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Bioresorbable composites prepared by supercritical fluid foaming.

Bone is a natural composite construct, with a gradient structure going from a loose interconnected cellular core to an outer dense wall, thus minimizing bone weight while keeping a high mechanical resistance. Due to this unique and complex structure, bone defects are difficult to replace or repair. Tissue engineering aims at providing artificial bone grafts. Several techniques have been proposed to produce porous structures or scaffolds, but, as yet, with no optimal solutions. This article focuses on bioresorbable ceramic-polymer composite foams obtained by supercritical fluid foaming. This flexible technique enables an adequate morphology and suitable properties for bone tissue engineering to be obtained. Composite scaffolds are biocompatible, allowing cell proliferation and differentiation.

Absorbable Implants↗

Simple surface modification of poly(epsilon-caprolactone) to induce its apatite-forming ability.

A biodegradable polymer coated with a bone-like apatite layer on its surface is useful as a scaffold for bone tissue regeneration. In this work, a poly(epsilon-caprolactone) (PCL) surface was modified by an O2 plasma surface treatment to form oxygen-containing functional groups. The plasma-treated samples were subsequently dipped alternately in an alcoholic solution containing calcium ions and one containing phosphate ions to deposit apatite precursors on the surface. The surface-modified PCL samples formed a dense and uniform surface bone-like apatite layer after immersion for 24 h in a simulated body fluid with ion concentrations approximately equal to those of human blood plasma. This surface-modification process is applicable to two-dimensional PCL plates and three-dimensional PCL meshes. In the resulting apatite-PCL composite, the apatite layer strongly adhered to the PCL surface and remained intact after a tape-detachment test. Therefore, this type of composite material will be a useful scaffold for bone tissue engineering.

Absorbable Implants↗

Hydrolytic degradation behavior of biodegradable polyetheresteramide-based polyurethane copolymers.

In this article, a new kind of biodegradable polyetheresteramide-based polyurethane (PEEA-U) copolymers were prepared by the melt polycondensation method from epsilon-caprolactone, 6-aminocaproic acid, poly(ethylene glycol), and toluene diisocyanate. The water absorption of PEEA-U was affected strongly by the reaction time and the content of chain extender; and the hydrolytic degradation behavior of the copolymers was mainly determined by the reaction time, chain extender content, and the pH value of the degradation medium. Also, DSC, (1)H-NMR, and inherent viscosity were used to characterize the degradation behavior of the copolymers.

Absorbable Implants↗

Fabrication and evaluation of porous 2,3-dialdehydecellulose membrane as a potential biodegradable tissue-engineering scaffold.

A simple, novel method to produce porous 2,3-dialdehydecellulose (DAC) membranes as a potential tissue-engineering scaffold has been developed from methylolcellulose by the simultaneous water-induced phase separation and sodium chloride salt leaching techniques, followed by oxidation with sodium periodate in water. Membrane pores increased in size with increasing weight or particle size of the sodium chloride salt. The porosity of the membrane was not affected by the salt particle size, but it increased with an increase in the salt weight to 60%. At higher salt weight percentages, no significant change in the membrane porosity was observed. The oxidation step had no effect on the membrane porosity or pore size. All membranes with a porosity value ranging between 87 and 93% showed interconnected porous structures. The use of these membranes as a potential tissue-engineering scaffold was evaluated with the use of human neonatal skin fibroblast cells. Confocal microscopy showed cell attachment and spreading on these membranes. Immunohistochemical tests revealed the presence of collagen type III and fibronectin, indicating that the cells were viable and formed the extracellular matrix. In conclusion, the DAC membrane supports cell adhesion and proliferation and hence shows potential to be used as a tissue-engineering scaffold.

Absorbable Implants↗

A long-term in vitro biocompatibility study of a biodegradable polyurethane and its degradation products.

