molecular chain of PLLA was bonded at the end of an organic chain in APTES through the amide bond formed between carboxy-groups in PLLA and amino-groups in ATPES. As a result, the hybrid membrane was formed. The PLLA in the membrane was an amorphous phase. By heating the membrane at 100 degrees C for 1 h, the PLLA in the membrane crystallized and some organic chains in APTES and amide bonds decomposed. Moreover, numerous pores of 0.5-1 microm in diameter were newly formed at the surface. When the membranes before and after heat treatment were soaked in simulated body fluid, the amount of silicon species in SBF released from the membrane after heat treatment was higher than that before heat treatment. A test of osteoblast-like cellular proliferation on the membrane showed that the membrane after heat treatment has much higher cell-proliferation ability than that before heat treatment.     

Poly(L-lactide)acid/alginate composite membranes for guided tissue regeneration

The barrier membranes for guided tissue regeneration (GTR) to treat bone defects have to satisfy the criteria of biocompatibility, cell-occlusiveness, space-making, tissue integration and clinical manageability. In this study a system constituted of a poly(L-lactide) acid (PLLA) asymmetric membrane combined with an alginate film was prepared. The PLLA membrane functions to both support the alginate film and separate the soft tissue; the alginate film is intended to act as potential vehicle for the growth factors to promote osteogenesis. The structural, morphological, and mechanical properties of the bilamellar membrane and its stability in culture medium were evaluated. Moreover, the feasibility of using the alginate membranes as controlled-release delivery vehicles of TGF-beta was monitored. Finally, the bacterial adhesion and permeability of Streptococcus mutans, selected for the high adhesive affinity, were monitored. The results showed that the surfaces of the alginate side, to be used in contact with the bone defect, were rougher than PLLA ones. When in contact with complete culture medium, the PLLA-alginate membrane retained its mechanical and structural properties for more than 100 days. Then, the degradation processes occurred but the membrane continued to be stable and manageable for 6 months. Growth factors such as TGF-beta can be incorporated into alginate membranes functioning as drug delivery vehicle, and retain the biological activity when tested in an in vitro model system. The obtained membrane acted as a barrier to the passage of S. mutans bacteria and showed to promote a lower bacterial adhesion with respect to commercial GTR membranes.     

Enhanced guided bone regeneration by controlled tetracycline release from poly(L-lactide) barrier membranes

With the aim of providing effective periodontal therapeutic modality, drug-releasing membranes for guided tissue regeneration (GTR) were developed. As GTR membranes, biodegradable barrier membranes composed of porous poly(L-lactide) (PLLA) films cast on poly(glycolide) (PGA) meshes were fabricated using an in-air drying phase inversion technique. PLLA was dissolved in methylene chloride-ethylacetate mixtures, cast on knitted PGA mesh, and then air-dried. Tetracycline, which is used in periodontal therapy because of its antibacterial activity and tissue regenerating effects, including osteoblast chemotactic effect and anti-collagenolytic activity, was incorporated into the membranes by adding it to PLLA solutions. The guided bone regenerating potential of tetracycline-loaded membranes was evaluated using release kinetics both in vitro and in vivo, biodegradation tests, and cell attachment tests. Homogeneous pores were generated both at the surface and in a sublayer of the membranes. The release kinetics of tetracycline depended mainly upon the hydrophilicity of tetracycline and the porosity of the membrane. The release rate further could be controlled by loaded drug contents. The release of tetracycline was appropriate for maintaining anti-microbial activity and for its tissue-regenerating potential. The membranes retained a proper degradation property, maintaining their mechanical integrity for the barrier function for 4 weeks. Tetracycline-loaded membranes induced increased cell attachment levels compared with those of unloaded membranes. Tetracycline-loaded membranes markedly increased new bone formation in rat calvarial defects and induced bony reunion after 2 weeks of implantation. These results suggest that tetracycline-loaded PLLA membranes potentially enhance guided tissue regenerative efficacy.     