The biological safety of degradation products from degradable biomaterials is very important. In this study a new method is proposed to test the cytotoxicity of these degradation products with the aim to save time, laboratory animals, and research funds. A biodegradable polyurethane (PU) foam was subjected to this test method. The PU had soft segments of DL-lactide/epsilon-caprolactone and hard segments synthesized from butanediol and 1,4-butanediiosocyanate. Copolymer foams without urethane segments, consisting of DL-lactide/epsilon-caprolactone, were tested as well. Accumulated degradation products were collected by degrading the foams in distilled water at 60 degrees C up to 52 weeks. Cell-culture medium was prepared from powder medium with this water. In different tests the cytotoxicity of this medium was established. The first signs of cytotoxicity were observed after 3-5 weeks of degradation. This accounts for both materials and reestablishes the good short-term biocompatibility of these materials. The PU showed more toxicity toward the end stages of degradation in comparison with the copolymer. This is probably related to the accumulation of degradation products of the urethane segments. In the in vivo situation the degradation of the PU and the metabolism and excretion of degradation products may differ. Therefore, long-term in vivo studies will have to establish whether these in vitro results are representative for the in vivo behavior of the degrading PU.

Absorbable Implants↗

Manufacture of multimicrotubule chitosan nerve conduits with novel molds and characterization in vitro.

Multimicrotubule chitosan conduits (M-conduits) were fabricated using novel molds and a thermal-induced phase-separation technique. Hollow chitosan conduits (H-conduits) with an inner diameter of 1-5 mm and a wall thickness of 0.2-1.0 mm were made, and then a novel mold composed of a styrofoam insulating pedestal with several holes and a stainless steel cover plate was used to make M-conduits. In brief, corresponding H-conduits were inserted upright into the holes of the styrofoam pedestal, and filled with chitosan solution, then rapidly covered with the precooled stainless steel cover plate, and then placed in a freezer. The styrofoam insulating pedestal enclosing the conduits could reduce the heat transfer through the side wall of the conduits. Gradual phase separation then occurred uniaxially in the presence of a unidirectional temperature gradient from the top end to the bottom end of the chitosan conduits. The phase-separated polymer/solvent systems were then dried in a freeze-dryer. The microtubule diameters were controlled by adjusting the polymer concentration and cooling temperature. In vitro characterization demonstrated that the mold-based multimicrotubule chitosan conduits possessed suitable mechanical strength, microtubule diameter distribution, porosity, swelling, biodegradability, and nerve cell affinity, and so they showed potential for application as nerve tissue engineering scaffolds.

Absorbable Implants↗

A new biodegradable polyester elastomer for cartilage tissue engineering.

The objective of this study is to assess whether a new biodegradable elastomer, poly(1,8-octanediol citrate) (POC), would be a suitable material to engineer elastomeric scaffolds for cartilage tissue engineering. Porous POC scaffolds were prepared via the salt-leaching method and initially assessed for their ability to rapidly recover from compressive deformation (% recovery ratio). Controls consisted of scaffolds made from other materials commonly used in cartilage tissue engineering, including 2% agarose, 4% alginate, non woven poly(glycolic acid) (PGA) meshes, and non woven poly(L-lactide-co-glycolide) (PLGA) meshes. Articular chondrocytes from bovine knee were isolated and seeded onto porous disk-shaped POC scaffolds, which were subsequently cultured in vitro for up to 28 days. POC scaffolds completely recover from compressive deformation, and the stress-strain curve is typical of an elastomer (recovery ratio>98%). Agarose gel (2%) scaffolds broke during the compression test. The recovery ratio of 4% alginate gel scaffolds, PLLA, and PGA were 72, 85, and 88%, respectively. The Young's modulus of POC-chondrocyte constructs and cell-free POC scaffolds cultured for 28 days were 12.02+/-2.26 kPa and 3.27+/-0.72 kPa, respectively. After 28 days of culture, the recovery ratio of POC-chondrocyte constructs and cell-free POC scaffolds were 93% and 99%, respectively. The glycosaminoglycan (GAG) and collagen content at day 28 was 36% and 26% of that found in bovine knee cartilage explants. Histology/immunohistochemistry evaluations confirm that chondrocytes were able to attach to the pore walls within the scaffold, maintain cell phenotype, and form a cartilaginous tissue during the 28 days of culture.

Absorbable Implants↗

Effects of a biodegradable polymer synthesized with inorganic tin on the chondrogenesis of human articular chondrocytes.

Recent study has shown that biodegradable polymers are attractive candidates for chondrocyte fixation and further transplantation in cartilage tissue engineering. Poly (glycolic acid) (PGA), a polymer of glycolic acid, is widely used in orthopedic applications as a biodegradable polymer. Organotin, lead, antimony, and zinc are catalysts commonly used in synthesizing PGA. Here, we investigated the biocompatibility of PGA, synthesized with and without inorganic tin as a catalyst in chondrogenesis of human articular chondrocytes in a micromass culture system. Significant enhancement of chondrocyte proliferation and expression of the collagen type II protein gene were observed in cultures treated with PGA synthesized with a tin catalyst. However, aggrecan gene expression was very similar to the control culture. Amount of collagen type II protein was also increased in the same group of cultured chondrocytes. In contrast, PGA without a catalyst caused overall inhibition of chondrogenesis. Despite several positive findings, extensive investigations are essential for the feasibility of this PGA(Sn) in future clinical practice.

Absorbable Implants↗

A method for the effective formation of hepatocyte spheroids using a biodegradable polymer nanosphere.

Cultures of hepatocytes in spheroid form are known to maintain higher cell viability and exhibit better hepatocyte functions than those in monolayer cultures. In this study, a method for the formation of hepatocyte spheroids was developed using biodegradable polymer nanospheres. The addition of poly(lactic-co-glycolic acid) nanospheres to hepatocyte cultures in spinner flasks increased the efficiency of hepatocyte spheroid formation (the number of cells in spheroids divided by the total cell number) as compared with hepatocyte cultures without nanospheres (control). The viability and mitochondrial activity of the hepatocyte spheroids in the nanosphere-added cultures were significantly higher than those in the control. In addition, the mRNA expression levels of albumin and phenylalanine hydroxylase, both of which are hepatocyte-specific proteins, were significantly higher in the nanosphere-added cultures than in the control. This new culture method improves upon the conventional method of forming hepatocyte spheroids in terms of spheroid formation efficiency, cell viability, and hepatocyte function.

Absorbable Implants↗

Biodegradable porous polyurethane scaffolds for tissue repair and regeneration.

Critical-size bone defects usually require the insertion of autogenous bone graft to heal. Harvesting of bone is traumatic and results in high morbidity at the donor site. A potential alternative to bone graft may be a bone substitute with adequate biocompatibility and biological properties produced from ceramics or bioresorbable/biodegradable polymers. In the present study, new elastomeric biodegradable polyurethanes with an enhanced affinity toward cells and tissues were synthesized using aliphatic diisocyanate, poly(epsilon-caprolactone) diol, and biologically active 1,4:3,6-dianhydro-D-sorbitol (isosorbide diol) as chain extender. The polymers were processed into 3D porous scaffolds by applying a combined salt leaching-phase inverse process. The critical parameters controlling pore size and geometry were the solvents and nonsolvents used for scaffold preparation and the sizes of the solid porogen crystals. Scaffolds prepared from the polymer solution in solvents such as dimethylsulfoxide or methyl-2-pyrrolidone did not have a homogenous pore structure. Many pores were interconnected, but numerous pores were closed. Irrespective of the high pore-to-volume ratio (75%), the scaffolds showed poor water permeability. The best solvent for the preparation of scaffolds from the polyurethane used in the study was dimethylformamide (DMF). The type of nonsolvent admixed to the polymer solution in DMF strongly affected the scaffolds' pore structure. The elastomeric polyurethane scaffold prepared from the optimal solvent-nonsolvent mixture had regular interconnected pores, high water permeability, and a pore-to-volume ratio of 90%. The osteoconductive properties of the 3D porous polyurethane scaffolds can be additionally promoted by loading them with calcium phosphate salts such as hydroxyapatite or tricalcium phosphate, thus making them promising candidates for bone graft substitutes.

Absorbable Implants↗

Effect of radio-opaque filler on biodegradable stent properties.