In vitro evaluation of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds for bone tissue engineering

A three-dimensional (3-D) scaffold is one of the major components in many tissue engineering approaches. We developed novel 3-D chitosan/poly(lactic acid-glycolic acid) (PLAGA) composite porous scaffolds by sintering together composite chitosan/PLAGA microspheres for bone tissue engineering applications. Pore sizes, pore volume, and mechanical properties of the scaffolds can be manipulated by controlling fabrication parameters, including sintering temperature and sintering time. The sintered microsphere scaffolds had a total pore volume between 28% and 37% with median pore size in the range 170-200microm. The compressive modulus and compressive strength of the scaffolds are in the range of trabecular bone making them suitable as scaffolds for load-bearing bone tissue engineering. In addition, MC3T3-E1 osteoblast-like cells proliferated well on the composite scaffolds as compared to PLAGA scaffolds. It was also shown that the presence of chitosan on microsphere surfaces increased the alkaline phosphatase activity of the cells cultured on the composite scaffolds and up-regulated gene expression of alkaline phosphatase, osteopontin, and bone sialoprotein.     

Development of guided bone regeneration membrane composed of beta-tricalcium phosphate and poly (L-lactide-co-glycolide-co-epsilon-caprolactone) composites

To create biodegradable and thermoplastic materials for guided bone regeneration, GBR, and guided tissue regeneration, GTR, membranes, composites of beta-tricalcium phosphate, TCP, and biodegradable polyesters, poly (L-lactide-co-glycolide-co-epsilon-caprolactone), PLGC, and poly (L-lactide-co-epsilon-caprolactone), PLCL, were prepared by a heat-kneading method. The composites maintained thermoplasticity and mechanical strength by formation of a chemical interaction between Ca on TCP and C=O on the lactide segment of PLGC or PLCL. The composites also indicated composite effects in pH auto-regulation property and elongation of biodegradation period, e.g., the composites maintained their mechanical strength up to 12 weeks after soaking in both physiological and phosphate-buffered saline, and the period was sufficient time to use for GBR and GTR membranes. Animal tests for GBR indicated that the present composite membrane successfully regenerated beagles' mandible defects 10 x 10 x 10 mm3 in size. These results suggested that the TCP/PLGC bioresorbable composites could be utilized for GBR and GTR therapy.     

Guided bone regeneration by poly(lactic-co-glycolic acid) grafted hyaluronic acid bi-layer films for periodontal barrier applications

A novel protocol for the synthesis of biocompatible and degradation controlled poly(lactic-co-glycolic acid) grafted hyaluronic acid (HA-PLGA) was successfully developed for periodontal barrier applications. HA was chemically modified with adipic acid dihydrazide (ADH) in the mixed solvent of water and ethanol, which resulted in a high degree of HA modification up to 85 mol.%. The stability of HA-ADH to enzymatic degradation by hyaluronidase increased with ADH content in HA-ADH. When the ADH content in HA-ADH was higher than 80 mol.%, HA-ADH became soluble in dimethyl sulfoxide and could be grafted to the activated PLGA with N,N′-dicyclohexyl carbodiimide and N-hydroxysuccinimide. The resulting HA-PLGA was used for the preparation of biphasic periodontal barrier membranes in chloroform. According to in vitro hydrolytic degradation tests in phosphate buffered saline, HA-PLGA/PLGA blend film with a weight ratio of 1/2 degraded relatively slowly compared to PLGA film and HA coated PLGA film. Four different samples of a control, OSSIX^TM membrane, PLGA film, and HA-PLGA/PLGA film were assessed as periodontal barrier membranes for the calvarial critical size bone defects in SD rats. Histological and histomorphometric analyses revealed that HA-PLGA/PLGA film resulted in the most effective bone regeneration compared to other samples with a regenerated bone area of 63.1% covering the bone defect area.     