The effect of the addition of a radio-opaque filler, barium sulfate (BaSO(4)), on the mechanical properties of a biodegradable amorphous polymer film (poly-lactic-co-glycolic acid, PLGA) was studied, as a function of degradation. With up to about 18% loading (v/v), the modulus of the filled polymer increases; beyond this concentration, agglomerates are formed. The filled systems are also radio-opaque, over a thickness range of 0.07-0.19 mm in stent form (helicoidal). These stents were then immersed in phosphate buffer pH 7.4 at 37(o)C for 2 weeks. The radial strength of stent was measured by using a compression test. It was found that filler-loaded stent (FS) increased in radial strength by about 4 times (14.95 +/- 1.20 N/mm) compared to the unfilled stent (UFS). However, both samples lost radial strength as the polymer degraded in buffer, but FS retained 60% (9.05 +/- 0.07 N/mm) of its strength after 2 weeks whereas only 36% (1.39 +/- 1.04 N/mm) was retained for UFS. Moreover, UFS lost its helical shape after 3 weeks. The findings have implications for optimization of degradable stent formulations.

Absorbable Implants↗

Comparative study between coral-mesenchymal stem cells-rhBMP-2 composite and auto-bone-graft in rabbit critical-sized cranial defect model.

Tissue engineered bone has become a bone substitute for the treatment of bone defects in animal research. This study investigated the osteogenesis capacity of coral-MSCs-rhBMP-2 composite with the auto-bone-graft as control. Coral-MSCs-rhBMP-2 composite were fabricated by coral (as main scaffold), rhBMP-2 (as growth factor), and MSCs (cultured from iliac marrow as seed cells). Critical-sized defects (d = 15 mm) were made on forty rabbits crania and treated by different composite scaffolds: iliac autograft (n = 8), coral (n = 8), rhBMP-2/coral (n = 8), and MSCs/rhBMP-2/coral (n = 8). The defects were evaluated by gross observation, radiographic examination, histological examination, and histological fluorescence examinations after 8 and 16 weeks. The results showed that repair of bone defect was the least in coral group, and significant ingrowth of new bone formation and incorporation could be seen with 77.45% +/- 0.52% in radiopacity in MSCs/rhBMP-2/coral group, which was similar to that in iliac autograft group (84.61% +/- 0.56% in radiopacity). New bone formation in MSCs/rhBMP-2/coral group was more than that in rhBMP-2/coral group. And osteogenesis rate in MSCs/rhBMP-2/coral group (10.23 +/- 1.45 microm) was much faster than that in rhBMP-2/coral group (5.85 +/- 2.19 microm) according to histological fluorescence examination. Newly formed bone partly came from induced MSCs in composite scaffold according to bromodeoxyuridine immunohistochemical examination. These data implicated that MSCs could produce synergic effect with coral-rhBMP-2, and the tissue engineered bone of coral-MSCs-rhBMP-2 is comparable to auto-bone-graft for the repair of critical-sized bone defect.

Absorbable Implants↗

Diffusion of bioactive molecules through the walls of the medial tissue-engineered hybrid ePTFE grafts for applications in designs of vascular tissue regeneration.