A poly(lactic acid)/calcium metaphosphate composite for bone tissue engineering

A new method to prepare PLA/CMP (poly-L-lactide/calcium metaphosphate) composite scaffolds was developed for effective bone tissue engineering. This novel sintering method is composed of pressing the mixture of PLA, CMP, and salt particles at 150 MPa for 3 min followed by heat treatment at 210 degrees C for 30 min. The scaffolds had a homogeneously interconnected porous structure without a skin layer, and they exhibited a narrower pore size distribution and higher mechanical strength in comparison with scaffolds made by a solvent casting method. The scaffolds were seeded by osteoblasts and cultured in vitro or implanted into nude mice subcutaneously for up to 5 weeks. The number of cells attached to and proliferated on the scaffolds at both in vitro and in vivo was in the order of; PLA by novel sintering < PLA/CMP by solvent casting < PLA/CMP by novel sintering. In addition, the alkaline phosphatase activity of and calcium deposition in the scaffolds explanted from mice were enhanced significantly for the scaffolds by novel sintering compared to them by solvent casting. The in vitro results agreed well with the in vivo data. Such a superior characteristic of the novel sintering method should have resulted from the fact that the CMP particles could contact directly with cells/tissues to stimulate the cell proliferation and osteogenic differentiation, while the CMP particles would be coated by polymers and hindered to interact with cells/tissues in the case of a solvent casting method. As the novel sintering method does not use any solvents it offers another advantage to avoid problems associated with solvent residue.     

Molded porous poly (L-lactide) membranes for guided bone regeneration with enhanced effects by controlled growth factor release

The aim of this study was to develop platelet-derived growth factor (PDGF-BB) loaded moldable porous poly (L-lactide) (PLLA)-tricalcium phosphate (TCP) membranes for guided bone regeneration (GBR) therapy. The membranes were designed to fit various types of bone defect sites. PDGF-BB-dissolved PLLA-TCP in methylene chloride-ethyl acetate solution was cast on a dome shaped metallic mold to fabricate a model membrane. The release rate of PDGF-BB, the osteoblast attachment test, and guided bone regeneration potential were evaluated with PDGF-BB-loaded PLLA-TCP membranes. Regular pores were generated throughout the membrane mainly due to phase inversion of PLLA-methylene chloride-ethyl acetate solution. A therapeutic amount of PDGF-BB was released from the membrane. The release rate could be controlled by varying the initial loading content of PDGF-BB. A significant amount of cells attached onto the PDGF-BB-loaded membrane rather than onto the unloaded membrane. Dome shaped bone formation was achieved in rabbit calvaria at 4 weeks. This indicated that restoration of bone defects to the bone's original shape can be made possible by using molded membranes, which guide bone regeneration along with providing sufficient spaces. Bone forming efficiency was increased remarkably due to PDGF-BB release from PLLA-TCP membranes. These results suggested that the PDGF-BB releasing molded PLLA-TCP membrane may potentially improve GBR efficiency in various types of bone defects.     

Biodegradable poly (lactic acid) microspheres for drug delivery systems

In connection with aim of maximizing the bio-availability of conventional drugs with minimum side-effects, new drug delivery systems (DDS) continue to attracted much attention. The controlled or sustained release of drugs represents one such approach, and in this regard report upon a study of DDS using biodegradable polymers which include poly (lactic acid) (PLA), poly (glycolic acid), and their copolymers (PLGA). Much attention is being paid to the controlled release of bio-active agents from microcapsules and microspheres made of biodegradable polymers, such as lactic acid homopolymers, as well as copolymers of glycolic acid. (11-21) Microcapsules or microspheres are injectable and able to provide pre-programmed durations of action, offering several advantages over the conventional dosage forms. This article reviews the results of a work program conducted in collaboration with a medical doctor upon DDS using biodegradable microspheres, such as PLA and PLGA.     

Fabrication of novel calcium phosphate/poly(lactic acid) fiber composites

Composites using high-modulus polylactic acid (PLA) fibers coated with calcium phosphate (CaP) were prepared using a cyclic precipitation technique. Scanning electron microscopy revealed that small nuclei of CaP formed after the first soaking cycle, while large quantities of CaP particles were observed after the sixth cycle. The amount of CaP deposited on the PLA yarn increased with deposition time in Ca(2+) and PO(4) (3-) solutions and number of cycles, and decreased with stirring rate during washing cycles. It was observed that around 35 wt % of CaP was deposited on the yarn surface after six cycles of cyclic-soaking. Based on the results, a heterogeneous nucleation and growth mechanism was proposed for the CaP deposition on the surface of hydrolyzed polyester. Composites comprising the coated fibers in a poly(caprolactone) matrix exhibited flexural moduli within the range of that of the cortical bone.