Strategies of better vascular tissue engineering may require delivery of soluble bioactive signals in cell culture medium to the cells in tissue-regenerating constructs. We measured the diffusivity and permeability of model tissue-engineering bioactive molecules such as water and heparin through the walls of both a hybrid ePTFE graft and a porcine carotid artery, a model vascular tissue. While diffusivities of H(3)-water and H(3)-heparin were measured as 3.9 x 10(-) (6) and 1.6 x 10(-) (6) cm(2)/s in the artery, respectively, under diffusional circulation of cell culture medium through the lumens of the carotid arteries, their corresponding permeabilities were 4.7 x 10(-) (5) and 2.0 x 10(-) (5) cm/s. On the other hand, diffusivities of H(3)-water and H(3)-heparin were also measured as 5.1 x 10(-) (6) and 4.7 x 10(-) (6) cm(2)/s, respectively, in the tissue-engineered hybrid ePTFE grafts; their corresponding permeabilities were 5.1 x 10(-) (5) and 3.7 x 10(-) (5) cm/s. The hybrid graft tissues were engineered by replacing the biodegradable, porous poly(lactide-co-glycolide) layers coated on the ePTFE surfaces with smooth muscle cell-derived tissues for 6 weeks. We analyzed the morphologies of the artery and the engineered hybrid ePTFE tissues with scanning electron microscopy and H&E stains. While the artery had its typical structure properties with layers of intima, media and adventitia, the tissue-engineered ePTFE hybrid graft had two layers of engineered tissues on the inner and outer surfaces of the ePTFE. There were no significant differences among the luminal tissue morphologies of the test samples from the effects of diffusion flow applications, with minor changes on their luminal surfaces. The results of water and heparin diffusion experiments indicated that these bioactive molecules were well transported from the cell culture medium to the tissue-engineering cells, enough to support tissue regeneration. We hope that these transport results may elucidate the transport behaviors of soluble nutrient molecules and biological signals through the vascular constructs under tissue engineering processes.

Absorbable Implants↗

Fabrication and in vitro characterization of porous biodegradable composites based on phosphate glasses and oligolactide-containing polymer networks.

Degradable porous composite materials for use as temporary bone replacement or tissue engineering scaffolds were produced using a methacrylate-modified oligolactide polymer network and phosphate invert glasses in the system P2O5-CaO-MgO-Na2O-(TiO2). Porous glasses with an open interconnective porosity were produced by a salt sintering process. Compressive strengths were significantly enhanced by polymer coating of the inner surface of the porous glasses or by fabrication of glass powder-reinforced porous polymer specimens. In vitro degradation in simulated body fluid showed a degradation pattern of the composites which could be modulated by the composition and resulting solubility of the incorporated glass phase. Cytocompatibility of the composites was investigated in a FDA/EtBr viability assay using an MC3T3-E1 osteoblast-like cell line and showed good biocompatibility of the materials in vitro.

3T3 Cells↗

Pores in synthetic nerve conduits are beneficial to regeneration.

Current opinion holds that pores in synthetic nerve guides facilitate nerve regeneration. Solid factual support for this opinion, however, is absent; most of the relevant studies assessed only morphological parameters and results have been contradictory. To evaluate the effect of pores, the rat sciatic nerve was either autografted or grafted with nonporous, macroporous (10-230 mum), and microporous (1-10 microm) biodegradable epsilon-caprolactone grafts. Twelve weeks later, the grafted nerves were resected, and the electrophysiological properties were determined in vitro. Subsequently midgraft-level sections were inspected, and peroneal nerve sections were evaluated morphometrically. Finally, the gastrocnemic and tibial muscle morphometrical properties were quantified. The microporous nerve graft performed much better than the nonporous and macroporous grafts with respect to most parameters: it was bridged by a free floating bundle that contained myelinated nerve fibers, there were more nerve fibers present distal to the graft, the electrophysiological response rate was higher, and the decrease in muscle cross-sectional area was markedly smaller. Hence, the present study demonstrates the beneficial effect of synthetic nerve guide pores on nerve regeneration, although with the caveat that not pores per se, but only small (1-10 microm) pores were effective.

Absorbable Implants↗

Preparation and assessment of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue engineering.

A portfolio of crosslinked chitosan:collagen blends was prepared, and their microarchitecture and water binding capacity were studied to investigate their application for adipose tissue engineering. Glutaraldehyde (GA) concentration had little effect on scaffold morphology or water binding capacity. However, the processing freezing temperature prior to lyophilization affected both. In vitro cytocompatibility of pre-adipocytes (PAs) was assessed for a candidate collagen:chitosan blend using two assays. Results confirm the viability of PAs on GA-crosslinked collagen:chitosan scaffolds. A rat subcutaneous pocket assay was employed to assess PA-seeded scaffolds in vivo. Animal tests proved that PA-seeded scaffolds were biocompatible, could induce vascularization, and form adipose tissue.

Absorbable Implants↗

A dual constant-composition titration system as an in vitro resorption model for comparing dissolution rates of calcium phosphate biomaterials.

It has been postulated that the in vivo resorption rates of calcium-phosphate bone-graft materials are closely related to their dissolution rates in demineralizing solutions having ionic compositions mimicking the acidic environment produced by osteoclasts. Thus, it should be possible to use an in vitro model to produce dissolution-rate data of calcium-phosphate materials as a starting point for predicting in vivo resorption properties. Direct pH measurements of the extracellular fluid from bone-resorbing cells showed that the pH was as low as 3. In the present study, a dual constant-composition dissolution system was used as an in vitro resorption model to compare dissolution rates of different calcium-phosphate materials. NIST standard reference hydroxyapatite (HA), dicalcium-phosphate dihydrate (DCPD), and calcium-phosphate cement (CPC) discs of known dimensions (6-mm d x 3-mm h) were allowed to dissolve at 37 degrees C in a solution that had an inorganic composition similar to that of serum ([Ca] = 1.15 mmol/l; [P] = 1.2 mmol/l; [KCl] = 133 mmol/l) and a pH of 3.0. A Ca ion-specific electrode and a pH electrode were used to control the addition of titrants to compensate for the increases in calcium and phosphate concentrations, respectively, in the demineralizing solution. The rate and stoichiometry (Ca/P molar ratio) of dissolution were obtained from the titration data. Each solid dissolved at an approximately constant rate during the dissolution process. The dissolution rates, expressed in mg cm(-2) min(-1), (mean +/- standard deviation, n = 5) were for HA: 6.58 +/- 1.22; DCPD: 21.0 +/- 2.6; and CPC: 8.21 +/- 0.73. DCPD dissolved three times faster than HA (p < 0.05). CPC dissolved 1.2 times faster than HA but the difference was not statistically significant (p > 0.05). This model can be used to study the rate and stoichiometry of dissolution of calcium-phosphate bone-graft materials and coatings under a wide range of mineral saturation conditions.

Absorbable Implants↗

In vivo efficacy of bone-marrow-coated polycaprolactone scaffolds for the reconstruction of orbital defects in the pig.

Alloplastic materials offer a number of advantages over bone autografts in the reconstruction of craniofacial defects. These include: lack of donor site morbidity, unlimited quantities of available material, and the possibility to conform exactly to the defect. An ideal bioresorbable material would degrade slowly, and have osteoconductive properties to allow replacement and remodeling by osseous tissue. This is seldom observed, the materials instead being replaced by fibrous tissue. Polycaprolactone (PCL), an FDA-approved bioresorbable polymer, has several properties that might make it suitable for reconstruction of craniofacial defects. The technique of fused deposition modeling (FDM) allows for the fabrication of highly reproducible bioresorbable 3D scaffolds. The nature of the fully interconnected pore network might enhance vascular ingrowth and osteoconductive properties. It was hypothesized that coating the scaffolds in bone marrow might enhance bone formation due to the osteoinductive nature of the bone-marrow mesenchymal cells. This study aimed to test these hypotheses in the pig model. Defects measuring 2 x 2 cm were surgically created in each orbit of eight Yorkshire pigs. The orbits were divided into three groups: Group 1 (n=4), no reconstruction (control); Group 2 (n=6), reconstruction with no coated PCL scaffolds; and Group 3 (n=6) reconstruction with bone-marrow-coated PCL scaffolds. The results were evaluated at 3 months by histological and histomorphometric analyses. The defects in Group 1 were covered with fibrous scar tissue. The shape of the reconstructed area was insufficient. The defects in Groups 2 and 3 were reconstructed correctly. In Group 2 the noncoated scaffolds showed 4.5% of new bone formation compared with 14.1% in Group 3, which is statistically significant (p<0.05). The entirely interconnected 3D polycaprolactone scaffold seems to be a promising material. It induces the bone ingrowth required for reconstructing craniofacial and orbital defects. Further long-term evaluations of these PCL scaffolds must be made in order to confirm these conclusions.

Absorbable Implants↗